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J ‘Hhkpijb- 463-31: THE MECHANISM OF VIRAL CARCINOGENESIS -- MOLECULAR CHARACTERIZATION OF LYMPHOMAGENESIS BY AVIAN RNA TUMOR VIRUS BY Mohammad Reza Noori-Daloii A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program 1981 ABSTRACT THE MECHANISM OF VIRAL CARCINOGENESIS -- MOLECULAR CHARACTERIZATION OF LYMPHOMAGENESIS BY AVIAN RNA TUMOR VIRUS BY Mohammad Reza Noori-Daloii Certain cancers in animals are caused by RNA tumor viruses. These viruses are useful agents for studying the oncogenic processes. The "chronic" viruses are one of the two major general classes of oncogenic retro- viruses. These viruses induce neoplasms only after a long latent period and do not contain known transforming genes. They also do not encode any oncogenic proteins and fail to transform fibroblasts in 23359. These viruses are likely to be responsible for the majority of naturally occurring neoplasms associated with RNA tumor virus (retrovirus) infection. The present study has focused on the molecular characterization of the bursal and metastatic lymphomas induced by chicken syncytialvirus (CSV - a member of the reticuloendotheliosis virus family), genetically and anit- genetically unrelated to ALV (avian leukosis virus). How- ever, both groups of these viruses are capable of inducing lymphomas in chicken with similar incidence and pathology. Lymphoid leukosis is bursa dependent B-cell lymphoma. Mohammad Reza Noori-Daloii By application of modern molecular biology techni— ques attempts were made to obtain insight into the mole- cular events involved in the mechanism of CSV-induced lymphomagenesis. The structure of newly integrated CSV- exogenous provirus DNA was characterized in more than 30 lymphoma tumors. The major findings are summarized below: (a) The structure of the unintegrated CSV DNA was analyzed. (b) The CSV proviruses of the primary bursal and metastatic tumors characterized by an EcoRI enzyme indicated that for the majority of the tumors the "TS" (referred to as tumor- specific) bands fall into six size classes, which suggest that CSV integration in at least six different sites of the chromosome can lead to development of tumors. (c) Each tumor contains from one up to four proviruses and many tumors carry a single provirus, suggesting that the pre- sence of a single provirus is sufficient for lymphoma- genesis. (d) tumors in infected males appear to have rela- tively fewer numbers of TS bands than those of the female. Also, deletions and other structural alterations appear to occur more frequently in the female birds. (e) All tumors examined display at least one distinct "TS" band and in the vast majority of tumors metastatic tumors appeared to have proviral DNA patterns identical to the primary bursal target; this is consistent with the clonal origin of tumors. (f) While in a few cases the complete structure of the provirus apparently may not be needed for neoplastic Mohammad Reza Noori-Daloii transformation, in many tumors the presence of apparently complete viral genomes seems to indicate that extensive deletion is not a requirement of transformation. In addition, deletions in the adjacent cellular sequence probably occurred in some of the tumors. (9) Analysis of the DNA of several infected birds at the preleukotic stage Suggests random integration of CSV viral genes into the host genome. (h) The present study indicates that in some of the tumors the CSV provirus and the adja- cent host sequences have undergone amplification. (i) Analysis in all samples including the uninfected con- trol shows the presence of a band which presumably repre- sents the endogenous REV sequence. Such a fragment has not previously been reported. (j) Finally and more import- antly the proviral DNA in nearly all tumors is integrated next to the "c-myc" (the progenitor sequence of the onco- gene of acute leukemia virus, MC-29) gene. These data are compatible with recent findings that ALV proviruses are linked to the "c-myc" gene in ALV induced tumors and strengthens the hypothesis that "c-myc" and its adjacent sequences are important in B lymphocyte transformation. The significance of these findings are discussed in the present thesis. © 1981 MOHAMMAD REZA NOORI-DALOII All Rights Reserved DEDICATION To the heroic revolutionary Moslem people of Iran, and to freedom-fighters and lovers of independence and justice around the world. ii ACKNOWLEDGMENTS I am grateful to Dr. H. J. Kung for his guidance and financial support during the course of this research. I would like to express my special thanks to Dr. L. Snyder for his friendship, guidance and careful reviews of the manuscript of this thesis. Also, my thanks to other mem- bers of my guidance committee, Drs. L. W. Mericle and P. Carlson. My thanks also to Dr. R. Witter and his associates for supplying the tumor tissues. Finally, my thanks to Drs. J. M. Bishop and H. Temin for providing me the cloned DNAs for the syntheses of radioactive probes. This work was conducted at the Biochemistry Department of Michigan State University and was supported in part by Public Health Service Grant CA 24798-01 and by a grant from Michigan State University foundation awarded to Dr. H. J. Kung. iii LIST OF LIST OF LIST OF TABLE OF CONTENTS TABLES O O O O O O O O O O O O O O O O O O FIGUES O O O O O O O O O O O O O O O O O ABBMVIATIONS O O O O O O O O O O O O O 0 CHAPTER ONE - INTRODUCTION AND LITERATURE REVIEW . I. II. In trOduc tion I O O I O I O O O O O O O 0 Literature Review . . . . . . . . . . . . A. B. CHAPTER TWO I. II. A Brief Summary on Retroviruses . . . Reticuloendotheliosis Viruses (REVS) Classification . . . . . . . . . . . l. Reticuloendotheliosis Virus Strain T (REV-T) . . . . . . . . Chick Syncytial Virus (CSV) . . . Spleen Necrosis Virus (SNV) . . . Duck- Infectious Anemia Virus (DIAV)............. On the Analysis of the Nucleic Acid Components in REV . . . . . . . . . . On the Comparison of REVs and Avian Leukosis Sarcoma Viruses (ALSV) . . . 1. Some Major Similarities . . . . . 2. Some Major Differences . . . . Extent of Phenotypic Mixing Between REVS and ALSVs . . . . . . . . . . On the Pathology of REVs . . . . . . On the Mechanisms of Retrovirus Repli- cation and Integration . . . . . . . £50030 0 o o - MATERIALS AND METHODS . . . . . . . Materials . . a . . . . . . . . . A. A Few Notes on the Materials . . . . B. Tissue Culture Medium and Most Common Buffers and Reagents . . . . . C. Restriction Digestion Enzymes . . . . D. Plasmid Growing, Amplification, Iso- lation and Transformation . . . . . . Methods . . . . . . . . . . . . . . . . . A. B. Induction of Lymphomas in Infected ChiCkens O O O O O O O O O O O O O 0 Cells and Viruses . . . . . . . . . . iv 44 45 45 46 TABLE OF CONTENTS (Continued) CHAPTER TWO - continued II. (continued) C. Preparation of Purified CSV Particles and 60- 708 RNA . . . . . . . . . . . . . 47 D. CSV In Vitro Infection, DNA Extrac- tion and Purification . . . . . . . . . 49 E. Extraction of High Molecular Weight DNA From Chicken Tissue . . . . . . . . . 51 F. Digestion of DNA Samples with Restric- tion Endonucleases, Agarose Gel Elec- trophoresis and DNA Transfer to Nitro- cellulose Filter Papers . . . . . . . . . 52 G. Hybridization, washing and Autoradio- graphy . . . . . . . . . . 54 H. Rehybridization of Filters . . . . . . . 55 I. Growth, Amplification and Isolation of Normal Plasmid and Recombinant DNA Clones . . . . . . . . . . 56 1. Growth of Cells and Amplification of Clones . . . . . . . . . . . . . . S6 2. Cell Lysis . . . . . . . . . . 56 3. Cesium Chloride Gradient Centrifu- gation and/or Hybroxyapatite (HAP) Purification . . . . . . . . . . . . . 57 J. Transformation of E. coli H8101 . . . . . 58 K. Further Purification of DNA for Molecular Hybridization Probe . . . . . . . . . . . 58 L. Labeling of DNA with P32 by Nick-Trans— lation (Preparation of CDNA Probe) . . . . 60 CHAPTER THREE - RESULTS . . . . . . . . . . . . . . . . 62 I. A Brief Background to the Biology and Experimental Design . . . . . . . 62 II. Strategies for the Identification of CSV Exogenous Proviruses in the Induced Tumors . . 64 III. The CSV Endogenous EcoRI Fragments . . . . . . 68 IV. The Mode of Integration of CSV-Proviruses . . 73 A. A Survey of Integration Sites of the CSV-Proviral Bursal DNAs . . . . . . . . . 73 B. Abundance of a Single Integration Site of the Proviruses in the Primary (Bursal) Tumors . . . . . . . . 79 C. CSV-Induced Bursal Tumors May be "Clonal" in Origin . . . . . . . . . . . . . . . 80 V. On the Mechanism of Deletion in CSV- Induced Avian Lymphomas . . . . . . . . . . . 88 CHAPTER V. VI. VII. VIII. IX. X. TABLE OF CONTENTS (Continued) THREE - continued (continued) A. A Comparison of the Structure of the Single "TS" CSV-Proviruses in Tumor No. 1 and No. 2 . . . . . . . . . . . B. The Proviruses Having Abnormal Structure . . . . . . . . . . . . . . Amplification of the Proviruses . . . Linkage of the CSV Provirus With the Mc-29 Related Endogenous Sequences ("C-myc") . . A. A Survey of the Linkage of the "TS" Bands by MC Probe . . . . . B. Appearance of Multiple Linkage of the CSV Provirus With the Mc- 29 Related Endo-Sequences . . . . . . . . . . C. No Apparent Linkage of the CSV Provirus With the Mc- 29 Related Endogenous Sequences . . . . . . . . . . . The Structure of Other "Potential Oncogenes" in the Tumors . . . . . . . . . . . . . . . The Relationship Between Primary and Meta- static Tumors . . . . . EcoRI Analysis of Infected Birds at What is Presumably a Preleukosis Stage . . . . . CWTER FOUR - DISCUSSION 0 O O I O C O C O O O O O I. The Structure and the Mode of Integration of the CSV-Proviruses . . . . . . . . . . II. The Clonality of the Primary and Metastatic Tumors . . . . . . . . . . . . . . . . . . III. Deletion in CSV Provirus in Avian Lymphomas IV. Analysis of Infected Birds at the Pre- leukosis Stage . . . . . . . . . . . . . . V. CSV-Endogenous EcoRI Fragments . . . . . . VI. Amplification of the CSV-Proviruses and Their Adjacent "C-myc" Gene in a Few Tumors VII. Linkage of the CSV Provirus with the Mc-29 Related Endogenous Sequences . . . . . . . VIII. On.the Mechanism of Viral Lymphomagenic Transformation and Speculations on "Cancer" Induction . . . . . . . . . . . . . . . . . Ix. Summary . . . . . . . . . . . . . . . . . . APPENDIX . . . . . . . . . . . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . . . . vi 88 94 95 96 96 99 99 103 103 112 116 116 117 118 120 121 121 123 125 137 139 149 Table LIST OF TABLES Page Identification of CSV provirus and "C-myc" locus by EcoRI-digestion . . . . . . . . . . . 75 The frequency of occurrence of the most common "TS" bands in 15 bursal tumors . . . . 73 The frequency of occurrence of the most common "TS" bands in bursal and metastatic tumors I O I O O O O O I O O I O O O O O O O O 8 2 vii Figure 10 ll 12 LIST OF FIGURES The haploid genomes of ASV (avian sarcoma virus), LLV (avian leukosis virus) and RAV-O (Rouse-associated endogenous virus) . The structure of RNA genome, linear and integrated retroviral DNAs . . . . . . . . Replication and integration scheme of a typical retrovirus . . . . . . . . . . . . The structure of unintegrated linear CSV DNA 0 O O I O O O O O O I O O I O O O O O O The structure of CSV'proviruses and the "C-myc" gene in lymphoma as analyzed by EcoRI-digestion analysis . . . . . . . . . EcoRI analysis of bursal DNAs from CSV- induced lymphomas . . . . . . . . . . . . . A comparison of the structure of the CSV proviruses in tumor No. 1 and No. 2 and Bgl II enzyme analysis of some lymphoma tumors . . . . . . . . . . . . . . . . . . Hypothetical structure of the CSV proviral DNA in tumor number 1 and tumor number 2 . The structure of CSV provirus in the No. 3 bursal DNA - the lack of linkage with "C-myc" . . . . . . . . . . . . . . . . . . The structure of other putative oncogenes in the lymphoma and normal tissues . . . . Demonstration of Clonality of bursal tumors and metastases by digestion of tumor DNAs With EC ORI en zyme O O I O O O O O O I I I O EcoRI restriction endonuclease analysis of DNA from CSV infected birds at preleukosis stage I O O O O O O I O O O O O O O O O O 0 viii 10 38 67 70 86 90 92 102 105 109 115 RAV-O SNV DIAV LLV ALSV MULV M-MULV Qt6 CEF "onc" "C-onc" LIST OF ABBREVIATIONS Avian Leukosis Virus Chicken Syncitial Virus Reticuloendotheliosis Virus Avian Sarcoma Virus Avian Myelocytomatosis Virus Avian Erythroblastosis Avian Myeloblastosis Virus Rous Associated Endogenous Virus Spleen Necrosis Virus Duck-Infectious Anemia Virus Avian Leukosis Virus Avian Leukosis Sarcoma Virus Murine Leukemia Virus Moloney Murine Leukemia Virus Marek's Disease Herpes-Virus Quail transforming virus Chicken Embryo Fibroblast viral oncogene cellular oncogene oncogene of MC29 virus oncogene of ARV oncogene of AMV oncogene of moloney murine sarcoma virus II Src fl "C-myc" endo endo "C-mos” LL EM «Cu ”LTR"(U3R-U gs ts VLds DNA CCCVD SSCT DNA ys ll TS" PR dZHZO FCS f.c DPA 5) oncogene of ASV the cellular progenitor sequence of the oncogene of MC29 virus endogenous MC29 endogenous REV cellular oncogene of M-MUSV lymphoid leukosis electro-microscope "common region" "Long Terminal Repeat" group specific temperature sensitive viral linear double-stranded DNA covalently closed circular viral DNA single stranded calf thymus DNA yolk sac intraabdominal or intravenous room temperature ultra-violet reverse transcriptase plasmid tumor specific Roginal poultry double-distilled water fetal calf serum final concentration Diphenylamine HSB DMEM BSA EtBr BHI DMSO PBS CsCl HAP BRL TPB RSB SSSSDNA 939 pol env High salt buffer Dubellco Modified Eagle Medium Trichloroacetic Bovine Serum Albumine DL-dithiothretol DL-a -Diaminopimelic acid (Sigma) Diethylpyrocarbonate Tetracylcine resistant Ampicillin resistant Etylene diamine tetraacetic acid Sodium dodeycyle Ethidium bromide Brain Heart Infusion Dimethylsulfoxide Phosphate buffered saline Cesium Chloride Hydroxyapatite Bethesda Research Laboratories, Inc. Triptose phosphate borth Resuspension salt buffer Salmon sperm single-stranded DNA Viral gene encoding the structure proteins of the viron core Viral gene encoding reverse transcriptase Viral gene encoding the envelope structure proteins which are the host range determinants xi CHAPTER ONE I. Introduction Some types of cancer in animals are caused by small viruses which have RNA as their genome material. These viruses, sometimes called retroviruses (as RNA tumor viruses) are widespread in nature. They are useful reagents for studying the oncogenic processes, for several reasons, including the rapidity with which they cause $2.!lEEQ trans- formation, the ease with which the virus can be titered, their high efficiency of tumor induction in infected animals, and their capability in many cases of transforming cultured cells. The oncogenic retroviruses can be grouped into at least two general classes since they appear to induce neoplastic transformation by at least two different molecu- lar mechanisms: these two classes are the ”acute" trans- forming viruses and the "chronic" transforming viruses. Retroviruses in the first class are capable of inducing neoplasm in the host animals after a short period of latency (i.e., 2-4 weeks post-inoculation) with efficiency near 100%. In addition, they are also capable of transform- ing embryo fibroblasts (l, 2, 3, 317) . The acute transforming viruses are probably laboratory products. The transform- ing ability of this class of viruses is apparently due to the presence in the viral genome of a specific trans- forming or "one” gene, probably of host origin. In recent years more than ten such specific viral transforming genes have been partially defined. They all have homolo- gous counterparts in the chromosomes of their hosts. The sequences are highly conserved throughout vertebrate evolution (1, 2, 4, 5, 6). Furthermore, under normal conditions these cellular ”one" genes are expressed in certain uninfected cells at a very low level (7, 8, 9). With the exception of ASVs (avian sarcoma viruses), acute transforming viruses are usually defective in their repli- cation functions, therefore requiring a genetically related replication-competent helper virus for infectivity and for replication. The helper, however, probably has little to do with the transformation process (5, 10). The second group of viruses (the "chronic” group), unlike the first, induce neoplasms only after a long latent period (i.e., 4-12 months post-inoculation of animals). Several studies indicated that these viruses lack known transfonming genes, do not encode any oncogenic proteins, and fail to transform fibroblasts in 21559 (4, 11). The mechanism of in yitgg oncogenesis by the second group of viruses, therefore, may be completely different from those of "acute" oncogenic retroviruses. Furthermore, some of the viruses in this group are pleiotropic and appear to have the potential for inducing different types of neoplastic diseases (5, 10). For example, avian leu- kosis virus induces mostly lymphomas, but occasionally leukemias and sarcomas as well. These viruses are likely to be responsible for the majority of naturally occurring neoplasms associated with retrovirus. infection (12). My studies have focused on the molecular characteri- zation of the bursal lymphomas induced by the second group of viruses. LL (lymphoid leukosis) is bursa depen- dent B cell lymphoma (12, l3, l4). Lymphoid tumor cells from LL tumors and also from transplants produce IgM but not other immunoglobulins led to the suggestion by Cooper 33.31; (14) that the virus transformation inter- feres with the normal intraclonal switch from IgM to IgG and IgA production, a normal pathway in B-cell differ- entiation (13, 14). The nature of this interruption of differentiation during leukemogenesis remains to be elucidated. Most recently it has been shown that following ALV (avian leukosis virus) infection in young chickens the "c-myc" gene (the cellular progenitor sequence of the oncogene of Mc29 virus) may be (at least partially) responsible for the induction of LL (15, 16, 17, 18, 19). These studies used chicken syncytial virus ("CSV", a member of REVs family). The main aim of the studies was to gain insight into the molecular event which lead to the development of LL in infected birds. By application of modern molecular biology techniques, including restriction enzyme digestion in conjunction with Southern blot analysis, the newly integrated CSV- exogenous proviruses in the DNA of lymphoid tumor cells were characterized. II. Literature Review A. A Brief Summary on Retroviruses It was first reported in 1908 (20) that "leukemia" could be transmitted in chickens by a filterable agent. Later in 1911 Peyton Rous described the isolation of the sarcoma virus (21). Subsequently many other retro- viruses have been isolated and identified in other animal species. However, they did not attract a great deal of attention until the past 20 years. These viruses, which now are collectively called "retroviruses" for their ability to reverse transcribe the genetic information, have recently attracted consider- able interest, especially since they can cause cancer in a wide variety of vertebrate species. Experimental studies have focused on their potential to induce a variety of neoplastic diseases as one type of cancer-causing agent, as well as in other related areas of interest. In recent years, with the hope of finding a simple and possibly general model for studying neoplastic diseases, they have become a subject of intensive studies. It should be mentioned that an additional advantage ‘ of the retrovirus system over other virus systems stems from the fact that the infection with these viruses does not lead to cell lysis; therefore, several different and stable virus-host cell interaction can be relatively easily studied. Retroviruses are very wide-spread among vertebrate species and may be transmitted both horizontally and genetically in susceptible animals. The family of Retroviridae share several defining characteristics and features including a similar archi- tecture of their virions, a unique and apparently diploid single-stranded RNA genome (see below), and a virion RNA dependent DNA polymerase (Reverse Transcriptase Enzyme), an essential constituent enzyme of these viruses (22, 23, 24). They also share many other properties, including the need for a DNA intermediate for viral replication prior to integration and the utilization of a host poly- merase for transcription of viral DNA (20). Based on their morphology or according to their natural hosts, they can be classified into two groups (which will be discussed later). Due to lnmited space, in this brief summary most of the detailed information regarding the common features will not be considered except for the structure of the retrovirus RNA genome (see below). For further details the readers are referred to the corresponding references listed above and especially the comprehensive review by Bishop (20) and others as well (318, 319, 320, 321) . Among the genome of all known animal viruses, the diploid feature of retroviruses is a unique characteristic, and consists of two identical molecules of single—stranded RNA joined via hydrogen bonding to one another at or near their é-termini (20, 25, 26, 27). The diploid genome may play a very important and essential role in transcrip- tion of intermediate DNAs from the viral RNA genome via reverse transcriptase; it may also expedite genetic recom- bination (28, 29, 322, 334). Several studies have shown that the "core" of retro- viruses contain a ribonucleoprotein complex composed of the viral RNA genome and possibly some low molecular weight RNA (30, 31), the enzyme reverse transcriptase (32), and two small highly basic proteins which bind to the single-stranded genome (32, 33, 34). The viral specific RNAs synthesized in the infected cells can be divided into two distinct pools; one pool of RNA is encapsidated and serves as the viral genome, whereas the other pool serves as mRNA for viral proteins (20). The genome of retroviruses share certain common features with eukaryotic mRNAs including polyadenylation at the 3-termini of both RNA subunits (35, 36), a "cap" structure at the 5-termini (37, 38) and a low level of internal methylation (37, 39. 40). Indeed, the viral genomic RNA can serve as a mRNA to synthesize virus specific proteins in an _i£ 233.12 reaction. The genes of some viruses have been partially mapped and ordered. The best characterized and adequately mapped example in this regard is the ASV (avian sarcoma virus) ge- nome (43, 44, 45, 46, 323) . The gene order for ASV is shown in Figure l-a (see below). These mapping experiments have mostly used deletion mutants and recombinant strains in conjunction with EM on hetroduplex molecules and RNA fingerprinting studies. For example, marker rescue experi- ments have proven the linkage between 221 and 223 genes (47). Also lg yitgg translation studies using viral RNAs (48) have shown the respective position as well as the order of ggg and pgl. Therefore, these and other related studies clearly indicated that the viral genome of a typical replication- , gag pol env src cx , A. ASV 5 I } % 1, w 3 polyA , gag pol env cx , B. LLV 5 § § 1 W 3 A,B,C,D polyA , gag pol env on . c. RAV-O s 1, i i AM 3 E polyA Figure l. The haploid genomes of ASV (avian sarcoma virus), LLV (avian leukosis virus) and RAV-o (Rous - associated endogenous virus). competent retrovirus, for example ASV, contains three genes reSponsible for the replicative machinery and structural proteins of the virus (41, 42). They often have, in addi- tion to these genes, a s52 gene, which is responsible for both transformation of fibroblasts in cultures and induction of sarcomas in infected birds (20, 41, 42). In addition to the ASV RNA structure, the haploid genomes of two other retroviruses, including LLV (avian lymphoid leukosis virus), and RAV-o (Rous associated endogenous virus) are illustrated in Figure 1. As shown in Figure 1, there are four subgroups of LLV, designated A to D (Fig. l-b). These subgroups are distin- guished on the basis of their serological and host-range specificities. Despite these distinctions, the genomes of all four subgroups share extensive nucleotide sequence homology (2 70%) (4). RAV-o, the endogenous virus, has an envelope property different from the other four subgroups of LLV and is identified as subgroup E (Fig. l-c). Several studies have shown that the RAV-o has little or no onto- genic potential (13, 244, 245). The haploid genomes of LLV and ASV are approximately 8 and 10 kilobases (kb) respec- tively (20, 240), and as mentioned earlier consist of three genes (Figure l): ggg,encoding group specific antigen; 22$: encoding the replication enzyme - reverse transcriptase; and Egg, encoding the envelope structure proteins which are the host-range determinants (20, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 335). As is also illustrated in Figure l, the genomes of RAV-o and, for example, LLV are very similar. The only nonhomologous region is the "3" region for "common region" (see below). cn and cx differ in the first 200—400 nucleo- tides next to the polyA sequence (20, 245, 246). These authors, as well as others (48, 49), have reported that the 9 region is located at the 3 end of viral genome adjacent to the polyA sequence, corresponding to about one kb in size termed "3? (defined as 2?, see Figure 1). This genetic re- gion is highly conserved in all exogenous viruses. Although some investigators have listed some debatable roles for this region, i.e. in virus tumorogenicity, its function remains elucidated, at least, in the avian system. This region does not appear to encode a protein (50, 241, 242). The viral replicative genes are themsevles flanked by directly repeated sequences termed "long terminal repeats" (LTRs). This feature of terminal redundancy has been shown in DNA of most retroviruses (51, 52, 53, 54, 55, 56, 57, 58). For example, in the ALV (avian leukosis virus)-integrated proviruses the "LTR" approximately 300 nucleotides consist of ”U3RUS" (designated as 3'5' in Figure 2) which signifies the unique sequence derived from the 3 end (03) and the 5 end (US) of ALV genomic RNA. Also, the ”R" region which is located between U3 and U5 is termed the short terminal redun- dancy (about 20 nucleotides) (16). The structures of the RNA genome and the linear and integrated avian retroviral DNAs are shown in Figure 2. 10 m7G 5' 3' ‘—- e~===a RNA genome polyA l3'5'}— {3'5'l free linear DNA LTR LTR u u integrated W W M Figure 2. The structure of RNA genome, linear and inte- grated retroviral DNAs. From a different perspective the retroviruses can also be grouped into either exogenous or endogenous viruses (59, 60). The genome of the latter class resides in the germ lines of many (if not all) normal host species. In addition, at least some of these species carry more than one strain of retrovirus in their genome. These sequences are persistently segregated as normal genetic elements via the gametes of the species, and their expression is con- trolled by genetic determinants of the host (61, 62, 63). The question of when and how these endogenous viruses ori- ginated and became a normal part of various vertebrate genomes is still a subject of speculation (61, 62, 63, 64, 65). Some of the exogenous viruses are transmitted hori- zontally or spread epigenetically among members of suscep- tible host species (59, 60). In most cases the genome of an exogenous virus is at least to some extent homologous to that of an endogenous virus in the species of origin (66). 11 This may be the basis for recombination between exogenous and endogenous viruses. It may be mentioned that there have been at least three major forms of antigenicity found _to be associated with retroviruses. These are the group-specific antigens (gs) which are common among related viruses which have all been originally derived from a single host species, type- specific antigens which are the most specific presently known serological subgroup-antigens, and interspecies anti- gens which are shared by otherwise unrelated viruses of different species (20). In regard to groupespecific anti- gens, Bolognesi (67) reported in 1974 that the "core shell" of the viruses is composed of several virus-coded proteins including the major locus for group-specific antigenicity. One widely used taxonomic tool, which is also an import- ant genetic marker for retrovirus classification, is the host range of these viruses, since in general not all viruses can enter a given cell. This is based on the find- ings that the susceptibility of avian cells to infection by different strains of viruses may be determined by the pre- sence or absence of specific cellular receptors. If the specific receptor for a given virus is present at the cell surface, then the virions may attach and penetrate (since normally virus particles absorb to even a resistant cell). The glycoproteins of the viral envelope facilitate both the absorption and penetration of the virus in an 12 acceptor-receptor relationship. Weiss (68) reported :hi 1976 that in the chicken susceptibility to virus infec- tion is a dominant trait specified by at least three separate genetic loci. Most oncogenic retroviruses can also be distinguished from one another based on the neoplasms they induce. Although all retroviruses share a similar virion architecture, earlier EM studies in 1972 by Dalton (69) using criteria including morphological features of viruses during the process of maturation and mature structure of the viruses have defined three major classes of retro- viruses, types A, B, and C. Type A particles are intra- cellular forms, and some of the viruses of this type are found within the cytoplasm of cells in which they may be precursors to the other two types (70, 71). Type B parti- cles are less common and morphologically are typified by the mammary carcinoma virus of mice. Type C particles constitute the major class . Most of the retro- viruses including leukemia, sarcoma and RE (reticuloendo- theliosis) viruses are classified as Type C particles, based on their certain morphological maturation and mature structural features (20, 70). In recent years a great deal of information on the genetics of retroviruses have emerged from many laboratories. In particular, many different types of mutants were isolated. These include nonconditional deletion mutants, i.e., repli- cation defective strains whose lesions can be complemented 13 by gene products specified by another virus (via mixed infection and appearance of phenotypic mixing, etc.), and conditional temperature sensitive (ts) mutants. Mutants in retroviruses can be acquired either naturally or by induction by several types of mutagens, or by measuring the transformation ability of retroviruses (4, 72, 73, 74, 75, 76, 77, 78, 79). The genetics of these mutants were then analyzed by complementation mapping and recombination analysis (4, 20, 334, 335, 336, 337). Conditional mutants were found to fall into three classes: class ”T" mutants which only affect transformation capacity of the virus, class "R" mutants only affecting viral replication, and class "C" mutants which affect both replication and transformation functions of retroviruses coordinately (20, 73, 80, 81, 82). These results have shed considerable light on the mechanism of viral replication, viral genome structure, and gene function, including the mechanism of the neoplastic transformation. Finally, the "protovirus hypothesis" about the origin of retroviruses proposed by Temin in 1964 (83, 192) merits dis— cussion. In 1970 Temin and his colleagues speculated that retroviruses might have evolved from ancestral genes from the genomes of animal cells; subsequently they proposed that the retroviruses are related to transposable genetic elements. These moveable genetic elements were first noted in the 19403 by Barbara McClintock in Maize. Recently, they 14 have been found in bacteria, yeast, and Drosophila. Some of the main structural features of these moveable elements are as follows. After transposition, transposable element DNA is flanked by a small direct repeat of cellular DNA (about 4-12 bp) next to an inverted repeat of about 2-1500 bp which belongs to element DNA. Within this overall structure there is great variation in both the structure of the transposable element DNA and the specificity of sequences of the cellular DNA. For example, the length of the repeated sequences varies among different elements. In addition, each element con- tains a different host sequence at each site of the inser- tion (or a different host sequence for each different in- sertion) (57, 58, 84, 85, 86, 87). Some similarities between the structure of retro- virus genomes and transposable elements have been observed. Recently Temin and his coworkers have studied the structure of the integrated proviruses by means of molecular cloning and DNA sequencing of the cell-virus junctions of several integrated proviruses of the SNV (spleen necrosis virus) strain of REV (57, 88). They found that the structure of these integrated proviruses were quite similar to those of transposable genetic elements. Similar results were recently obtained for other retro- viruses (50, 89, 90, 91), including proviruses of Moloney Musine sarcoma virus in mink cells (Dhar gt 31. (91)), 15 mice mammary tumor virus in rat cells (Major 31; g” (89)) and RAV-o, an endogenous virus in chicken cells (Ju gt 31. (see 58)). Again, in all cases, a direct repeat of host cell DNA sequences (of 4-6 bp for different proviruses), was bounded by the inverted repeats of the viral DNA (3-12 bp in different viruses) and the latter was located next to a direct repeat of viral DNA, also termed the LTR for ”long terminal repeat" (340-1200 bp). Furthermore, cloned circular unintegrated DNAs of the viruses have shown comparable results (57, 92). These results also have indicated a great specificity in the sequence of the viral DNA integration site, and no specificity in the sequences of the cellular DNA integration site. All the above mentioned showed a structural resemblance between the retroviruses and moveable genetic elements found in both prokaryotes and eukaryotes. Temin prOposed that the resemblance reflects an evolutionary relationship between the retroviruses and cellular transposable elements. On the other hand, it is possible that these similarities may be the result of evolutionary convergence (i.e., similar function) and so forth. They, therefore, hypothesize that the retroviruses and the moveable element may share a common ancestor. During evolution a virus may have formed from the "elements" acquiring additional information needed, i.e., the gene for RNA-dependent DNA polymerase and the means for packaging the RNA transcript etc. This hypothesis is at present speculative. 16 B. Reticuloendotheliosis Viruses (REVs) Classification Ample evidence clearly indicates that infectious type-c retroviruses are causative agents for many naturally occur- ring tumors in vertebrates, including birds, rodents, etc. (21, 93, 94, 95, 96). REVs are more recent isolates of the avian type-c retroviruses (97, 98). The REV group consists of several members such as REV-T, CSV, SNV, DIAV (see below) plus many others. These members were isolated from turkeys (99, 100, 101, 102, 103), chickens (104), and ducks (97, 105, 106, 107), respectively. They are very closely related to one another in their genomic sequences (108, 109, 110), their structural proteins (111, 112), the antigenicity of their DNA polymerase (113, 114), their group-specific anti- gens (112), and their morphology (115). Experimentally these viruses can cause a variety of characteristic lesions including lymphoproliferative disease, markedly enlarged liver and spleen, depression of cellular immune response, etc. in several infected birds such as chickens, ducklings, turkeys, quail, goslings, guinea keets, and pheasants (83, 98, 99, 116, 117, 118, 119, 120). However, their patho- genicity differs from one to another (97). For example, while SNV and some other strains of REV are highly patho- genic for ducks and chickens, the DIAV and CSV were found to be less effective in inducing diseases (97) in the birds tested. The following four members of the group have undergone relatively extensive studies (97, 109, 121, 122, 123, 124) 17 and are worth describing in more detail. 1. Reticuloendotheliosis Virus Strain T (REV-T) REV is the name of both the species and one of the members of the species REV-T (now known as a mixture of REV-T and REV-A, see below). REV-T, the prototype of REVS, originally was isolated by Twiehaus in 1958 from the tumors of an adult turkey and appears to be carried by a natural parasite of turkeys which causes leukosis-like lesions or "turkey leukosis" disease, since its antibody occurs among some flocks of turkeys (99, 101, 102). Because of the historical importance of REV, I think it worth mentioning very briefly the original circumstances of the isolation. In October, 1957, a dying adult turkey was submitted to the diagnostic laboratory (Department of Veterinary Pathology, Kansas State University) due to occurrence of an unusually high mortality among the commer- cial flock. The disease was not completely diagnosed but lymphoid leukosis was suggested due to gross enlargement of the liver and Spleen, a good indication of LL disease. Further research by the same team enabled them to isolate REV in 1958. Because of insufficient data it took them and other investigators until 1966 before they finally estab- lished that the disease was believed to be avian reticulo- endotheliosis (101, 102). In 1964 the virus was named REV-T virus (99). Since then the virus (REV-T) has been distributed to several laboratories and has been subjected 18 to extensive studies. During the subsequent passages 13‘31139 (cell culture) or 13‘3123 (i.e., chickens) in different laboratories (83, 104, 124, 125, 126, 127, 128, 129) some of the prOpertieS of the original virus have apparently changed. Therefore, stocks prepared by others people including Cook (104), Fisher and Thompson (83) and Sevoian 33 31. (99) no longer have the same patogenicity as the original stock (124). In 1964 Sevoian 31 31. (99) observed that this virus was able to cause a consistent rapid fatal and marked RE following inoculation into chicken embryos by the yolk sac route. When it was inoculated into one to three day old chicks, they exhibited several different lesions, including markedly enlarged liver (sometimes 20 times the normal Size), tumorous nodules in the Spleen of some and a high rate of mortality of relatively young chickens, Japanese quail, and turkeys. These investigators described the experimental disease as lymphomatosis and considered the virus to be clearly oncogenic. Subsequently, in 1975 Schat 31 31. (103) reported a naturally occurring lymphoproliferative disease, with some evidence of REV involvement in three flocks of Japanese quail in Mexico. The tumor lesions were found mainly in livers and spleens, i.e., enlarged to 10 times the normal size. It Should be noted that earlier in 1967 Léliger 31 31. (119) reported a similar recurring lymphoproliferative disease in Japanese quail. 19 REV-T appears to be the only member of the group capable of transforming fibroblasts and bone marrow in cultures (97, 98, 99, 130, 131, 132, 133, 134, 135) based on studies on Japanese quail fibroblasts. Franklin 33 31. in 1974 (131) reported that REV-T can transform chicken bone marrow cell (13_!1!3L Furthermore, they were able to isolate an REV-transformed cell line (chicken bone marrow cell) from the REV infected bone marrow chicken cells. These all apparently Showed infinite growth poten- tial typical of transformed cells. Recent studies indicate that oncogenic infectious REV is a mixture of two components, a replication-defective transforming virus (i.e., REV-T) with an extensive deletion of the replicative genes, and a non-oncogenic associated helper replication-competent entity (i.e., REV-A) which provides the virion polypeptide to the REV-T virus (133, 134, 135). The REV-A appears to be present in the original virus stock at about 100 fold over the REV-T component. In addition, REV-A, like other members of non-defective REVS (nd REVS), is unable to transform cultures of bone marrow cells. It can be propagated in fibroblast cultures and induce plaque formation (cell death), but no foci are formed (22, 23, 97, 104, 117, 118, 120, 133, 135, 136, 137, 138, 139, 140, 141, 142). Hoelzer 3E a_l_. (135, 144) reported that REV-A fails to induce RE following injection into chickens. 20 Ample evidence strongly suggests that the transforming ability of REV appears to be independent of the helper virus. Although several investigators (116, 117, 118, 120) have reported that at least some of these "nd-REV" strains can cause marked depression of the cellular immune response, it is not clear whether the induced tolerance is a direct result of viral immunodepression. Rup 31 31. (143) and Hoelzer 33 31. (144) observed that "REV-A" induces a progressive runting disease in infected birds, and is capable of inducing immunosuppression. These observations led them to suggest that "REV-A“, in addition to providing functions required for "REV-T" replication machinery in the mixture stock, may also have some effect on Ithe extremely high virulence capacity of REV. Recently Witter and Crittenden (145) and Grimes _1 31. (139) have provided some preliminary evidence on the occur- rence of LL in chickens which were inoculated with CSV (chick syncytial virus) Strain of nd-REV. The studies presented in this thesis provide the first biochemical information about the disease caused by CSV strain of REV in infected chickens. The study was undertaken to investigate, at the molecular level, the mechanism of CSV-induced lymphomas in chickens. The bio- chemical analysis of many tumors confirms that CSV, like ALV, is responsible for the induction of bursal lymphomas in the inoculated chickens. The detailed information is presented in the next chapters. 21 2. Chick Syncytial Virus (CSV) Although chickens may not be the primary host species for nd-REVS, in 1969 Cook (104) isolated a virus from Marek's disease tumor suspension in chickens which contained nerve lesions. At that time this virus stock was thought to be Marek's disease, but subsequent studies showed thatthe virus was a member of the REV group based on serological close- ness of this virus to REV-T. This virus, capable of pro- ducing syncytia in chicken embryo fibroblasts (CEF), was then designated as chick syncytial virus "CSV" (104). Witter 31 31., in 1970 (124), reported that this virus was nonpathogenic for chickens. Subsequent studies, how- ever, suggested that this may not be true (see pathogenicity of REVS). In 1969 Cook (104) and in 1973 Purchase 31_31. (97) isolated CSV from many sources of chicken blood (104). They found that this virus, including isolates from DIAV (see below), does not produce RE in infected chickens but can induce nerve lesions. In addition, they too have found that CSV produced syncytia in CEF cultures. 3. Spleen Necrosis Virus (SNV) In 1959 Trager (146) isolated a virus from duck in- fected with malaria organism Plasmodium lophurae which produced a rapid fatal disease involving the enlargement as well as the necrosis of spleen. The investigator called it "spleen necrosis virus" (SNV). This virus has been 22 Studied by a number of investigators, especially Temin and his coworkers (see the sections on replication and inte- gration). 4. Duck-Infectious Anemia Virus (DIAV) In 1969 Corwin and McGhee (105) reported an anemia of ducklings which was induced by a filterable agent present in malarious-plasma (106, 107). Ludford 33 31. (1947) in 1972 named the virus "duck-infectious anemia” virus. C. On the Analysis of the Nucleic Acid Components in REV Several studies have been carried out in recent years to determine the genetic relatedness of the two components of REV. For example, Breitman 33 31. (134) reported a 50-708 and a 40-603 RNA for REV-A and REV-T respectively. The monomer for the former sedimented at 348 and the latter at 288. Several more recent independent studies have Shown that the REV-T has a 5.5 to 6-kb or 28-308 RNA genome, and the genome Size of the REV-A was found to have a molecular' weight of 8.7 to 9.3-kb (134, 135, 148, 149, 150). From these and other related studies the following conclusions for REV can be drawn: REV-T appears to be Similar to murine sarcoma virus, feline sarcoma virus (151, 152, 153, 154, 155) and the avian acute leukemia viruses including Mc-29 (avian myelocytomatosis virus), AEV (avian erythroblastosis 23 virus) and avian sarcoma and leukemia virus MH2 (152, 156, 157, 158) in that all of them require a helper virus for replication. All of them have a size smaller than the helper viruses, but are genetically related to their asso- ciated viruses (133, 134, 145, 152, 153, 154, and the above references as well). Indeed, the 288 of REV-T is quite Similar to the genome size of those of AEV, MC-29 and MH2 (152, 156, 157, 158). AS mentioned above, the smaller Size of these defective acute transforming viruses is due to an extensive genetic deletion in their replicative genes. (For example, about 55% of the helper genome Size is missing in the REV-T, see Reference 134.) That is why (to my know- ledge) all of these groups of acute leukemia retroviruses, without exception, fail to replicate in the absence of their related associated helper viruses, since Simply no virus particles can be released (133, 135). In addition, in all of them the transforming ability apparently is due to the presence of a Specific "onc" transforming gene in their genome, possibly of host origin. Because this is a primary interest of this study, I will discuss REV-T a little more in regard to this feature even though there is relatively limited data available. Breitman 33 31. in 1980 (134), using hybridization studies, Showed that about 70% of the 6 kb genome Size of REV-T has common sequences with its associated helper "REV-A" by which it could accommodate 24 only about 45% of the 9 kb, the estimated genome Size of the helper. What are those ca. 30% unique sequences which are only present in the REV-T genome? Presumably like other avian acute viruses mentioned above, they may be responsible for REV-T acute leukemogenic potential 13,3133 and its transforming ability 13‘31333. Most recently Hu 33 31. (150) partially characterized REV-T using hetroduplex mapping analysis, the genetic structure of REV-T and the location of the transforming- Specific sequences in the virus genome. Like others, they reported an extensive deletion in the gag-pol region, in addition to a contiguous stretch (1.6 to 1.9 kb) of REV-T specific sequences located in the 33! region (REV-T lacked the 33! sequences). These REV-T unique sequences, unlike those of other avian acute leukemia viruses, were not contiguous with the gag-pol deletion (150). There is also no homologous sequences or antigenic cross-reactivity between REV-T and other avian acute leukemia viruses (42, 49, 109, 114, 159, 160, 161, 162, 163, 164, i 165, 166, 167, 168). REV-T are genetically unrelated to ASV (i.e., the sarc transforming gene of ASV) (149). There- fore, the REV-T unique sequences may represent a new class of avian retrovirus transforming gene. Finally, Moloney sarcoma virus was postulated to be derived from a recombinational event between the host DNA and corresponding related helper virus (7, 169). At present 25 it is not known whether this could possibly be the case for REV-T, although Simck and Rice (151) have observed REV-T specific sequences in normal chicken DNA, yet their nature remains unproven. D. On the Comparison of REVS and Avian Leukosis Sarcoma Viruses (ALSV) REVS are Similar in some respects to ALSV, yet differ- ent by several criteria. 1. Some Major Similarities AS mentioned earlier REVS are Similar to ALV in the characteristic structure and mode of replication (42, 97, 101, 109, 123, 160, 162, 163, 164, 165, 166, 170, 171). Also, tumors induced by both viruses are similar with respect to latent period, organ distribution, and surface IgM production (145). 2. Some Major Differences The two classes are genetically and serologically unrelated to each other (97, 101, 102, 108, 109, 113, 121, 122, 164, 165, 171, 172). They can be distinguished by several other criteria including differences in their reverse-transcriptases (113, 164, 165) , the extent to which their viral nucleic acid hybridize with host cell DNA (108, 151, 172) and their buoyant density (123, 160, 173) . EM studies showed that the two groups of viruses also differ morpho- logically (98, 101, 102). 26 Although it is known that the natural host for ALVS of subgroup A to E are chickens and, as mentioned earlier, may be turkeys and possibly ducks for REV, neither class is entirely host specific Since both can infect several and similar species of birds in addition to their natural host. The reverse transcriptases (DNA polymerases) of these two groups of retroviruses differ in that the REV poly- 2+ 2+ to those merase has a preference for Mn instead of Mg of avian leukosis sarcoma viruses and ALSV (166, 174, 175, 176, 177, 178, 179). Also unlike ALSV the primer for REV is a ”cellular” tRNApro (180). These properties of REV DNA polymerase are reminiscent of those of mammalian type-C retroviruses. In addition, the size of REV enzyme is also similar to those of mammalian type-C retroviruses (114, 166, 181). The finding of Specific cross-reaction between mammalian type-C and REV DNA polymerase, in addition to amino acid sequence homology such as relatedness between P303 of the two groups, common immunological determinants in their major core proteins, and sharing the tRNAPro as their primer etc. (111, 174, 180, 182, 183) may imply the existence of common evolutionary relationship and origin between the two classes of retroviruses, a "common progenitor" in their evolution as proposed by these and other investigators (174, 266). They have suggested that, either REVS have been trans- mitted horizontally for a relatively long period of evolution or originated from relatively recent infection 27 of birds with a type-c "endogenous" mammalian retrovirus which now can infect avian species as well. The progenitor of mammalian type C-endogenous retrovirus, if it exists, remains to be identified. Many retroviruses have most of their genomic sequences existing in the normal host DNA in a quiescent state. This is true for ALVS. However no REV sequences existin unin- fected avian cells or at most 10% of the sequences of REV were present in the DNA of normal chicken, pheasant, quail, turkey, and none in uninfected duck (108). Collett 33_31. in 1975 (172) and Simek 33 31. (151) in 1980 confirmed the earlier observation but the former found 3 . 8% of the REV sequences in uninfected duck and the latter observed less than 10% of REV sequences in normal duck and goose DNA and about at most 15% of the REV sequences in uninfected chickens. The conclusion from these studies is that there are no endo- genous REV sequences present in normal chickens. However, in the present studies, the presence of presumptive REV endogenous loci was detected in at least all three different uninfected chicken inbred lines studied; although, at present, it is not clear whether this endogenous REV sequence is Specific only for these three chicken lines or exists in all chickens. Furthermore, most recently Simek 33 31. (151), using hybridization experiments performed at low stringency, detected no REV-related sequences in various mammalian DNAS, 28 which makes the proposal of REVS being a mammalian type-C retrovirus less attractive. Further work will be required before any definite conclusion in this regard can be derived. E. Extent of Phenotypic Mixing Between REVS and ALSVS AS mentioned earlier, REVS and ALSVS are genetically unrelated to one another. There is no or very little (less than 4%) nucleic acid sequence homology between their respective RNAS. They have no physiological intraction (109). Therefore, given these and Similar data it is logical that there is no complementation or homologus (legitimate) recombination between these classes of viruses (161). Furthermore, earlier studies in 1973 by Halpern 33 31. (164) indicated that these two groups of viruses cannot undergo phenotypic mixing with each other. However, subsequent studies conducted by Vogt 33 31. in 1977 (184) demonstrated the presence of a biological intraction between these two apparently distinct Species of retroviruses. They reported that coinfection of CEFS with both REV and ASV generated an ASV pseudotype which carries envelope - glycoprotein determinants of REV. In addition, the pseudotype had a host range different from any known ASVS, although the viruses could be neutralized by anti-REV antiserum. These observations of phenotypic intraction clearly suggested that the REV glycoprotein can be incorporated in the ASV envelope. The low efficiency might be due to varying degrees (from Slightly to moderately) of acytophatic effect in CEFS 29 caused by REVS, thus contributing in varying degrees to the difficulty of the forming of an effective pseudotype. In the same year studies which agreed with Vogt's were reported by Sawyer and Hanafusa (185) . The CEFS were super- infected by the Bryan strain of Rous sarcoma virus (an ASV strain which lacks the envelope gene, therefore being un- able to direct synthesis of its envelope glycoprotein) and with two REV strains, REV-T or SNV strain of REV. They found that this led to the formation of an infectious pseudotype which was similar to that of Vogt.33 31. This indicated that these strains of REV can be complemented by a strain of Rous sarcoma virus (RSV) which provides the envelope glycoprotein, although with much lower effi- ciency than by an ALV. One might ask whether or not phenotypic mixing can also take place for other REV virion components such as 231 or g3g genes. So far all tests conducted for this purpose, either by these workers or others, have failed. Therefore, from our knowledge to date, the extent of REV and ALSV intraction and physiological complementation appears to be limdted to the envelope glycoprotein. F. On the Pathology of REVS Since questions of whether naturally occurring diseases are induced by REVS in many avian species (except perhaps turkeys and possibly wild waterfowl) remain unanswered, only the experimental disease can be described and for this 30 purpose at least chickens, Japanese quail and turkeys were found to be suitable experimental hosts. From several experimental Studies, it is now clear that depending upon the strain used REVS produce several types of pathology in many birds, including chickens, turkeys, duck, and Japanese quail (97, 98, 99, 172). In the follow- ing paragraphs some examples are mentioned. In 1966 it vwas reported (102) that even very low doses of virus resulted in grossly enlarged peripheral nerves. Initially, it was not clear whether the diseases induced were of neoplastic or of hyperplastic origin (101, 186). Subsequent studies Showed that REV can induce neo- plastic diseases; formation of tumorswas observed in the liver tissue of infected birds (173 in 1971). Paul 33 31. in 1976 observed that, following infec- tion,1esions were found in several tissues including liver, Spleen, heart, intestines, and peripheral nerves which consist mainly of lymphoreticular cells (187). In addition these authors also were able to isolate a virus from turkeys with Similar features to that of REV-T. This virus was not Marek's disease herpesvirus (MDHV) since the disease was experimentally reproduced with cell free preparation, whereas MDHV is a cell-associated virus. The infected tissues of both naturally affected and the experimentally infected turkeys were free from ALSV antigen. Thus, this virus does not belong to the ALSV group either. 31 Finally Witter and Crittenden in 1979 (145) Sometimes observed mortality following infection. The chickens ino- culated with CSV-strain of REV developed a reasonably high incidence of lymphoid neOplasms between the 17th and 37th week post-inoculation. No mortality occurred prior to the 17th week even with high doses of virus. The lesions were commonly found in the liver and bursa of Fabricus, but in certain individuals the tumors also developed in the Spleen, gonads, kidney, and lungs. All of these lesions caused by REV infection have been observed in relatively young sus- ceptible birds and the mortality depends on the virus dose and the age of the bird at the time of inoculation. Lastly, from several studies the REV induced diseases are distinguishable from proliferative diseases induced by other viruses such as ALVS and MDHV which both typically induce tumors composed of reticular and lymphoid cells in liver, spleen and other visceral organs in suitable birds (101, 124, 145, 188). As far as what is known about natural infection by REVS and their natural hosts in addition to turkeys (101, 190) Carlson H.S. 33 313 (247) and Schat 33 31. (103) also reported that these viruses have been associated with naturally occurring lym- phomas in quail as well. Therefore, chickens may not be their natural hosts. In fact, no naturally occurring disease has been described in chickens. The observation 32 that REVS are poorly transmitted horizontally among chickens also indicates that chickens may not be their natural hosts. However, Fisher and Thompson in 1967 (83) and Thompson 33‘ '31. in 1968 (189) suggested that these viruses may be transmitted vertically or by an insect vector, from one chicken to another. Their suggestion requires further verification. Thus, despite their widespread (as judged by several reports) work, the present evidence seems to suggest that natural hosts of REV are turkey and probably wild waterfowl (98, 101). In addition, the epidemiology of REV infection in different birds is not clear. It is true that they can be transmitted horizontally among differ- ent avian Species, but the possibility of their vertical transmission remains somewhat obscure. Furthermore, it is not clear by what means they maintain themselves in natural avian populations. Obviously further work is required to learn about their possible or real role of causing diseases in natural bird populations and to provide means for pre- venting their possible economical damages in loss of poultry. For instance, one may be able to select genetically resistant birds for control of the lymphoid disease etc. G. On the Mechanisms of Retrovirus Replication and Integration The mechanism of retrovirus replication is poorly under- stood. Studies on REV replication have suggested that they 33 may replicate through a DNA intermediate similar to other known retroviruses such as ALSV (137, 163, 172, 191). In 1971 Temin (l92),and subsequently others, provided stronger evidence for the presence of a DNA intermediate in retrovirus replication (20, 97, 108, 114, 124, 164, 171, 172, 191, 209, 210, 211). In recent years several features of the complex stages of replication have been studied in- cluding the kinetics of formation, cellular location of the synthesized viral DNA and some of the several inter- mediate types of viral DNA (193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208). Also, several studies have Shown that the nonintegrated viral DNAS of several retroviruses, for example ASV, MULV (murine leukemia virus) (125) , and SNV strain of REVS (198) , were infectious in their proper recipient cells using DNA transfection assays (212, 213, 214). It has been observed that the linear infectious viral DNA had 21 5-10 fold higher Specific infectivity than either the closed circular or integrated infectious viral DNA (198, 209, 210, 215). Also, infectious viral DNA did not appear in infected sta- tionary cells (216) suggesting some cellular influence on the formation of infectious viral DNA (216, 217, 218). The general mode of replication in retroviruses (also called "t" strand RNA viruses) are Similar. Some of the major points can be outlined as follows. Early after infection, using virus-coded reverse- transcriptase, many complete viral linear double-stranded 34 (VLds) DNA molecules are copied from the viral genome in the cytoplasm of susceptible cells (20, 108, 212, 213, 219, 220, 221, 222, 223, 224). A fraction also can be found in the nucleus. Only a portion of these molecules become stably integrated into the host genome (20, 198, 225, 226, 231). The rest of the nonintegrated viral DNA eventually disappears, possibly by being diluted out during cell growth (204, 227). The structure and kinetics of synthesis of "'-" strand and "+" strand (the latter identical in polarity to viral DNA) of the unintegrated linear viral DNA have been studied (196, 197, 201, 208, 219, 228). From these studies a rela- tively clear picture for the synthesis of the "-" strand has evolved, yet the molecular events for the "+" strand synthesis remains to be understood. In the ASV and MULV, the "minus" strand of the linear DNA is continuous and of full genome Size, whereas the "+" strand is segmented and composed of fragments smaller than the genome Size. These researchers have concluded that the mode of synthesis in these two strands may be different (196, 197, 201, 208,.219, 228, 229). It should be mentioned that several studies indicated that in the SNV-strain of REV, both strands (" -" and "+") are of genome size linked by ribonucleotides and the majority of both types were found to be continuous (209, 210, 227). One additional point on the formation and structure of .infectious DNA of SNV Should be made. At about 18-24 hours 35 after infection of CEFS, nonintegrated infectious DNA were found and persisted for more than 14 days after infection. These viral DNAS consist of either a double stranded linear DNA (as the major portion) found in both cytoplasm.and nucleus, or as closed circular DNA detected primarily in the nucleus, both of a Similar Size of 6 x106 daltons (198, 209, 210, 215). Although ample evidence indicates that integration is an essential event for viral replication (211, also for detailed review see reference 20), little knowledge exists about the Site of integration and the molecular mechanism by which the viral DNA integrates into the chromosomal DNA of the host cells. However, recent studies provide some insight into this process. The proviral DNA is colinear with viral RNA and with linear unintegrated viral DNA. Both viral DNAS seem to be terminally redundant (202, 203, 230). It has been reported that the terminal redundancy in SNV Strain of REV is about 600 bp (54). Also, retro- viruses are stably integrated into the host cell DNA (198, 231). For example, Steffen and Weinberg (232) have reported that the retrovirus is stably integrated for many hundreds of cell generations and possibly never excised from the chromosome. 13‘31333, REVS lose their virulence after serial passages (124, 164). In general, soon after infection, viral IHEAS integrate into cellular DNA at a large number of sites, Very likely in a random fashion (180, 193, 202, 225, 230, 36 232, 233, 234, 235), and they express a different level of the viral information (193, 225, 233). SNV integration appears to occur at a Specific site located at the termini of the viral DNA, similar to the integration of phage Mu (236) , but different from SV40 which contains no specific viral sequence to constitute the junction with cellular sequences following integration (237). Infection of chicken or duck embryo fibroblasts results in some cell death. The production of REV in infected cells occurs in two phases. The first phase, which lasts between 2-5 days, is "the acute infection,” and virus production is accompanied by extensive cytopathic effects and cell death, possibly as a consequence of multiple random integration which either leads to the overproduction of viral components or possibly inactivation of required cellular functions. The surviving cells from this phase proceed into the second phase, "the chronic phase," in which the viruses are produced without cell death and cytopathic effects (193, 198, 209, 225, 230, 233, 234, 235). Infected cells in this phase persistently maintain and multiply at the same rate and with similar morphology to the uninfected (normal) cells, and therefore, can be established in cell culture (124, 173, 188, 191, 193, 225, 233). Kang and Temin (108) have found that in chronically SNV-infected chicken cells, there are on the average 3-5 SNV DNA copies integrated .per genome, and. the infected cells contain both infectious 37 and noninfectious SNV proviruses (193). However, recently Simek and Rice (151) obtained evidence for the presence of about 12 copies of REV-related sequences in infected bone marrow cell-cellular DNA. Battula and Temin (225) and Fritsch and Temin (198) observed that infectious DNA of SNV is most probably inte- grated at a single Site in the DNA of chronically infected CEFS (chicken embryo fibroblasts). They also obtained evidence indicating that the virus DNA was not tandemly integrated in the host chromosomal DNA. Finally, although the nature of precursor (i.e., whether the linear or circular DNA or both etc.) involved in the integration is not clear, the covalently closed circular viral DNA (CCCVD) was considered to be an inter- mediate in the process of integration (145, 199, 238), based on analogies to the "Campbell model" for phage A DNA inte- gration (239). A simple replication scheme of a typical retrovirus (i.e., LLV) is illustrated in Figure 3 (see below). As mentioned earlier, early after 13’31333 infec- tion, using virus-coded reverse transcriptase, the viral RNA genome is copied into linear double stranded DNA in the cytoplasm. These viral DNA sequences are then trans- ported into the nucleus where a fraction of them are con- verted into supercoiled form. Thereafter, integration of viral genes into host chromosome commences. The viral genes are transcribed by the host RNA polymerase into viral genomic and messenger RNAS. They are then translated into 38 viral polypeptides. The virions are assembled and released into the medium. The whole replication cycle takes about 24 hours (336, 337, 338, 339, 340, 341, 342). RNA genome reverse transcription linear DNA (cytoplasm) transport linear and supercoiled DNA (nucleus) integration pgovirus DNA transcription L viral RNA and mRNA translation viral pofypeptides l 2.1.2.122 Figure 3. Replication and integration scheme of a typical retrovirus. CHAPTER TWO Materials and Methods MATERIALS A Few Notes on the Materials The following are common buffers, reagents, solutions, etc. frequently used in this research; they are listed as their final concentrations unless otherwise stated. All four radioactive labeled isotopes were purchased either from New England Nuclear (NEN) with specific activities of 700 to 3200 a/mmole, or from ICN Chemical and Radioisotope Division with specific activities from 429 - 2800 a/mmol. All isotopes usually were used within a few days after their arrival. Restriction digestion endonucleases were purchased from Bethesda Research Laboratories Inc. (BRL), Miles Biochemicals, ICN Chemical and Radioisotope Division and New England Biolabs, Inc. They were stored at -20°C in proper buffers until used. In general all enzymes were used according to the supplier's instructions (also see below). Some EcoRI enzymes used in this study were a generous gift of Dr. Revzin (Biochemistry Department, ,Michigan State University). 39 40 High TE Buffer: 20 mM Tris-HCl - pH 7.2-7.4, 10 mM EDTA - pH 7.0; Low TE Buffer: 10 mM Tris-HCl -.pH 72.-74., 0.1 mM EDTA - pH 7.0; 2XSET (for extraction of high mole- cular weight DNA from chicken tissue): High TE + 1/20 volume of 20% SDS; SET Buffer: 10 mM Tris-HCl - pH 7.5, 5 mM EDTA, 1% SDS; DNA Buffer (1x): 100 mM NaCl, 50 mM Tris-HCl - pH 8.2, 1 mM EDTA - pH 7.0; 333 (1x) Buffer: 100 mM NaCl, 10 mM Tris-HCl - pH 7.4, 10 mM EDTA; leEAC (buffer for agarose gel electrophorosis): 40 mM Tris-HCl, 20 mM Na-Acetate, 18 mM NaCl - pH about 8.15, 2 mM EDTA; Oligode] - Column High Salt Buffer (HSB): 0.5 M NaCl, 0.1% SDS, 10 mM Tris-HCl - pH 7-7.6, 1 mM EDTA; Oligo[dT]- Loading Buffer: 0.1 M NaCl, 1 mM Tris-HCl - pH 7.4, 0.2% SDS; Oligo[dTl-Washing Buffer: 0.1 M NaCl, 10 mM Tris-HCl - pH 7.4, 0.2% SDS, 0.5 mM EDTA; Elution Buffer (for polyA Containing RNA): 1 mM Tris-HCl, 0.5 mM EDTA, 0.2% SDS; Blot Denaturation Buffer: 0.3 M NaOH, 1.5 M NaCl; Blot Neutralization Buffer: 3 M NaCl, 0.5 M Tris-HC1 - pH 7.04, Concentrated HCl (37%) - 37.5 mlll; Blot Washing Buffer: 0.1 X SSC, 0.01% SDS (leSC is 0.15 M NaCl, 0.015 sodium citrate); Southern Transfer Buffer (annealingybuffer): 7.5 mM EDTA, 100 mM Herpes - pH 7, 50% Formamide (99%); 3xCCS 2cx SSC, single-stranded calf thymus DNA (8858) of 2 mg/ml, 10 mg/ml yeast t RNA, l/10 of the volume of Dextran Sulfate (i.e., 10 gm/100 ml), 1x 100x Denhardt's; Denhardt's Buffer: 0.02% polyvinyle pyrolidone (Calbiochem), 0.02% 41 Ficoll (Sigma), 0.02% BSA (Sigma) was soaked at 68°C about Six hours; Tris Acetate Buffer (for extraction of DNA from seaplaque agarose: 40 mM Tris, 5 mM Na-Acetate, 1 mM EDTA which was adjusted to pH 7.6 using HCl; 10x Nick- Translation Buffer: 500 mM Tris - pH 7.5, 100 mM M9504, 10 mM DTT, 500 ug/ml BSA; G-50 Column Buffer: 0.3 M Nacl, 2 mM EDTA, 20 mM Tris-HCl - pH 7.4, 0.2% SDS; DNA Poly- merase 1 Storage Buffer; 50 mM KPO4 - pH 7, 0.25 mM DTT, 50% glycerol; Synthesis of the Long CDNA (endogenous reaction): 0.1 M Tris-HCl - pH 8.1, 30 mM DTT, 3 mM MgACZ, 2 mM dNT ps (all 4), [P32]-dcTP, 1 mg/ml purified virus, 0.02-0.03% Triton x-100 (This proper concentration was determined for individual bath of virus);SYnthesis of Short CDNA (exogenous reaction): 10 ug/ml purified 705 RNA, 10 mg/ul calf thymus DNA, 0.05 M Tris-C1 - pH 8.1, 0.002 M DTT, 0.008 MgClz, 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dTTP, [P321-dcTP reverse transcriptase; 1% DPA (Di-phenyl amine): For 100 m1 - 1 gm DPA, 96.5 ml glacial acetic acid (HOAC), 2.75 ml concentrated H2804 which was kept at 4°C; Reverse Transcriptase Test for Virus Production (reaction mixture for 500 ul - the following ingredients were premixed): 12.5 ul 2 M Tris-Cl pH 8.1, 5 ul 1 M MgAC 100 pl 0.1 M dTT, 225 ul 0.0% Triton X-100, 25 ul 2! 1 mg/ml poly rA, 2.5 ul 1 mg/ml oligo [dT], 12.5 pl 4 M NaCl, 17.5 ul H20 (Then, 100 pl H20 containing i.e., 100 uCi 3H-dTTP was added and finally equal volume of reaction mixture and purified virus (1-2 ug) was mixed and incubated 42 at about 37°C for 6-15 hours.); Stopper Buffer for Reverse Transcriptase Assay: 0.1 M Na pyrophosphate, 800 ug/ml calf thymus DNA or yeast t RNA, 20 mM EDTA, TCA to final of 10%. Tissue Culture Medium and Most Common Buffers and Reagents DMEM Medium (for 4.5 liters): 66.87 gm powdered DMEM H-21 (or -16), 18.5 gm.NaHCO3, lx pen-strep 10K units per ml Gibco 20 ml/bottle, 1x Fungigone, Gibco 20 ml/bottle adjusted to pH 6.8 before filterization, 425 m1 DMEM, 50 m1 TPB at 10%, 25 m1 FCS at 5%; 333 (pH 2.2, for one liter): 8 gm NaCl, 0.2 gm KCl, 1.15 gm NaHPO4, 0.2 gm KH2P04 was declared before use; Cell Freezing Medium: for 100 ml - 60 ml DMEM containing 10% TPB and 5% FCS, 20 ml FCS and 20 m1 DMSO; 10x Tris-Celucose: for one liter - 80 gm NaCl, 3.8 gm KCl, 10 gm glucose, 30 gm Tris-base, 17.5 mi concentrated HCl (37%), 900 ml d2 H20 was stirred until everything dissolved - then was adjusted to pH 7.2- 7.4; 0.1x RSB: 1 mM Tris-HCl - pH 7.4, 1 mM NaCl, 0.15 mM MgC12; 1x RSB - pH 7.4: 10 mM Tris-HCl, 10 mM NaCl, 1.5 mM MgC12. Restriction Digestion Enzymes 1. ENDO R. EcoRI a) 333. Assay Buffer (1x): 100 mM Tris-HCl (pH 7.2), 5 mM MgC12, 2 mM 2-mercaptoethanol, 50 mM NaCl. b) Miles Biochemicals. Assay Buffer (1x): 100 mM 43 Tris-HCl (pH 75.), 50 mM NaCl, 10 mM MgC12. 2. ENDO R. Bam H1 a) 333. Assay Buffer (1x): 20 mM Tris-HCl (pH 7.0), 100 mM NaCl, 7 mM MgC12, 2 mM 2-mercaptoethanol. 3. ENDO R. Bgl II a) 333. Assay Buffer (1x): 20 mM Tris-HCl (pH 8.0), 7 mM MgC12, 7 mM 2-mercaptoethanol. 4. ENDO R. Hind III a) 333. Assay Buffer (1x): 20 mM Tris-HCl (pH 7.4), 7 mM MgC12, 60 mM NaCl. b) 133: Assay Buffer (1x): 60 mM NaCl, 6 mM MgC12, 10 mM Tris-HCl (pH 7.4), 100 uglml BSA. 5. ENDO R. Kpn l a) 333. Assay Buffer (1x): 6 mM Tris-HCl (pH 7.5), 6 mM NaCl, 6 mM MgC12, 6 mM 2-mercaptoethanol. 6. ENDO R. Pst 1 a) 333. Assay Buffer (1x): 20 mM MTris-HCl (pH 7.5), 10 mM MgCl 50 mM (NH4)4SO4, 100 ug/ml, BSA. 2: 7. ENDO R. SOCl a) Bio Labs. Assay Buffer (1x): 6 mM MgC12, 6 mM Tris-HCl (pH 7.4), 6 mM 2-mercaptoethanol, 100 ug/ml BSA. b) Biotec. Assay Buffer (1x): 20 mM NaCl, 7 mM MgC12, 10 mM Tris-HCl (pH 7.5), 6 mM 2-mercaptoethanol, 100 ug/ml BSA. 8. ENDO R. Sal 1 a) 333. Assay Buffer (1x): 8 mM Tris-HCl (pH 7.6), 0.2 mM Na 6 mM MgCl EDTA, 150 mM NaCl. 2’ 2 IC 10 151' r—a "U ["1 ['4 I'll (In 955 44 b) Biotec. Assay Buffer (1x): 10 mM Tris-HCl (pH 7.9), 150 mM NaCl, 7 mM MgC12, 6 mM 2-mercaptoethanol, 100 ug/ml BSA. 9. ENDO R. Sma l a) 333. Assay Buffer (1x): 15 mM Tris-HCl (pH 8.0), 6 mM MgC12, 15 mM KCl. 10. ENDO R. Xba 1 a) 333. Assay Buffer (1x): 6 mM Tris-HCl (pH 7.4), 100 mM NaCl, 6 mM M9012. ll. ENDO R. Xho l a) 333. Assay Buffer (1x): 8 mM Tris-HC1 (pH 7.4), 150 mM NaCl, 6 mM MgC12, 6 mM 2-mercaptoethanol. Plasmid Growing, Amplification! Isolation and Transformation: D-glucose (Mallinckrodt) 20%, Thymidine (Sigma): 2 mg/ml, DPA (Sigma): 20 mg/ml, Tetracycline (Sigma): 1.5 mllml, Ampicillin (Sigma): 4 mg/ml, Thiamine-HCl (Vitamin B1 from Sigma), Casamino acids (Difco) 20%, Ampicillin:_ 78.125 mg/ml in DMSO, L-Broth Agar (1%, Bacto-agar "Difco”), L-Broth agar (1%) selective plates (i.e. Tet and Amp plates), Chloramphinicol: 15 mg/ml (Sigma), washing buffer: 10 mM Tris, pH 8, 1 mM EDTA, lysozyme (Sigma): 10 mg/ml in (25% sucrose + 0.05 M Tris, pH 8.0), 10T np-40 in 10 mM Tris pH 8.0, sucrose buffer: 25% sucrose, 10 mM Tris pH 8, 1 mM EDTA, protenase K (Fungal): 25 mg/ml, SDS: 10 and 20%, T10E1 Buffer: (10 mM Tris, pH 7.4, 1 mM EDTA) pH 7.4, csc12: (Grad 1, 99+%, 45 Sigma), EtBr (Sigma), Dowex AG50W-XB Na+ cation exchange resin, Source: Bio Rad. Dowex Resin AG50W-X8 (hydrogen form), Convert: Daw ex 50/H+ to Dowex 50/Na+, NloT10 buffer, pH 7: 10 mM NaCl, 1 mM EDTA, 10 mM Tris pH 7.6, Phosphate buffer, pH 6.8: 54 M, Alkaline SDS: 0.2 N NaOH, 1T SDS, Phenol water and Tris saturated, chloroform (Mallinc- krodt). II. METHODS* A. Induction of Lymphomas in Infected Chickens There were three strains of chickens from which the tumors were derived: line 1515 x 7, line 72 and line 6.3. The inoculation was given either through’ yolk sac route to'S day old embryos or by intra-abdominal inoculation to day-old chickens. The parent stocks were free of infec- tion with non-defective REVS, lymphoid leukosis virus, and Marek's disease herpesvirus (MDHV) antibodies, as determined by routine tests for the respective antibodies conducted by Dr. Witter and his colleagues (at the Reginal Poultry Research Laboratory, East Lansing, Michigan). At 17 weeks post hatching, selected male and female chickens were carefully transferred to plastic canopy isolators for a long term observation, the residual chickens were necrOpsied. Five birds were killed at 90 *In this section many abbreviations are used. See abbre- viation lists and materials. 46 days and the remainder at 138 to 285 days post-inoculation. Certain tissues were examined histologically by Dr. Witter. Control tissues from birds which received no injection were also obtained. Tumorous and representative non-tumorous tissues (from infected and normal animals) were taken for DNA extraction (see subsequent sections) and were transferred into vessels containing liquid nitrogen and then stored at -70°C until use. B. Cells and Viruses Spafa, chicken embryo fibroblast (CEF) and Qt6 (a quail cell line derived from a chemically induced fibrosar- comas by Moscovici 33 31. (248)) were cultured as previously described (198). The virus for all experiments presented here was the CSV strain of REV (104) (designated chick syncitial virus) which was propagated in CEFS, cloned three time by end-point dilution, and used as cell-free fluids from the 14th tissue culture passage. Virus stocks, supplied by Dr. Witter, were free of exogenous lymphoid leukosis virus based on appropriate complement-fixation tests (249) on C/E cell cultures 14 days after inoculation. Approxi- mately 2.5 - 7.5 x 103 FFU (focus forming unit) per chicken or embryo was administered. Cells were propagated at 37°C in medium DMEM (Dubellco Modified Eagle Medium) supple- mented usually with 3-5% calf serum and occasionally with 5% fetal calf serum and 10% tryptose phosphate broth 47 vol/vol). Cells were passaged about every two days when 3-uridine labeled they appeared near 100% confluency. H or unlabeled virus media (see below) were harvested at four hour or 24 hour intervals. Also, several times cellular preparation of virus were prepared and both cellular and cell-free preparations of virus were frozen at -70°C or liquid nitrogen until use. Virus production was monitored by measurement of the reverse transcriptase (R.Tn) activity (a quick method to monitor the presence or absence of virus) in the cell-free culture media from infected cells (250). All virus stocks used for infections or other purposes (i.e. viral RNA iso- lation) contained a high level of R.Tr. activities. In most of the infections the virus media were concentrated before use. The concentrated viruses were used for several purposes including preparation of hybridization radio- labeled probe, 13‘31333 infection, etc. C. Prepara3ion of Purified CSV Particles and 60-7OS RNA CSV virus stock media which contained a high level of R.Tr. activity were purified as follows. Using low speed centrifugation (8K rpm for 30 min.), the cell debris were removed, the pelleted viruses were then layered onto 25-55% sucrose gredient in 1x STE (see materials) and were centrifuged in a SW27 rotor at 25K rpm for 12-15 hours at 4°C. Then 1 m1 fractions were collected and monitored by 3H cpm incorporation in an R.Tr. assay. Virus containing 48 fractions were pooled and diluted with 1x STE and were centrifuged at 25K rpm for 2-3 hours at 4°C. The pelleted viruses were resuspended in proper solution (i.e., low TE). 60-7OS CSV RNA was isolated as follows: H3-1abeled or unlabeled purified virus was treated with proteanase-K to a final concentration of 100 ug/ml to remove the protein and lyzed by adjusting to a final concentration of 1% SDS and before extracting at least twice with freshly distilled phenol saturated with distilled water. The extraction pro- ceeded as described in the section on DNA extraction. The RNA extract was sedimented directly in a sucrose gradient and further purified, usually by two successive passages through oligo [dTl-cellulose columns, thus separating the viral RNA from contaminating ribosomal RNA (polyA). The chromatography on oligo-[dT] cellulose (251, 252) was done as follows. Briefly heat denatured purified RNA was passed through a well prepared column. The elution was performed under proper conditions, the RNA then adjusted to a final concentration of 0.25 M Na acetate, pH 5 and ethanol preci- pitated. For detailed information about chromatography on oligoldt]-cellulose see references 253, 254, and 255. The purified RNA as well as virus was used for preparation of radio-labeled hybridization probes (see below). It should be mentioned that mainly at the beginning of the present studies when the cloned DNA fragments (see Nick-translation section) were not available, using CSV or ASV purified viruses or purified RNAS, CDNA reps 49 (representative of RNA genome) were prepared by the follow- ing ways: (a) In the "exogenous" R.Tr. reaction highly 32] dCTP (400 ci/mmol) was incorporated into Specific [a-P Short segments (about 300 nucleotides long) by purified AMV RNA-dependent DNA polymerase (reverse transcriptase) using purified heat denatured (60-708) RNAS (from CSV and ASV) as template. Oligomers of calf thymus DNA about lOOO-fold by weight was used for priming the reaction. Using this procedure relatively complete and uniform trans- cription of RNA was synthesized. (b) In the "endogenous" R.Tr. reaction, the polymerization was carried out at high concentrations of dNTPS and at proper concentrations of NP*-40 to disrupt the purified virus particles (from CSV and ASV). This procedure is most efficient in synthesizing CDNA of large size (256, 257). D. CSV In Vitro InfectionnyNA Extraction and Purification PrOper cells, usually primary or secondary chicken embryo fibroblasts of about 50-70% confluency, were infected with CSV virus stock, the same stock which was used for 13 3133 infection. The linear, unintegrated CSV DNAS were isolated from the cytoplasms of CSFS infected with CSV viruses 2-4 days earlier by the procedures of Shank 33 31. (258). Cells were harvested by washing with 1x Tris glucose or PBS and *A non-ionic detergent which can break the cell membrane, but leave the nuclear membrane intact. 50 then were trypsinased, centrifuged and finally further washed with lx Tris glucose for at least three times, resuspended in 0.1 x RSB (5-6x106/m1) to swell for 20 minutes on ice. This was followed by lysing the cells with NP-40 (to a final of 1%, NP-40 is a non-ionic deter- gent which binds to the protein, thus removing both cell and cell membrane, etc. (258)). In most experiments Hirt fractionation (259) was used to separate the unintegrated DNA from integrated DNA (3.8 and H.P)*. This procedure entails coagulation of the high molecular weight DNA, thereby separating them from the low molecular weight DNA. The Hirt supernatant and pellet fractions were then treated with 250-500 mg/ml predigested pronase at 37°C for 30-60 minutes. The digested samples were extracted twice with phenol saturated with 100 M Tris-HCl (pH 7.3) (203), followed by similar procedures described in the in zigg DNA extraction (next section). The unintegrated linear DNA obtained from 0.8 - 1x107 cells was used per restriction enzyme digestion reaction. In several cases the high molecular weight DNA were isolated from chronically infected cells for the analysis of integrated viral DNA. In these cases cells were trans- ferred 2-3 times and harvested more than three weeks after *H.S (Hirt supernatant) and H.P. both are viral nuclear DNAs; the former is unintegrated and the latter is inte- grated high-molecular weight DNA. Sl infection. The procedure for the isolation was described previously by Battula and Temin (260). Briefly, cells were pelleted and washed 2-3 times with Tris glucose (as mentioned above) and resuspended in DNA buffer (see matere ials) at a final concentration of 2-5x107 cells per ml. The cells then were treated with pronase and lysed by SDS. The DNA was extracted as mentioned in the extraction of DNA from in 3332 infections. E. Extraggion of_High Molecular Weight DNA From Chicken Tissue Frozen tissues (about 0.5 - 1 g/sample) were homo- genized in a glass barrel with a loose teflon pestle and to the mixture was added 15 ml buffer*. The tissues were dispersed by homogenization (1-2 stroke) using a motor- driven glass Dounce homogenizer. The DNA in the homogen- ate was then digested with 250-500 ug/ml of self-digested pronase, or occasionally with 25-50 ug/ml proteanase K for 2-3 hours at 37-40°C in the presence of 1% sodium dodecyl sulfate (SDS). The digested preparations were then ad- justed to 0.1 M of NaCl and extracted at room temperature (r.t) with phenol (equilibrated with 50 mM Tris HCl pH 7-8)/ chloroform (1:1) until the aqueous phase became clear. It was then extracted once with chloroform, adjusted to a final concentration of 0.25 M NaCl and followed by precipi- tation with 2-2.5 volumes of ethanol either at -20°C for *(ZXSET = high TE (10 mM Mris HCl pH 7.5-7.8, SmM EDTA, 1% SDS)) + l/20 volume of 20% SDS + 50 ug/ml pronase. 52 overnight (O/N) or 30-60 minutes at -70°C. The resulting precipitates were collected by centrifugation and dissolved in 5-10 ml sterile double distilled H20. Some (but not all) samples were further purified, i.e. by treating with RNAase (i.e. pancreatic RNAase and T1 RNAase) for 30-60 minutes at 37°C. The corresponding samples then were deproteinized by extraction with phenol: CHCl3 and followed as above (258). The concentration of DNA samples were then measured by Diphenylamine (DPA) which reacts only with DNA, thus permitting a precise measurement of DNA concentration. Alternatively the concentration of DNA was determined by optical density (absorbance) at 260 nm (one Optical density unit at 260 nm was taken as 50 ug/ml DNA). The DNA ob- tained by the above procedure were relatively highly puri- fied, i.e., the ratio of absorbance A260/A280 of DNA samples were invariably greater than 1.85. All DNA samples were adjusted to a final concentration of .5 - 1 mg/ml and were stored at -20°C before use. F. Digestion of DNA Samples with Restriction Endonucleases, Agarose Gel Electrophoresis and DNA Transfer to Nitro- Cellulose Filter Papers Except for minor modifications (see below), digestion of DNA were carried out under conditions described previously (258). Briefly, high-molecular weight DNAs (usually 25 ug DNA/lane) were digested with several restriction endonucleases in the buffers recommended by the 53 suppliers (see materials) and incubated at 37°C for two hours. To test the extent of completion of each EcoRI enzyme* digestion, small aliquots (corresponding to 5% of total volume of each sample) were taken out and mixed with 0.5 - 1 ug PBR 313 or lambda (A) DNA. They were incubated in parallel at 37°C for two hours. The aliquoted samples and the markers were then electrophoresed in 0.8% agarose gels (261, 262). EcoRI enzyme contains only one site in PBR 313, so when all of the PBR 313 present in the aliquoted samples was converted from the circular to the linear form and appeared on EtBr stained gel as a distinct band, then the digestion was considered complete. Di- gested samples usually were precipitated with ethanol. The precipitate was then dissolved in dZHZO or leEAC (see materials). {Robe sure they were resuspended, samples were allowed to stand at r. t fornot more than 3 hours. In most cases the bromophenol blue was added in each sample, and sub- jected to electrophoresis on 0.8% agarose gels (Seakem), in leEAC buffer pH 8.1-8.2, for 12-16 hours at 40-60 mA. In parallel with DNA samples in separate lanes, fragments of A DNA generated by digestion with EcoRI or Hind III restriction enzymes were used as molecular size markers. Following electrophoresis, gels (0.6-0.8 cm thick) in each sample were migrated about 16-16 cm. DNAs were *The principal enzyme employed in this study. 54 stained with EtBr (1-10 ug/ml in distilled H20) for about 30 minutes at r.t and bands were visualized by u.v. light induced fluorescence. The DNAs were then denatured in gigg for 30-60 minutes at r.t and neutralized either at r.t. for 1.5-2 hours or at 4°C for 6 hours to overnight. The resultant DNA samples were then transferred onto nitro- cellulose filters for 1-3 days in 6 to 10x SSC buffer, a modification of the method originally described by Southern (263). The filters with DNA samples attached were then air dried and baked at 80°C for 2-3 hours in vacuo to immo- bilize the DNAs onto the filters. G. Hybridization, washing and Autoradiography Filters with DNA immobilized were preannealed for 6 hours to overnight at 37°C. The main purpose for this was to saturate the filter with carrier DNA to eliminate non- specified binding of the probes. Hybridizations using appropriate radioactive probes were carried out in 50% formamide, 3X SSC, 1X Denharts (.02% bovine serum albumin, .02% polyvinylpyrolecone, .02% Ficoll, see Reference No. 280) as described in materials, usually at 37-41°C (258). The radiolabeled hybridization probes were prepared mainly by Nick-translation (264). However, in some cases exogenous or endogenous reverse transcriptase reactions using purified RNA or virus was used to synthesize CDNA 6 probes (see corresponding sections). About 1x10 cpm of the P32 labeled specific probe in 0.5-0.8 ml hybridization 55 buffer for each standard size filter was used. Hybridiza- tions were for 12-15 hours in the presence of dextran sulfate and about 60 hours in the absence of this reagent in the hybridization buffer. I preferred the latter method and used it for most of the cases. Following hybridization to remove unannealed CDNA: filters were washed using a slight modification of the procedure described by Shank 22,2i; (258). Briefly, fil- ters first were soaked three times at r.t with 2X SSC, each time about 20 minutes, and then were incubated in 0.1x SSC, 0.1% SDS at either 41 or 50 in a shaker incu- bator with at least two changes of solutions, each about 45 minutes, and finally rinsed three to five times in 0.1x SSC for one minute at r.t. Autoradiography was performed as follows. The dried filters were exposed to x-ray film in the presence of Dupont Lighening plus x-ray intensifying screens in close contact with each other (265, 266) and stored at -70°C for various times of the exposure of the films. Films were developed in developer's solution. The developer and fixer were purchased from Eastman Kodak Company, U.S.A., and were used according to their instructions. H. Rehybridization of Filters Certain filters, in addition to hybridization to the radioactive probe, were washed and rehybridized to a second probe. Rehybridization was usually accomplished by first 56 eluting the former probe bound to the filter with low TE (see materials) at 70-75°C for 3-5 minutes. The filter could then be hybridized with a second probe as described before. I. Growth, Amplification and Isolation of Normal Plasmid and Recombinant DNA Clones* 1. Growth of Cells and Amplification of Clones Briefly, to prepare small and fresh cultures, E, ggli strain HBlOl from stock plates, with and without PBR 322cm: A dgwes was grown at 37°C shaker either in M-9 + supple- ments or occasionally in MZYDT (which mainly is used for normal and inserted A growing) medium. After cultured cells reached 0.D 600 = 0.5 - 0.6 (as measured by spec- tronic 20, Bush and Lensh), solid chloramphenicol was added to these logarithmic growing cultures. Small cul- tures then were transferred to large cultures and like- wise at similar growth phase (as above), dry chlorampheni- col was added for further plasmid amplification (to a final concentration of 150 ug/liter culture). The large cultures were then incubated overnight and the 0.D 600 was then remeasured to make sure that it had not dropped, thus no lysis had occurred. 2. Cell Lysis Earlier in the course of the present study, the cells usually were lysed by treatment with a proper amount of *Plasmids containing the oncogenes for Mc29, AEV and SNV DNA. Also chimeric A DNA of the oncogene of the endogenous AMV sequence. 57 lysozyme to destroy the cell coat, under proper condi- tions, and cellular lysis was completed with the addition of SDS to the final concentration of 1%. This was fol- lowed by a slight modification of the Hirt fractionation method (259) pronase treatment, extraction by phenol/CC13H, and, as mentioned earlier in cases which the upper phase was still cloudy, the extraction was repeated. The solu- tions then adjusted to 0.25 MNaCl and ethanol precipitated as described before. 3. Cesium Chloride Gradient Centrifugation and/or Hybroxyapatite (HAP) Purification Nucleic acids from PBR 322 and the recombinant were further purified using CSCl gradient or hydroxyapatite chromatography (269). Using this procedure (268, 269, 270) the yield for plasmids was about 600 and 120-150 pg per liter respectively. Alternatively, the alkaline extraction method was used. Basically, in this procedure the growth and amplification methods were the same as above except the extraction of plasmid was carried out under alkaline condi- tions. After nucleic acids were precipitated by ethanol, CSCl-EtBr gradient was employed to separate supercoiled plasmid from chromosomal DNA. To remove the EtBr used in the gradient from the plasmid DNA, Dowex50W—X8 (Na+) cation exchange chromatography was used. Using this rapid isolation procedure up to 1.5 mg of purified DNA per liter HBlOl-PBR 322 culture was obtained. The detailed proce- dures of this as well as for transformation of E. coli 58 HBlOl were provided by Dr. Horowitz (Department of Bio- chemistry, Michigan State University). J. Transformation of E. coli HBlOl The transformation procedure was a modification of the procedure described by Villa-Komaroff 35 El. (271). Briefly, proper amounts of competent cells of E; coli strain of HBlOl were prepared. The plasmids were diluted in DNA dilution buffer (see materials) and mixed gently with the above cells. Following the procedure the trans- formation tubes were transferred to fresh plates. In PBR 322 DNA transformants showed up in 24-36 hours and the recombinant plasmid transformants at least 48 hours follow- ing incubation at 37°C. The number of transformants was counted in each plate and the efficiency of transformation was calculated as the number of transformants per ug DNA. Usually about 105 - 8x105 transformants per ug PBR 322 DNA and much less recombinant plasmid were obtained. To insure that no contaminants had occurred of Tetr (tetra- cycline resistant) or Ampr (ampicillin resistant) colonies H3101 was usually included as control. In addition, PBR 322 DNA was used to evaluate the efficiency of the transformation. K. Further Purification of DNA for Molecular Hybridization Probes To obtain highly purified DNA for hybridization probes DNA samples purified through CSCl-EtBr gradient were 59 treated with RNAase at a concentration of 100 pg/ml at 37°C for 30-60 minutes followed by treatment with proteanase K at a concentration of 100 pg/ml at 37°C for one hour. DNA samples were then extracted and precipitated as described before. When necessary, a G-SO column was employed to remove the digested RNA from the plasmid DNA. In some cases the inserts were further purified from PBR 322 or A vectors by a preparative gel after digestion with proper enzymes. For this purpose low-melting (sea plaque) agarose was used (M. Bishop, H.J. Kung, personal communication). Briefly, using the proper enzyme in each case, the restric- tion fragments were localized and visualized in gel by illuminating with a short wavelength u.v. light. Using a sharp razor the appropriate region of the gel containing the band of interest was removed and sometimes resuspended in a large volume of 1x TEAC (see materials) and adjusted to a final concentration of 0.1 M NaCl, and incubated at 65°C for about 30 minutes. After melting, the solution was extracted by 3/4 volume of r.t. phenol freshly pre- saturated with an equal volume of 100 mM tris pH 8, 10 m MEDTA, and was immediately vortexed for 30-60 seconds. This solution was extracted 2-3 times with n-Butanol to remove ethidium bromide and concentrated 4 to 8 fold. The agarose free solution containing the DNA of interest was then precipitated and further processed as described earlier. Recovery was usually 50-60%. It may be mentioned that in 60 some probes, i.e. PSNv-sal-I 60B, and PMC-pst, both with and without PBR 322 DNAs, were used for nick-translation assays. L. Labeling of DNA with P32 b‘ Nick-Translation (Preparation of CDNA Probe) There are several published procedures (264, 272, 273, 274) in which the nick-translation reaction is used for labeling DNA substrates to high specific activity. For this purpose throughout the course of this study the procedure of Maniatis 25.2i- (264) was used with minor modification (see below). Briefly, relatively random nicks are introduced by the action of DNAase 1 into unlabeled DNA (3'-hydroxyl-termine). At low temperatures (14-16°C) each nick is then utilized to elongate the DNA strand in a non- discriminatory fashion by E, 22;; DNA polymerase l (275, 276). The polymerization thus gave rise to a relatively uniformly labeled DNA molecule. Generally 0.25 - 0.5 ug purified DNA (from SNV DNA and Mc-29, AMV, AEV and ASV oncogenes) was labeled in a reaction mixture of a total volume of 25-50 ul. The reaction mixture contained no more than 50-100 picograms of DNAase, and 2.5-4 units of E. 92;; DNA polymerase 1. After the polymerization reaction the reaction mixture was loaded onto a Sephadex G-50 column to separate the nick-translated DNA from free nucleotides and fractions were collected. The leading peak (the incorporated one) was pooled. The fractions then were extracted and preci- pitated as described before, except in most cases yeast t RNA 61 at 5-10 pg/ml was added as a carrier for ethanol preci- pitation. To denature DNA, all nick-translated probes prepared by this procedure were heated for five minutes at 85-100°C prior to the hybridization reaction. To make labeled CDNA, in cases where only one radio- active nucleotide was used, generally 2 to lO-fold excess of the three unlabeled dNTPs (dATP, dGTP, dTTP with concen- tration of 6-30 pM) over the single labeled ([a-P32] dCTP, which usually contained 400 ci/mmol specific activity and concentration of 3 uM) was used. The DNA specific activity was calculated from the percentage of input radioactivity incorporated. Using proper conditions, described above, usually high efficiency of labeled nucleotides incorporated were gained, especially when all labeled dNTPs were employed (between 30-55%). The following probes were synthesized: SNV (1.5x107 cpm/pg), ASV (2x107 7 7 cpm/pg), AMV (1.2x10 7 cpm/ug), AEV (1.5x10 cpm/pg) and Src (2x10 cpm/pg). CHAPTER THREE RESULTS I. A Brief Background to the Biology and Experimental Design A total of 23 birds from three different inbred lines (1515 x 71,72 and 6.3) and all free of common avian patho- gens were inoculated with CSV strain of REV (see experi- mental procedure) either by the yolk sac route into five- day-old embryos or by intra-abdominal or intravenous routes into day old chicks. About five months following infection, the chickens started to develop a high incidence of lym- phomas, principally involving the bursa of Fabricus. The chickens with lymphomas were sacrificed. To study the target tissues at the preneoplastic stage, five of the in- fected chickens at 90 days of age were also sacrificed to serve as controls. A total of about 85 tumorous and non-tumorous tissues including bursa, liver, spleen, kidney, thymus, gonad, proventriculus, duodenum, and brain were collected. The tumors were usually associated with bursa, liver, gonad, and in a few cases, also with kidney, duodenum, thymus, and proventriculus. Portions of certain tissues were saved for histologic and pathologic studies. The remaining 62 63 portions were used for DNA and RNA analysis. They were stored at -70°C until use. With such relatively high numbers of tumors, it is possible to conduct a meaningful analysis of CSV-induced tumors. In addition, a cell line "RP-13” (developed from tumor number three by K. Nazerian at RPRL) was included in these studies. High molecular weight DNAs were extracted from tumors induced by CSV-strain of REV and from several controls,* and then digested with several endonucleases under conditions for complete DNA digestion. The digested samples were then analyzed in 0.8% agarose gel and hybridized to radiolabeled hybridization probes. Briefly, the hybridization probes were prepared by nick-translation (264) and occasionally from purified virus particles or purified viral-RNA (256, 257). The probes include the clone PSNV—sal-I 60B, which carries the entire genomic sequence of SNV strain of REV cloned in PBR 322 and CDNA rep (a probe representative of ASV genome), and four other probes carrying principally the oncogene sequences of Mc-29 ("MC"), AEV ("erb"), AMV ("myb") and ASV ('Src"), respectively. Details of the procedure are presented in both the legends of the figures and the Methods section. *Examples of controls included both the unintegrated linear and circular CSV DNA, isolated from acutely infected cells: unintegrated chicken DNA obtained from uninfected birds including all inbred lines used in this study; and many non-tumor CSV-infected tissue DNAs (e.g., DNA from brain tissue) as judged by the lack of tumor histology. 64 II. Strategies for the Identification of csv Exogenous Proviruses in the Induced Tumors Digestion of proviral DNA with restriction enzymes which have more than one cleavage site in the proviral DNA produces two types of virus-specific fragments, these are (a) the junction fragments which, in addition to viral sequences, also contain cellular sequences of the host DNA linked to one another. The size of these fragments varies among proviruses according to the position of the enzyme recognition sites in flanking cell DNA. The other type. is (b) the internal fragments which are composed solely of viral sequences. For an undeleted viral DNA (i.e., normal proviruses) the (b) type of fragment should be common to all proviruses, regardless of the integration site; generally, proviruses are colinear with the RNA genome and the unintegrated DNA (202, 203, 230). Most of the studies described here are based on EcoRI-digestion analyses of the structure of csv proviruses in the induced tumors. To facilitate data analysis, it was important to construct a partial enzyme map for the unintegrated csv DNA, since no data regarding csv DNA was available. Uninte- grated linear and circular DNA extracted from csv-acutely infected cells (e.g., 2-3 days after infection) was digested with several enzymes, electrophoresed and hybridized to SNV* probe. *SNV and csv strains of REVS are genetically very closely related to each other and their genomes share greater than 80% sequence homology (108, 109, 167). 65 Some of these results are illustrated in Figure 4. As shown in lanes a and b, EcoRI enzyme does not cleave the CSV DNA, since the unintegrated linear CSV DNA displays the same migration pattern before and after EcoRI digestion. From these data, the full-size CSV DNA was estimated about 9.3 Kb. Lanes c and d display the sac-l digestion pattern of two different preparations of unintegrated CSV DNA. In both cases a fragment of 8.3 Kb is generated. These data suggest the presence of either one or two sac-l sites in CSV DNA. Further analysis is required to distinguish between the two possibilities. Lanes e and f show CSV DNA fragments generated by XhoI and ng restriction enzyme respectively. The upper band present in both lanes corres- ponds to about 9.3 Kb (the intact CSV DNA size). These data suggest that both enzymes probably do not have any cleavage site in the CSV DNA. The possibility that they may have cleavage site(s) on or near the termini cannot be ruled out by this data. In addition to the upper band, XhOI (lane e) also generates two fainter fragments of 5.6 and 3.9 Kb in size. The nature and source of these frag- ments is presently unclear. The possibility that the virus stocks used for this infection may be a mixture of two or more cloned CSV viruses carrying different XhoI sites can- not be ruled out. Overall comparison of the partial CSV DNA restriction data obtained by this study with that of linear SNV DNA obtained by Temin and his coworkers (88, 203, 275) indicates 66 Figure 4. The structure of unintegrated linear CSV DNA. The unintegrated linear CSV DNA was extracted from the cytoplasms of chicken embryo fibroblasts (CEFs) two days after infection by CSV, according to the procedure of Shank e_t_ g. (258) . csv DNA (obtained about 1x107 cells per lane) was digested with several enzymes (see below) and subjected to electrophoresis in 0.8% (W/V) agarose gels and transferred onto nitrocellulose filters all as previously described (18, 258). Virus specific DNA was detected by filter hybridization with nick- 32 translated P -labeled pSNV-Sal 60B DNA (generously 1 provided by Dr. H. Temin of the University of Wisconsin). This probe carries the entire genomic sequence of SNV. Molecular size marker, EcoRI-generated fragments of A DNA, was run in parallel with other lanes and are indi- cated at the left side of the figure: lane (a) undi- gested, (b) digested with EcoRI, (c) digested with Sac l, (d) same as (c) except using a different prep of CSV DNA, (e) digested with Xhol, and (f) digested with XbaI. 67 Kb 203 Figure 4. 68 that despite some similarities (e.g., lack of EcoRI enzyme sites), they are not identical. This is consistent with earlier findings by Keshet 2; 3;. (203). Since EcoRI has no cleavage sites within the CSV proviral DNA, it also does not cut the endogenous Mc29 loci; therefore, it was used extensively throughout the course of this study. As discussed above, the size of the DNA fragment carrying the provirus generated by EcoRI enzyme is deter- mined by the flanking cellular sequences. Each distinct band thus should represent one provirus. More importantly, the size of these EcoRI fragments would serve as a basis for comparing integration sites in different tumors. III. The CSV Endogenous EcoRI Fragments When EcoRI enzyme was used to cleave the high molecular weight DNA from the tumorous tissues, and the digested DNA was hybridized to a cloned SNV DNA probe (see figure legends and method section), in all cases tumor-specific ("TS") bands of different size appear, which is indicated by dots in Figures 5A and 6B (see below). In addition to these ”TS" bands, in all samples including the unintegrated control (lane u), a band ca. 12.5 Kb (indicated as endoREV in the figures) is detected. This band presumably repre- sents the endogenous REV sequence (end ). Such an EcoRI REV fragment has not previously been reported, although earlier liquid-hybridization studies indicated a low level of 69 Figure 5. Ehe stgucture of CSV proviruses and the "c-myc" gene in lymphoma as analyzed py EcoRI-Digestion Analysis. Avian bursal lymphomas were induced by injections of end point-purified, helper-independent chicken syncitia viruses (CSV; a member of the REV family) either intra- abdominally into day-old chicks or by the yolk sac route into 5 day old embryos of lines 151571, 72 and 6.3. After a latent period of ca. 20 weeks, birds which develop- ed lymphomas were sacrificed and the tumor tissues col- lected (145). Both the virus stock and the tumor sam- ples were shown serologically and biochemically to be free of avian leukosis viruses. The DNA was extracted from the tissues, digested with EcoRI, analyzed by 0.8% agarose gel electrophoresis and transferred onto nitro- cellulose filters, all as previously described (18, 258). Filter hybridizations were carried out in 50% formamide and 3X.SSC (1X SSC = 0.15 M sodium chloride - 0.015 M sodium citrate) at either 41°C (panel A to C0 or 50°C (panel D). The radiolabeled hybridization probes were prepared by nick-translation (264) of the following two pBR-322 based DNA clones: (a) pSNV-Sal I 60B: this clone which carries the entire genome of SNV was used to de- tect the REV sequence in tumor DNAs (the REV probe). SNV and CSV are genetically closely related and their genomes share > 80% sequence homology (108, 109). And (b) pMC-pst (generously provided by J.M. Bishop's lab): o.o¢n~_ m ousmflm >um 02 < a .. Iona . . nundfimu .77.... a“ IMM . w .. 31“) 1.ll>mm . Y. , ... ._ _lmomooao =m=oo on N _ :_ e. 71' this clone which carries principally the oncogene se- quences of MC-29 virus was used to detect the "c-myc” gene (the MC probe). Panel A,B,D,E: EcoRI digested bursal DNA samples from an uninfected bird (lane U) or from tumorous birds (1 to 6). Panel C: The linear, unintegrated CSV DNAs isolated from the cytoplasms of chicken embryo fibroblasts infected with the CSV viruses 48 hours earlier by the procedures of Shank EE.E$' (258). Lane a and b represent DNA samples before or after EcoRI digestion. ~Allex’cept panel E are autoradio- grams of the filters afterjhybridizations with either the REV or MC probes (as indicated on the top of the gel). Panel E shows the ethidium.bromide (EtBr) stained DNA pattern of the gel used to prepare the filter shown in panel D. The molecular size markers (in Kilobases, Kb) shown on the side of the gels correspond to the migra- tion pattern of the EcoRI-digested phage lambda DNA (shown in lane A of panel E), which were included in each gel run as an internal size standard. Also, the tumor specific bands ("TS") are indicated by dots which are not detected in the DNA from.normal tissue of the same or uninfected birds, in this figure as well as other. 72 Awmocflusoo. m mun—9.9m «3.9: «n a. ...L_ m: no bum 73 homology (lo-20%) between REV and the normal chicken chromo- some (108, 109, 167). The endogenous sequence, however, is only weakly hybridizable to the REV probe (presumably due to the divergence of these two sequences) and it is possible to differentiate the endo- and exo-genous (see below) REV sequences by conducting the probe hybridization under more stringent conditions. As illustrated in Figures 5D and GB, under the stringent conditions (50°C), the hybridization to endo is significantly reduced to REV the background level (see especially the uninfected control in lane. u (Figures 5D and 6B) , whereas the intensity of the TS band including the ca. 13.2 Kb (see below) band of numbers E and LE are not affected. This analysis confirms the exogenous origin of all the TS bands (see below). IV. The Mode of Integration of csv-Proviruses A. A Survey of Integration Sites of the csv-Proviral Bursal DNAs. As mentioned above, when the DNA from tumors was analyzed by EcoRI enzyme, in addition to the REV endogenous (which appeared only under less stringent conditions), addi- tional bands appeared, referred to as tumor-specific (TS) bands, of differing sizes (Figures 5A and D, Figures 6A-B, also see below). They are indicated by dots. The csv, "TS" bands were found in all tumors examined which strongly im- plicates csv proviruses in the induction of lymphoma tumors. 74 The REV proviruses of the primary bursal tumors characterized by EcoRI enzyme indicated that csv inte- grates at several sites, and for the vast majority of the tumors the "TS" bands fall into six size classes suggest- ing that csv-integration in at least six different sites in different birds can lead to development of a tumor. Size classes are described as follows: Class A (ca. 13.2 Kb) Class B (ca. 12.8 Kb) Class C (ca. 12.5 Kb) Class D (ca. 10.8 Kb) Class E (ca. 10.3 Kb) Class F (ca. 9.9 Kb) Representative samples are shown in Figure 5A (lanes 1-6). The identity of the "TS" band in lane 6, which has a mobility similar to endo can be verified under stringent REV hybridization conditions (Figure 5D) where the endo REV disappeared, yet the intensity of the "TS" band (exogenous origin) is not affected. However, is a few bursal tumors, several "TS" bands with molecular-weights smaller than the six common size classes were found and are illustrated in. Figures 6A-B (lanes 7, ll, 12 and 18). Some of the pro- viruses (if not all) in these tumors are shown to be defec- tive. This will be discussed in subsequent sections. As summarized in Table 1 and tabulated in Table 2 (see below) Class D (ca. 10.8 Kb) appears to be most prevalent among a total of 15 analyzed bursal tumors (which cary a total of 19 "TS" bands) and occurs in at least 32% of the tumors. It is followed by Class C, A, and B which occurred in 26, 21 and 11% of the tumors, respectively. The other 75 O .. .. = .. I I I I .H. 8 8 8 8 8 8 8 ..H H m.oa m.oa ooa z baa a amemH .m.~a .~.ma .m.~a .~.ma m a C m 8 8 8 8 8 8 8 9 co m boa : we a m.oa .m.NH m m 8 8 8 8 8 M Q 2 8 o ..H H m m m = m oma m bx Hma m.m .m.oa .m.~a mm b 8 8 8 8 8 8 m o N H s 8 1H ooa z mom a» m.o ~.ma ~.ma m o O .. I I m I I .H m o o .—.. 8 8 8 m m o N. 8 8 8 8 8 8 —H ooa m «ma ma = m.~H m.~a m e O m : = = : I I = G O C H = = a = I I = M O O H : = = 3 I I s 1H a m m.o .m.m om m mma m> 5x HmH II .m.oH .m.~a m m cod 2 NmH a an m.oa m.oa m N oo 2 how oa aaxnama o.oa . o.oa m a cmamfimm Axum Anamov mcoHu mmcflq GUS >mm mmsmmfla Hoofisz onuazq . mod IommcH Hmucmumm p coxOflnu Hamo H0539 mo ousmEmmumleoom no a musom .cowumomMOIHmoom an msooH :oafilot pom msufl>oum >mo mo cowumoflmfiucmoH .H manna + .. .. s : II .. Hmmamk m m.H w m.a v + m cm a : m.m .~.> .m.m .~.> cm ma om z and a» a a o.~a .~.ma a pa = = : oz m.~H m ma o . oam a HememH .. I. am ma e.m .m.ca v.m .m.oa om = t = a m.NH .~.ma m.ma .~.ma o o w 8 8 8 8 8 8 .H cod a ova : a m.cH m.oa m va cod a and a mu ~.mH «.ma n ma 0 = .. .. = II II Hm m m 8 8 8 8 8 8 8 «H ooa : t a a m.~H m.m .m.~a m NH N 8 8 8 H w“ 8 8 ..H cm a «cm a bx Hma m.m m.m .m.> m Ha m.oa m N 8 8 8 8 8 ~ m o N H s 8 .H OCH 2 ova m we «.ma m.oH .~.ma m oH nonEmm Axum Anzac. mcoofi mmcwq 02 2mm mosmmfls Hone—oz moo ca . mmm IommcH Hmucmumm .c v HHmU H0358 mo oucmEmmuthmoom m0 a mason .Aposswucoo. .H manna 77 moauoao Odo amp m oucfl momsua> mo coaumHsoocfl 0mm xaom u m» «mxowno 0H0 mop oucw momsuw> mo coflumasoocw msoco>ouucw no AnswEOpbmumwucw u mam .muwxuosloo man can umuuwz .uo mo onEOM uh «mama "2a oouosccoo cowumsHEm o HMOflmoHonummoumwn co comma .omuowumo memo twat o: "In .cowuommu coflumuwowuomz cw cohonEm monoum one ‘HJ: .nmcwEHoumn uoc ”oz '0 0) .mosHm> mumfiflxoummm maco mum mmx m.oH M unmmeIHmHsooHoE mocmn ems: on» no meow mo ouflm wound on man can Amuwmm ommoloHfix. mom :H so>flm mum pawn awe: mo unmfloS “masomaozo omad .somamm .x .Ho an m .02 when mo Hofisu meonmsaa on» Eouw OmmoHobmv .msfia Hamo 6 ma malmm .Houucoo Oouoomcwss co m can mascos woman I am .msHsOfiwuco>oum u m u a .swmwo u Hm .Escmnoso u o .hocowx u M .uo>wa u A .mmnsn u m .qmmm mo cmwuonmz n was Oman mm Hogan: .Omcom u o .msahnu ”mmsmmflu mo monsomm m.m II a In .o.HH .m.~a nmm O : = I I s I I I I m 0 = : II : II II Cm MN 8 z s m \n .. I I : Gm N N s N = .. .. I I .. cm H N I = .. mun .. I I .. cm 0 N + z om a» HamemH u- abooEma on ma soamfimm xom Amamc; _mcoflu mmsfia Us >mm mosmmws Hoossz on» so 00¢ loomcH Handmade o n coxownu Hamu Means mo ousmEmMHmIHmoom m0 m «Doom .ooaoaoaoo. .a oaooa 78 Table 2. The frequency of occurrence of the most common "TS" bands in 15 bursal tumors.* Frequency of occurrence of Size** individual TS bands "TS" Band (Kb) (%I A 13.2 21 B 12.8 11 C 12.5 26 D 10.8 32 E 10.9 5 F 9.9 5 *There are a total of 19 "TS" bands detected in these 15 bursal tumors. **Due to the large sizes of the fragments, the molecular- weights 2 10.8 Kb (kilobase) are only approximate values. 79 two classes, E and F, are present in 5% of the tumors. These data (see Tables 1 and 2) also indicate that CSV induced tumors contain up to four proviruses and while there are several tumors that have only one TS band (examples can be seen in Figure 5A, lanes 1-2, 4-6, as well as Figure 6B, lanes 13-14); in many cases more than one TS band was found. Examples are shown in Figures 5A and 6A-B, lanes 3 and 7-11, 12, 18, respectively. B. Abundance of a Single Intecration Site of the Proviruses in the Primaryp(Bursal) Tumors As summarized in Table l and shown in Figures 5 (panels A and D) and 6 (panels A and B), six out of fifteen bursal tumors were found to carry only one "TS" band. Therefore, about 40% of the bursal tumors acquired a single REV provirus. The simplicity of the provirus pattern in many bursal tumors facilitates a detailed structural analysis of the unique "TS" band. Among these six bursal tumors carrying only one "TS” band, again class D and also class A, appear to be most prevalent. Each occurs in two bursal tumors. They were followed by classes B and E, which in each occurred one bursal tumor. In the remaining two classes (C and F) apparently no unique bursal ”TS" band was detected (Table 1). These data strongly suggest that the presence of a single provirus is sufficient for oncogenic transformation. 80 C. CSV-Induced Bursal Tumors May be "Clonal" in Origin A tumor is defined as ”clonal" when it consists pre- dominantly of a homologous population of cells, i.e. when all the cells in a given tumor are derived from a single transformed and infected cell. If the tumor is derived from a mixed population of a few initially infected cells, it is referred to as ”semi-clonal". All bursal tumors examined display at least one distinct "TS" band. This is consistent with the Clonality origin of tumors. However, the mere appearance of a "TS" band is not sufficient to claim the Clonality origin of a given tumor, but in this study, the fact that in different birds "TS" bands with different sizes were found, whereas a single band is usually found in a given tumor, is most suggestive of the clonal origin of CSV-induced tumors. In addition, similar analysis of the DNAs obtained from other CSV-infected birds at the preneoplastic stage (see preleukosis section), with only one exception, do not show a "TS" band, but rather a ‘ l"smear" pattern is obtained, as compared to tumors from terminal neoplastic stages with a distinct "TS" band. This also strongly supports the idea of clonal origin of CSV-inducted tumors. It, therefore, is quite likely that the initial integration took place at multiple sites in the cellular genome, of a large number of cells, but only a fraction of the infected cells were transformed. The transformed cells are characterized by a distinct band which indicates the location of the integrated provirus. 81 Recently the clonal nature of tumors has been reported in the ALV induced tumors [Neel EE‘EE. (l6), Payne 2; El; (17), Fung Egygi. (18), Neiman 2; 2i- (19)], murine leukemia virus induced tumors (Steffen 23 3;. (232)), mouse mammary tumor virus induced carcinomas (Cohen g3 Ei- (234), Cohen and Varmus (276)), and bovine leukemia provirus (Kettman gg‘gl. (277)). EcoRI analysis of a total of 31 tumors (i.e., bursa, liver etc.) shows that each of these tumors contains at least one ”TS" band. Table l compiles all the data concerning each of the 31 tumors including the inbred lines employed, the route of infection, the age and the sex of the bird and the nature of the "TS" bands. It also includes the data from uninoculated control birds and cell line RP13. As summarized in Table 1 and tabulated in Table 3, the latter table shows comparable data to those previously listed in Table 2. However in this table, in- stead of only bursal tumors, all analyzed tumors (i.e., including metastatic tumors in visceral organs) are con- sidered in the calculation (for detailed information see Table l). A brief comparison of Tables 2 and 3 shows similar results in regard to the distribution of tumor appearances among the six common size classes of TS bands. For example, the class D type band is again found to be the most prevalent class (i.e., 30% of all tumors carry such a band). Similar information for other size classes can be seen in Table 3. 82 Table 3. The frequency of occurrence of the most common "TS" band in bursal and metastatic tumors.* Frequency of Occurrence of Size** Individual TS Band "TS" Band (Kb) (%I A 13.2 18 B 12.8 12 C 12.5 28 D 10.8 30 E 10.3 4 F 9.9 8 *A total of 31 tumors (including bursa, liver, kidney, duodenum, thymus, gonad, and proventriculus) carrying a total of 50 "TS" bands were analyzed. **Due to the large sizes of the fragments, the molecular- weights a 10.8 KB (kilobase) are only approximate values. 83 Results in Table 1 also indicate that CSV DNA, at least in some tumors, was not tandemly integrated into the chicken chromosomal DNA. All EcoRI fragments were smaller than the dimer size of normal CSV DNA (i.e., smaller than the 2 x 9.3 or 18.6 Kb), although this con- clusion is based on the assumption that no deletions occurred in some proviruses (i.e., tumors No. l and No. 2, See next sections, so that this conclusion is valid for at least these tumors. Data from Table 1 also indicate that if one makes a comparison between lymphoma tumors in male and female CSV- infected chickens, the tumors of infected males appear to have relatively fewer numbers of TS bands than the female analyzed tumors. For example, in males, in most cases, one, sometimes two, and occasionally three TS bands were found, whereas in several female tumors more "TS" bands were detected. Furthermore, a closer look in Table l indi- cates that all the TS bands found in tumors in infected males fall into one or more of the following size classes: A, B, C, and D. The other two common size classes (E and F) in- cluding the smaller size TS bands were not found. This observation may be significant, especially since all TS bands which presumably contain smaller proviruses are pre- sumably defective and at least a few proviruses which must be defective (see subsequent sections) are found only in tumors of female infected birds. 84 The significance of these apparent sex differences, i.e., the presence of fewer integrated CSV proviruses and the lack of all smaller TS bands in males, is at present unclear. However, it is possible that for some unknown reason the female birds may tolerate more infection and accommodate more CSV proviruses in the chromosomes of their target tissues. The appearance of smaller TS bands only in the female birds is quite unlikely to be the result of chance and may suggest that, at least, that the occurrence of deletion and structural alterations are much more fre- quent in the female birds than the males. Although there may be some deletions in the proviruses present in tumors of the male birds, a possibility which, at least for some tumors cannot be ruled out from the present study such occurrence appears to be statistically rarer in the tumors of males as compared to those of females. As for the route of infection (i.e., yolk sac infection into 5 day old embryos versus intra-abdominal or intraven- ous infection into day old chicks) as judged from Table 1 data, apparently no major differences, i.e. in the sites of tumor development have been observed. This is consistent with the idea that bursa are specific targest for CSV-tumoro- genesis. It should be mentioned that many infected non-tumor DNAs as well as DNA from uninfected chickens as controls were analyzed by the same method. All of them are uniformly negative for the presence of "TS" bands. Examples of these 85 Figure 6. EcoRI analysis of bursal DNAs from CSV- induced lymphomas. DNAs from 17 different birds were analyzed using proce- dure as described in the legend of Figure 5 and methods section. Panel A: EcoRI digested bursal DNA samples from an uninfected bird (lane U) and from tumorous birds (1 to 11). For all samples in this panel, filter hybridi- zation was carried out at 41°C. Panel B: similar as panel A except hybridization was conducted at 50°C. Both panels A and B were hybridized to PSNV-sal 1603 DNA probe (see materials and methods). All except lane U (uninfected bird) and lane 18 (90 days post-inoculation) were bursal samples of tumorous birds sacrificed at 3 20 weeks post-inoculation. Panel C: EcoRI digested bursal DNA samples were hybridized to PMc-pst (as described in the legend to Figure 5). Lanes 12N, 15N and 5N are brain DNAs which serve as control samples. 86 .,. .... o _ mm b. you: w wusmah >wm 87 ...~_ m00.¢-.n.~- Awwscflucoo. o musmflm _ a. o. ..y. zozm.z.~._- 02 88 are illustrated in Figures 5 and 6 (lanes U). V. On the Mechanism of Deletion in CSV-Induced Avian Lymphomas By characterization of the internal fragments of the proviruses, possible deletions can be revealed. This type of analysis can be aided significantly by the availability of cloned TS fragments. While these experiments are under way, several tumors which carry single TS band patterns, using EcoRI enzyme, were selected and subjected to further restriction enzyme analysis (see below). A. A Comparison of the Structure of the Single "TS" CSV Proviruses in Tumor No. l and No. 2 The limited different number of "TS" bands suggests that integration of the viral DNAs at a limited number of sites is conducive to transformation, a conclusion consis- tent with previous studies of the LLV induced tumors (15, l6, 17, 18, 19). A close examination of the TS band pat- terns, however, reveals that the size variation of some of these bands is probably caused by deletions or other struc- tural alteration of the cellular or viral sequences. If the TS bands result from a simple integrative recombination involving the REV DNA and the "c-myc" gene which is the cellular progenitor sequence of the oncogene of Mc29 virus without other structural alterations, then the EcoRI-TS band should have a size equal to the sum of the viral DNA and the end MC fragment. This study shows that this is not 89 Figure 7. A comparison of the structure of the CSV proviruses in tumor No. l and No. 2 and Bgl II enzyme analysis of some lymphoma tumors. Chromosomal DNAs extracted from tumor No. 1 (left lane) or from tumor No. 2 (right lane) were digested with restriction endonucleases (Biolabs and Biotech) as indi- cated at the top of the gels. The samples were analyzed in 0.8% agarose gel and the Southern-filter hybridiza- tions with the REV probe were conducted at 50°C as des- cribed for Figure 1D. Panel B: Bursal DNAs from CSV- induced lymphomas extracted from tumors No. 3 to 6 were digested with Bgl II restriction endonuclease and were analyzed as before (i.e., as in panel A). 90 Kb XhoI XbaI Ba'nHI HindIlI SmaI zoo—E; .l g1; - i-o- i? » L“ 4 Kb 20.8— 7.2— . .8 5%: I - ..- 3.2— B ’ Mafia ’7 7 91 the case. This is especially obvious in samples 1 and 2 as well as in several other tumors (class D) and even some other tumors which have an even smaller size than class D tumors (i.e., classes E and F), yet are greater than or equal to the complete CSV genome size. In all these examples the TS bands are actually smaller than the endoMC fragment. This result indicates that a deletion or structural alteration (leading to the generation of a new EcoRI cleavage site) must have occurred. Further digestion analyses of the proviruses of samples 1 and 2 (Figure 7-A, left and right lanes, respectively), using other enzymes, including those which cut the viral DNA internally to generate the "signature" fragments (e.g., BamHI), indicated on the top of the gels, are shown in Figure 7 (panel A). In all cases (XhoI, XbaI, BamHI, HindIII, and SmaI), individual patterns consistent with the presence of a complete REV genome are displayed by these two proviruses. These results not only confirm the identity of these two proviruses, but also suggest that the extensive deletions which gave rise to the "shortened" 10.8 Kb band, if they occurred, probably involve the sur- rounding cellular sequences. Several bursal tumors which carry either one (i.e. tumer numbers 5 and 6) or more (i.e., tumor numbers 3 and 4) EcoRI TS band patterns, were also analyzed by BglII diges- tion. As illustrated in Figure 7-B, like EcoRI, this enzyme also possibly has no cleavage site present in the 92 .mocmsqmm dzn Hoasaamo on» mouocmo mafia >>m3 one .moososqmm dzo Hmasaamo man do ouwm omm>moao Hmoom “0m coaumooH manwmmom mnu mumoflGCfi mmamcmfiuu ofiaom ..m com d. mocmsqom «zo HmHsHHmo pouoaop oaowmmom oumowpcw mmHmGMfluu some .m Hones: Hofisu can a 0005:: MOSS» ca «29 Houw>oum >mu wow «0 muouozuum HoOfiuonuomhm .m musoflm 8 V rm . . 5 am am . a . ..., ()4 x ,.-. 2; Vb_=._oo _m .gsno. «.36... >8 III III 93 CSV-proviral DNA. This is based on the observation that, at least, in tumors number 4 and 5 a large TS band was found which is comparable in size to those generated by EcoRI enzyme using the same tumors (i.e. , for comparison see Figure S-A). The relative size differences in the B9111 generated fragments are also similar to their EcoRI enzyme corresponding patterns (i.e., in both enzymes each TS band in each tumor differs ca. 0.3 Kb in size from one another). As mentioned earlier, based on this preliminary BglII enzyme data, if one assumes that this enzyme does not cleave the proviral DNA, these data support the idea of the occurrence of deletions in surrounding cellular se- quences rather than in the viral sequences in those tumors. This is illustrated in Figure 8. If one assumes that EcoRI sites are located in the places indicated by' in Figure 8, then the possible BglII sites occur either in (l) or (2) in the surrounding cellular sequences, in such a space way that the BglII enzyme generates fragments slightly larger (i.e., ca. 0.3 Kb) than the EcoRI enzyme. But the relative sizes of the "TS" bands among different tumors generated by EcoRI or by BglII should be similar. This is in general the case (i.e., compare lanes 4 and 5 in Figures S-A and 7-B). However, the small fragment in tumor number 3 and the lower two fragments in tumor number 6 is not consistent with above speculation. It should be stressed that, at present, this is the subject of speculation and other alternatives 94 cannot be ruled out. To test the above hypothesis, and to verify the exact nature (or identity) of BglII fragments, and to identify possible deletions, etc., further work is badly needed. For example, one should construct the complete 22 33359 map for the viral DNA, then use those enzymes which cut the viral DNA internally (e.g. BamHI) in these and other tumors. As listed in Table l, the vast majority of the tumors fall into one of the six size classes. Since even the smallest size class (F) is larger than a complete CSV genome, these data are consistent with the notion that there are no extensive deletions in the integrated proviral DNA and unlike reports in ALV systems (l6, 17, 18, 19) suggest viral sequences may be somewhat important in the maintenance of transformation. However, a definite con- clusion, at least for some tumors, at present is not possible and further work is warranted. B. The Proviruses Having Abnormal Structure As summarized in Table l, eight tumors (from four birds) in addition to common size classes "TS" bands also contain TS bands with molecular weights smaller than the CSV pro- viral DNA indicating deletion or structural alteration of the proviruses has occurred. As can be seen in Figure 9-A, all four tumors from bird number 3, and tumor number 7 (Figure 6-A) carry a 6.6 Kb TS band. Tumor number 12 (Figure 6-B) also shows a 2.8 Kb TS band. In two bursal 95 tumors (No. 11 and No. 18) in Figure 6-B (lanes 11 and 18) only defective TS bands were detected in which a signifi- cant portion of the viral genetic material was deleted or rearranged. These data indicate that in these few cases the complete structure of proviruses may not be needed for neoplastic transformation since in these occasions lym- phomas were induced, apparently. However, deletions or major internal alterations may not be necessary as a require- ment for tumorogenesis since, as discussed earlier, the majority of the tumors probably carry apparently non- deleted proviruses. Again, DNA analysis of the tumors, i.e. using multiple "cutter" enzymes such as enzymes which cleave proviral DNA at several sites) will shed considerable light in this issue. Also, analysis of viral RNA in infected tumors will establish whether or not the viral gene pro- ducts are required for the maintenance of transformation. VI. Amplification of the Proviruses One of the striking features of the results of these studies is the unusual intensity of the hybridization sig- nal of some of the "TS" bands (e.g., samples No. l, 2, 18) especially the 10.8 KB band in sample 1 as shown in Figure 5-D. The total amount of the chromosomal DNA loaded on each lane, as judged by the ethidium bromide stains, is comparable (Figure 5-E). In addition, the DNA concentration was determined by DPA (diphenal-amin) assays and the same 96 amount of DNA (about 25 ug/lane) was used in all samples. The high intensity of the 10.8 Kb TS band in sample 1 is revealed by both REV and Mc probes (Figures S-A and B, also see below). In other words, the amplification (at least for sample 1) occurs for both REV and Mc ("c-myc") sequences. This observation, together with the larger size of this band as compared to the unintegrated viral DNA, suggests that amplification of the REV provirus and its surrounding cellular sequences has occurred. This ob- servation is being verified using other methods, for example, Southern-bolt analyses of the tumor DNA using a- globin gene sequence as an internal control and using cloned DNAs in a reconstruction experiment. VII. Linkage of the CSV Provirus With the Mc-29 Related Endogenous Sequences ("C-myc") A. A Survey of the Linkage of the "TS" Bands by Mo Probe Recent studies by Hayward 23 El- (15) and Fung 22.2l- (in press, 18) have obtained evidence showing that following ALV infection in young chickens, ALV provirus is integrated adjacent to a specific cellular gene, the "C-myc" gene (which is the endogenous proto-onc Mc-29 virus). This sequence is highly conserved and present in all vertebrates (2, 9, 279). These observations have strongly implicated the Mc-29 endogenous sequences in the induction of lymphoid leukosis (LL). These results raise the interesting 97 possibility that the "C-myc" gene is a key target in lympho- magenesis and its product is responsible for inducing trans- formation. Since the CSV strain of REV is also capable of inducing LL-like tumor "lymphomas" with similar latency and patho- logy (118, 120, 145), it was of considerable interest to determine whether in LL-like tumors induced by the CSV strain of REV the virus has integrated adjacent to "C-myc" gene. To examine this possibility and to test for linkage between CSV proviruses and " -myc" gene, the structure of the CSV proviruses in the induced tumors were characterized. Tumor DNAs were digested with EcoRI enzyme as described before and were hybridized to a probe synthesized from a cloned DNA PMo-38 which specifically carries the Mc-29 oncogene sequence. As shown in Figure 5-B which contained the same bursal DNAs as those of Figure 5-a, the TS bands (also indicated by dots) detected by the Mc probe, except sample 3 (see footnote), match well with the corresponding fragments identified by the REV probe in Figure 5-A. (The band marked as endoMC corresponds to the endogenous proto-onc MCV ("C-myc") gene which is present in both the normal and the tumor tissue (18, 54)). Each tumor appears to have one TS band co-hybridizing to both probes!’ Such cross *In tumor No. 3 the "TS" band shows apparently no linkage between REV provirus and onc MC gene although one "TS" band carrying REV sequence comigrates with the endoMC fragment, but only coincidentally. 98 hybridization is not due to homology between REV and Mo sequences since the unintegrated CSV DNA (lane R) is not detected by the Mo probe. More extensive analysis on the other samples with the Mc probe shown in Figure 6-C and corresponding analysis by the REV probe are shown in panels A and B of the same figure. In all of these samples each tumor contains at least one "TS" band cohybridizing to both probes. These results strongly suggest that in tumors obtained from 16 birds out of 17, at least one REV provirus is integrated next to the " -myc" gene on one of the two homologous chromosomes and strongly implicates the latter gene in lymphocytic transformation. Also, all types of controls used in this study showed only an endo MC Figure 6-C is the result with bursal DNA obtained from an band. Lane U of uninfected bird treated similar to injected animals (No. 23 in Table 1). DNA samples in lanes 12N, 15N and 5N were obtained from brains from the corresponding birds, which were infected, but in which no tumors developed. In all cases, as expected, no TS band was detected. These samples are used here as controls. As illustrated in these figures, and summarized in Table l, a total of 14 bursal DNAs were cleaved and hybridized with thelk3probe; among these bursas from eight birds. Interestingly, all "TS" bands detected bybkzprobe match well with the corresponding frag- ments identified by the REV probe. Five of the remaining contained two to three CSV proviruses detectable by the REV 99 probe. Only one TS band in each case seems to have inte- grated next to the "C-myc" gene. On exception can be found in the bursal tumor (bird no. 3). In this tumor the CSV DNA does not appear to be linked at all to endoMC (see below). Also listed in Table l is the data concerning the secondary tumors. In all of them, the CSV-DNA and the "c-myc" genes are found to be linked. B. Appearance of Multiple Linkage of the CSV Provirus With the Mc-29 Related Endo—Sequences As mentioned earlier in most of the tumors where mul- tiple CSV-proviruses are present, only a single csv-frag- ment is linked to the "C-myc" sequence. However, in Figure 6-C and lane. G in Figure ll-A more than one provirus appeared to be linked to the "C-myc". The most straight- forward interpretation is that these tumors consist of two or more independent tumors coalescing together and each carries a CSV-provirus integrating adjacent to the "C-myc" gene, but at a slightly different position. C. No Apparant Linkage of the CSV Provirus with the Mc-29 Related Endogenous Sequences Among the lymphoma tumors analyzed only one bird (No. 3) seems to show no linkage between the CSV proviruses (as detected by REV probe) and the Mc-29 related endogenous sequences (as judged by Mc probe hybridization. As illus- trated in Figure 9, in all four tumors (bursa, liver, kid- ney, and duodenum) in chicken No. 3, identical proviruses were detected using the REV probe. When the similar samples 100 were hybridized to the Mo probe apparently no "TS" band was detected. The upper TS band seems to co-migrate with the endoM band, but this is coincidental. The bursal DNA C of this bird was further analyzed by BglII enzyme. When hybridized to the Mo probe, only BglII endoMC was detected, and the small BglII-fragment detected by the REV probe (see Figure 7-B, lane 3) was not apparent. This suggests that no bursal ”TS" bands co-migrate with the endoMC. No further tests were carried outwj-th other tumors of this bird. From the present study it seems unlikely that the pro- virus bands co-mdgrate with endo in the primary target MC tissue (bursa), and data based on other enzyme digestions (i.e., BglII) also do not show apparent linkage to endo A cell line which was developed by K. Nazarian MC' (of RPRL) from bird No. 3 also does show a similar pattern‘ to the other tumors of this bird. This is shown in Figure 9 in the lane marked RP for Regional Poultry. At present it is not clear why the exogenous CSV proviruses in this in- fected bird, unlike the rest, appeared not to be linked to endo sequences. One possibility is that the proviral MC DNA is linked to another yet to be identified transforming gene. If this is the case, it would suggest that more than one cellular gene has the potential to activate lymphocyte- transformation. Cloning of the "TS" band and structural analysis of the downstream sequence should provide insight into this possibility. 101 Figure 9. The structure of CSV provirus in theNp. 3 bursal DNA — the lack of linkage with "c-myc”. DNAs samples extracted from bursal (B), liver (L), kid- ney (K), and duodenum (D) tumors of No. 3 bird were di- gested with EcoRI enzyme and hybridized either with SNV strain of REV probe or with Mc probe. Also included in this study, the sample from the cell line RP13 derived from the liver tumor of this bird (supplied by Dr. K. Nazerian). Lane RP represents the RP13 DNA sample. 102 -‘- ONIOMC Figure 9 103 VIII. The Structgre of Other "Potential Oncogenes" in the Tumors To study whether other known cellular oncogenes are associated with tumorogenesis, EcoRI cleaved tumor DNAs were hybridized to the following probes: a) ASV CDNA rep (a probe representative of the ASV genome b) PBR322-STC (P-src) c) P-"erb"-pubII d) "myb"-¢4A EcoRI subcloned DNA Probes b, c, and d carry principally the oncogene sequences of ASV, AEV and AMV, respectively. Details of the proce- dure are presented in both the legends of the figures and method section. SacI digestions were also used to confirm some of the data. The results are presented in Figure lO-A,B. All bands in this figure represent the endogenous sequences. In all cases patterns identical to the uninfected controls were obtained, suggesting that these genes are not altered by CSV proviral DNA integration. IX. The Relationship Between Primary and Metastatic Tumors Using the same method as previously described, CSV- provirus DNA has also been analyzed in more than 15 meta- static tumors (e.g. "secondary" and "tertiary" targets). Several examples are shown in Figure 11 (panels A-F). Each panel shows tumors from one bird (as numbered at the top of 104 Figure 10. The structure of other putative oncogenes in the lymphoma and normal tissues Southern blot analysis of EcoRI (panel A) and Sac-l (panel B) digested chromosomal DNAs of tumor and non- tumor tissues. The radiolabeled hybridization probes were prepared as described in method section of the following four DNA fragments as indicated on the top of the gel: (a probe representative of (a) ASV(‘31)NA rep the ASV genome), (b) PBR 322-SrC (P-SrC); (c) P-erb- pvuII, and (d) myb-¢4A EcoRI subcloned DNA. Probes b, c and d carrying principally the oncogene sequences of ASV, AEV and AMV respectively. The latter three cloned DNAs 03,c and d) were generously supplied by Dr. D. Sheiness and Dr. J. M. Bishop (University of California,5an Francisco). Lanes T, B, G and K refer to DNA samples from thymus, bursa, gonad, and kidney, respectively. Lane U is the sample obtained from the uninfected bird. Lanes 10T, 14K, 9T are samples obtained from the non- tumor tissues from their corresponding infected birds. The remainder of the samples were obtained from the tumor tissues from several infected birds as indicated on the top of each lane. All bands in this figure repre- sent the endogenous sequences. 105 on oaaoam 106 Figure 10 (continued) 107 the gels; also for more information see Table l), which were analyzed by EcoRI enzyme using both the REV probe (the left lanes in each panel) and the Mc-29 probe (the right lanes in each panel). These results clearly demon- strate similar patterns in most of the metastatic tumors when compared to their corresponding bursa (primary target). (For examples see panel A (except line C, see below, B, C, F, etc.) Panel F is an especially distinct example in this regard. However, in a few cases some minor variation has been observed. For example, differences are apparent in panel E (bird No. 6) as well as in bird No. 10 (see Table 1). In addition to similar TS bands found in the bursa, one addi- tional band in the DNA of the liver was observed. Further- more, in bird No. 11 (panel D) it seems that the reverse is true, that is, in the bursal tumor of this chicken one addi- tional band was observed when compared to its corresponding liver tumor. Despite these minor differences in all of them, as is clear in Figure 11 (except panel F or bird No. 3 which was discussed earlier), similar "TS" bands were linked to the "c-myc" sequences. The single TS from the tumors of animal No. 11 linked to the "c-myc" gene warrants discussion, since among all tumors analyzed, it is the first occasion which the only single CSV-provirus which appeared to be linked to the "c-myc" is probably extensively deleted (ca. 3.3 Kb). 108 Figure 11. Demonstration of clonality of bursal tumors and metastases by digestion of tumor DNAs with EcoRI enzyme. DNAs were analyzed using procedure as described in the legend to Figure 5 and methods section. Panels A, B, C, D, E, and F show the results of digestion of a serious of tissues from tumor-bearing chickens with EcoRI enzyme. The tumor samples and the probes used are indi- cated on the top of the gel. Lanes B, L, G, K, and D represent DNA samples isolated from the bursa, liver, gonad, kidney and duodenum, respectively. "TS" bands are indicated by dots. 109 Ha whomam 110 302592.50. z ous—WE -.-“... 0.21 --H I.. .... coco 5-, m.-.. o x .4 ram 111 As mentioned earlier, despite these few cases where deletions and other structural alterations possibly have occurred, these data strongly suggest the clonality of the metastatic lesions with a primary bursal target, followed by migration of the primary clone to secondary sites. This usually occurs without structural alterations of the DNA, since in almost all of them TS lxmds were obtained which were similar (if not identical) to those in their corresponding primary target. The tumor of No. 14 shows a rather unusual feature as illustrated in Figure ll-A (lane G). While a similar TS band was found in both bursa and liver of this bird, the DNA obtained from the gonad (G) of this chicken shows four TS bands, all of which are different from the single TS band detected in the other tumors. All appear to be linked to the "C-myc" sequences. It is possible that the proviruses may have undergone extensive structural alteration in the process of migration, or during the development of metastatic potential. It is also possible that, during metastasis, a process, yet un- identified, of selection or adaptation was involved which possibly required such alteration. Also, the question as to how all four bands are linked to "C-myc" is not clear. As mentioned earlier, one interpretation would be that this tumor consists of four independent tumors coalescing to- gether, and in the bursa or liver all four "TS" bands may be possibly linked to "C-myc", but with similar sizes, 112 so that one single "TS" band shows up. But during the process of migration to the gonad, each of the four groups of related cells probably has undergone a slightly differ- ent structural alteration, so each carries a CSV-provirus integrating next to the "C-myc" gene, but at slightly different positions. Therefore, all four "TS" bands showed up. An apparently more likely possibility is that the bursa and liver only contain one "TS" band and during the process of migration the virus spread to form four "TS" bands. A1- ternatively, for a reason not yet understood, the gonad actually may also have been a primary target. X. EcoRI Analysis of Infected Birds at What is Presumably a Preleukosis Stage DNA samples taken from bursal tissues of five birds in which the animals were sacrificed 90 days post-inocula- tion (i.e., at preleukosis stage), were assayed with EcoRI, in parallel to other tumors, by the same method. Almost all of them did not show any distinct ”TS" bands. Some background of radioactivity, however, could be detected. In Figure 12 the first three lanes correspond to the chicken No. 18, whereas each of the remaining lanes represent indi- vidual samples from different birds. All the samples were taken from bursa, unless otherwise labeled. When individual tumor DNAs from this group of birds were compared with the DNA from normal tissues, i.e., lanes 188 and U [which are DNA samples obtained from spleen of bird No. 18 and bursa 113 from a non-inoculated bird (No. 23), see Table 1)], in all cases except lane 18 "smear" patterns were apparent. Lane 18 is the only tumor of the family in which three distinct TS bands, all smaller than 8.0 Kb, were detected. They all hybridized to Mc probe, indicating linkage to the "C-myc". The small sizes of the bands clearly indicate that the CSV- proviruses are deleted. Since at this stage most bursas do not show signs of tumor development, the presence of "TS" bands in No. 18 is indicative of a bird with an unusually rapid tumorogenic process. 114 Figure 12. EcoRI restriction endonuclease analysis of DNA from CSV infected birds at preleukosis 22292- DNA samples taken from bursal tissues of five birds in which the animals were sacrificed 90 days post-inocu- lation (presumably at preleukosis stage), were assayed with EcoRI enzyme as described in the legend to Figure 5. On the left hand lanes of this figure the result ob- tained from hybridization using REV probe and on the right hand lanes the autoradiograms of filters after hybridization with Mo probe are illustrated. The first three lanes (18$, 18, 188) correspond to bursa (B) and spleen (S) DNA samples from the bird No. 18. The re- mainder of the samples represent individual bursal DNA isolated from different birds. Lane U represents DNA from uninfected birds. 115 Figure 12 CHAPTER FOUR Discussion By application of modern molecular biological techni- ques,attempts were made to obtain insight into the mole- cular events involved in the mechanism of CSV-induced lymphomagenesis. The structure of the newly integrated CSV-exogenous provirus DNA were characterized in many lymphoma tumors. The major findings are discussed below. I. The Structure and the Mode of Integration of the CSV-Proviruses A partial enzyme map for the unintegrated CSV DNA was constructed and is in general similar to that of SNV DNA. The full-size of CSV DNA was determined to be about 9.3 Kbp. EcoRI enzyme does not cleave the CSV DNA so was most useful in characterization of the integration patterns of the viral DNA in tumor cells. The CSV pro- viruses of the primary bursal tumors characterized by EcoRI indicated that, for the majority of the tumors, the "TS" band falls into six size classes which suggests that CSV-integration in at least six different sites in the chromosome can lead to development of a tumor. However, these different sites appear to be located in similar chromosomal regions (see below). It may be mentioned that 116 117 molecular cloning of the CSV DNA and its adjacent se- quences will be useful in characterizing the exact nature of integration sites in different tumors. Among all bursal tumors,class D (ca. 10.8 Kb) appears to be most prevalent and occurs in at least 32% of the tumors. Each tumor contains from one up to four pro- viruses. Many tumors carry a single provirus suggesting that the presence of a single provirus is sufficient for oncogenic transformation. Many tumors from different birds appeared to have "TS" bands of the same size. The fact that all tumors harbor proviral DNA sequences confirms the viral etiology of the neoplasm. A comparison between lymphomoid tumors in male and female CSV-infected chickens shows that the tumors of infected males appear to have relatively fewer numbers of ”TS” bands than those of the female. Deletions and other structural alterations also appear to occur more frequently in the female birds than the male. At present, the signi- ficance of this observation is unclear, although this may implicate the involvement of sex-dependent factors, e.g. certain hormones in the induction of chromosomal altera- tion recombination processes of the tumor cells. II. The Clonality of the Primary and Metastatic Tumors In the vast majority of tumors analyzed by EcoRI enzyme, metastatic tumors appeared to have proviral DNA 118 patterns identical to the primary bursal target indi- cating a precursor-product relationship between the pri- mary bursal tumors and the secondary tumors. In the present study there is no evidence to support Neiman's assertion that amplification and/or major structural alter- ations of proviral DNA are required for metastasis (l9) . The clonality origin of CSV-induced tumors is reminiscent of the LL tumors induced by LLV (15, l6, l7, l8, 19, 232, and 277, 278) mouse mammary carcinomas induced by mouse mammary tumor viruses (234). III. Deletion in CSV Provirus in Avian Lymphomas Present data on the analysis of CSV-induced tumors summarized in Table 1 indicate that in a few cases the complete structure of provirus may not be needed for neo- plastic transformation, since "shortened" genomes were detected which were not in conformity with the map of a complete provirus. On the other hand, the presence of apparently complete viral genomes in many other tumors seems to indicate that deletion is not a requirement of transformation. This is especially obvious in tumors No. 1 and 2. The detailed restriction enzyme digestion data confirm the presence of a full-size CSV genome in these tumors. Although this result excludes extensive deletion of the proviral DNA of these tumors, minor structural alterations could not be revealed by this analysis. In addition, deletions in the adjacent cellular sequence 119 probably occurred. The reasons for believing this are as follows: If the "TS" bands result from a simple integrative (i.e., reciprocal) recombination involving the CSV DNA and the "c-myc" gene without other structural alterations, then the "EcoRI-"TS" band should have a size at least equal to the sum of the CSV-viral DNA and the endoM fragment. However, in both tumor No. l and No. 2 C as well as many other tumors, the "TS" bands are actually smaller than the endo fragment (ca. 12.5 Kb). This MC result, therefore, suggests that the extensive deletions which gave rise to the "shortened" "TS" bands above, prob- ably involve the surrounding cellular sequences. This observation is intriguing, since in other retrovirus- induced neoplasms (e.g. ALV), no such observation has pre- viously been reported. Another difference between CSV and ALV is that the extent of deletions and other structural alterations in the CSV-proviral DNA is lower than for the ALV provirus in ALV-induced lymphoma tumors (15, 16, l7, l8, 19). For example, it is found that at least 30% of the ALV pro- viruses were deleted (Fung §£.El°r 18) in lymphoma tumors and some tumors 90% of the proviral DNAs were deleted. A significant portion of the CSV-DNA sequences in induced tumors may have a role in the formation of lym- phomas. Whether the apparently intact CSV sequences are being transcribed or not awaits further RNA analysis. Based on the high frequency of deletion of the proviruses 120 in ALV induced tumors, Fung g; _E. (18) and Payne E; El- (17) proposed that cells which contained proviruses carry- ing deletions in the viral sequences would not be able to express the viral antigens (e.g., the Egg and gag) on the cell surface. Therefore, these cells are able to escape the host immune surveillence. Selective growth of such cells may lead to tumor formation. It is possible that the transcription of the CSV proviral DNA is interrupted by minor structural alteration which escaped deletion. If so, the present data can be reconciled. IV. Analysis of Infected Birds at the Preleukosis Stage An EcoRI enzyme analysis of the DNA of several in- fected birds at presumably the preleukosis stage in nearly all cases did not show any distinct EcoRI "TS" bands. In- stead "smear" patterns were apparent. This is consistent with the random integration of viral genes into the host (180, 193, 202, 225, 230, 233, 234, 235, 313). These data suggest that the initial E2 3319 infection by CSV- proviral DNA results in a large number of CSV-DNA inte- gration in many cells and therefore no distinct ”TS" bands are detected. After a long time the selected clonal trans- formed cells with characteristic "TS" band, multiply and develop into tumors. These observations may suggest that each tumor rises from the selective growth of a homogeneous 121 population of cells. This reinforces the view of a clonal origin for these tumors. V. CSV-Endogenous EcoRI Fragments The endogenous sequences partially homologous to REV sequences detected in this study (EcoRI fragments ca. 12.5 Kb) have not previously been reported, although earlier liquid-hybridization studies indicated a low level of homology (lo-20%) between REV and the normal chicken chromosome (108, 109). This REV endogenous sequence, how- ever, is only weakly hybridizable to the REV probe (pre- sumably due to the divergence of the two sequences). At present, it is not clear whether this endogenous REV sequence is specific for the three inbred lines used for this study or is ubiquitous in chickens. Further studies are required to determine the origin of this endogenous sequence. VI. Amplificagionvogthe CSV-Proviruses and Their Adjacent "C-myc" Gene in a Few Tumors The observation that the REV provirus and its adjacent cellular sequences may have undergone amplification and deletion in some of the tumors (i.e., tumor No. l and No. 2) is intriguing in light of the recent findings that retro- viral DNA has a transposable element-like structure (55, 57, 88, 91, 281) and shows behavior observed for the pro- karyotic transposable-elements, including deletion formation, insertional mutagenesis etc. (89, 275, 282, 300) also see 122 Literature Review). It is not clear how such structural alterations may contribute to the transformation process, if indeed they do. Detailed structural analysis of the molecularly-cloned "TS" bands should provide insight into the molecular mechanism of the recombination and its possible function in transformation. Amplification is not a general biological phenomenon. Among the few genes coding for proteins which have been shown to be amplified as a normal process of development are the rRNA genes in the ooctes of Xenopus (283, 284) and genes for chorion proteins in ovarian follicle cells of Drosophila (285). As to the amplification of viral genes there is only the report by Jaenish 23 3;. (286, 287, 288) in Moloney leukemia virus (M-MULV)-induced thymic lymphomas in inbred strain of mice. They showed that the development of leu- kemia in this mouse strain was accompanied by an increase of M-MULV specific DNA copies only in the target tissues (spleen and thymus). For example, the infected animal contained 2 or 3-4 copies of proviruses per haploid genome in the preleukosis and leukosis stages, respec- tively. Therefore, this viral amplification at the leu- kemic stage appeared to be related to leukemic transforma— tion. The observation of somatic DNA amplification only in target cells is consistent with the present study. While the molecular mechanism for amplification is not clear, 123 it is logical to assume that when a large amount of a protein is needed in a very short time, some genes may somehow undergo amplification. The possible amplifica- tion of CSV and "C-myc" genes in a few CSV-induced tumors may suggest that in these tumors it is related to lympho- magenesis. If so, such relatedness is yet to be elucidated. VII. Linkage of the CSV Provirus with the Mc-29 Related Endogenous Sequences The "C-myc” gene has recently been implicated in the induction of LL in young chickens, following ALV infec— tions (Hayward 35 91°! 15). This is discussed in the coming pages. To study the specific involvement of the "C-myc" locus in avian lymphomagenesis and to find whether in the CSV-strain of REV the "C-myc" or any other ”C-onc" (cellular transforming gene) is involved, the structure of CSV proviruses in many induced tumors have been charac- terized. The biochemical data presented in this thesis shows that in nearly all CSV-induced tumors one CSV pro- virus is specifically integrated next to the "C-myc" gene (on one of the two homologous chromosomes). This strong correlation implicates the latter gene in lymphocytic transformation. As indicated above, while in most tumors where multiple CSV-proviruses are present only one "TS" band was found to be linked to the "C-myc" sequence, in a few cases all proviruses appeared to be linked to such a sequence. The latter observation can possibly be explained 124 by the notion that these tumors consist of two or more independently selected transformed clones coalescing to- gether (i.e., semi-clonal). Each carries a CSV-provirus integrated next to the "C-myc" locus but at a slightly different position. Among all CSV-induced lymphoma tumors analyzed only in one tumor, the provirus doesrufizseem to be linked to "C-myc". At present the reason for this observation is not clear. However, it is possible that the CSV-proviral DNA in this tumor is linked to another yet to be identi- fied cellular transforming gene "C-onc." If so, it would suggest that more than one cellular gene has the potential to activate lymphomacyte transformation. To verify (or test) this hypothesis, it will be of prime interest to clone TS bands from this tumor and determine the nucleo- tide sequence of the adjacent regions. The proviral DNA adjacent to the "C-onc" provides a handle for molecular cloning of the fragment and identifying the putative oncogenes. To further examine whether other oncogenes are linked to CSV, provirus CDNA probes specific for several different oncogenes (e.g., Src, erb, and myg) were used. In all cases patterns identical to the uninfected (controls) were obtained, indicating they are perhaps not involved in the lymphomagenesis. This finding of linkage between CSV- proviruses and "C-myc" (and only "C—myc" but not other known oncogenes) strengthens the hypothesis that the 125 "C—myc" and its adjacent sequences are important in B- lymphocyte transformation. The significance of this hypothesis is discussed in a subsequent section. VIII. On the Mechanism of Viral Lymphomagenic Trans- formation and Speculations on "Cancer" Induction Several studies have shown that the genome of ALV is very closely related (about 85% homology) to that of genetically transmitted retroviruses of chickens. In certain inbred chicken lines , these viruses are released spon- taneously, as exemplified by Rous-Associated Virus type 0 (RAV-O) (290). RAV-O is not oncogenic (291). However, Crittenden 25 3;. (292) have observed pathologically iden- tical lymphomas to those induced by ALV, but at very low levels, in flocks of uninfected chickens free of exogenous ALVS. From several studies, including hybridization studies, the major region of non-homology between ALV and RAV-O has been localized and has been found to be concentrated at the 3' end of the genome of the virus, the gay and the "C" (for common) regions (245, 256, 293, 294, 295, 296). Recombination studies between RAV-O and RAV-60 (a subgroup E recombinant for exogenous and endogenous viral genes which carries only the C region of the exogenous source) suggest that $31 region is not involved in neoplastic transformation (297, 298). Thus, the C region was con- sidered to be the region to be associated with oncogenesis. In the past two years considerable information re- garding the role of C regions has been obtained. For 126 example, Robinson 22.21: (299) reported that several RAV-60 strains induced leukemia at the same rate as the exogenous virus. Also, recently Crittenden 23.21- (298) reported that the efficiency of lymphoma induction, using ALV for infection, is highest when the virus carries the "C" region from exogenous viruses. Based on these obser- vations, Tsichlis and Coffin (242) hypothesized that in ALVs,region "C" plays an essential role in oncogenecity and therefore is required for LL induction. As mentioned earlier (see literature review) the viral genes are bounded at both ends of the provirus by a long terminal repeat (LTR) sequence, comprising in regions UB-R-US. Several independent nucleotide sequence analyses and EE XEEEQ transcriptional studies indicated that the U3 region, which is present at both termini of the proviral DNA contains both the putative promoter for eukaryotic RNA polymerase II and the initiation site for transcrip- tion located downstream from and next to promoter-like sequences (50, 55, 91, 241, 301, 302). However, this region has not been shown to code for any protein thus far (50, 241). Recently, based on these and several other observa- tions (see below), a model ("oncogenesis by promotor in- sertion") was suggested for the induction of LL in young chickens following ALV inoculation (15, l6, 17). The essence of the model is described below. Following infec- tion the virus integrates next to the cellular oncogene 127 and the proviral sequence located at the LTR region pre- sumably carries a "strong" viral promoter (as discussed above), able to activate the cellular oncogene ("c-one“) located downstream from the integrated proviral DNA (in theory it can be any type of known and/or unknown "C-onc" gene(s)). In the case of lymphoid tumors induced by ALV and also by CSV (present study), the "C-onc" is the same as the "C-myc" gene. It is known that Mc-29 causes myelocytomatosis, fibro- sarcomas and carcinomas in infected birds (303) and also transforms both the haematopoietic cells and fibroblasts $2.!£E£2 (19, 193, 304). However, association of Mc-29 virus with B-lymphomas has never been reported. In the present study and others (15, 18) the "C-myc" gene is involved in the induction of lymphomas. The reason for this is unclear, although it can be argued that the trans- forming gene of Mc-29 ”myc" (the exogenous source) and the endogenous corresponding gene "C-myc" may have some minor differences in their sequences such that only the "C-myc"v is able to be associated with lymphomogenesis. Alterna- tively, it is possible that both "C-myc" and "myc" genes are identical, but in Mc-29 the latter gene is ex- pressed as a fusion protein which also contains amino acid sequences encoded by the viral replication genes (i.e., as gag-onc fusion protein). E3 yiyg, the gag encoded protein of the fusion proteins apparently is not needed for 128 lymphomogenesis. The "C-myc" product is not found to be linked in such a fashion which may possibly explain the difference in their target cell-specificity. It is also not clear why, to date, only the " -myc" gene and not other cellular transforming gene(s) (i.e., from AEV or AMV) is involved in lymphomogenesis, since these acute viruses also transform haematopoietic cells both 23 yiggg and EE yiyg (2, 314). Again, if the induc- tion of lymphomas is possibly due to some unique feature of "C-myc" or due to the "C-myc" itself and nota fusion pro- duct, then it would not be surprising that "C-onc" genes of viruses such as AMV and AEV, despite their common target with Mc-29, like Mc-29, have not been implicated in lym- phoma induction. The promoter-insertion model is attractive since normally the initiation of viral transcription starts from the left U3 (located at the 5' termini, as a portion of LTR region) and, as mentioned earlier, since the same U3 sequences are present at 3' end of the integrated proviral DNA (the right U3). Since the promoter sequences must also be there, it is likely that transcription may start also from the right U3 and if so it may continue to a potential oncogenic cellular gene, allowing the expression of such ” -onc", thereby triggering the transformation process. If so, that should also generate transcripts consisting of both viral and cellular information. Indeed 129 Hayward 2E.2l- (15) found a 30-300X higher expression of "C-myc" gene in infected birds than the normal birds and detected novel RNAS carrying both viral and "C-myc" infor- mation. These findings further strengthen this model. The similarity in the mode of action of two geneti- cally unrelated viruses in the induction of similar lymphoma tumors is in support of the promoter-insertion model, since the REV termini (LTR) , which is about 600 nucleotides, (see Ref. 54) also contains similar structural features to that of ALVS (i.e., putative promoter and initiation site for trans- cription). Thus, the "C-myc" and its adjacent sequences are important in B-lymphocyte transformation; and both systems possibly have a general pathway in lymphomagenesis. The observation that so many ALV proviruses in lymphoid tumors are defective is also quite supportive of the above model since, despite many extensive deletions in the proviral DNA in LL tumors, all carry at least one LTR sequence (16, l7, l8, l9), and in all cases the induction of tumors has occurred very efficiently. These findings strongly suggest that at least most of the ALV—proviral genetic information (i.e., the expression of viral RNA) may not be required for the maintenance of neoplastic trans- formation, which indeed was reported to be the case (15, l6, 17). However, in CSV this is not yet established. Studies on the expression of "C-myc" in CSV-induced tumors 130 should provide more insight into this issue. Indeed, experiments by my colleagues are in progress to study the transcription of the "C-myc" sequence in tumors induced by both CSV and ALV systems and, also, to compare the nucleo- tide sequences of their respective integration sites, which should shed light on this question. Also, Ea yiyg injec- tion of CSV LTR sequences into bursa and the construction of a chimeric DNA (e.g., CSV + Src sequences) using Ea XEEEQ transfection assays, should provide insight in this regard. If in the above cases tumor formation and trans- formation occur, respectively, then the possibility is ruled out that other CSV DNA sequences are requires for the induction of lymphomas. The "promoter insertion" model has-gained further support. Using transfection experiments, Blair gg‘gl. (6, 305) reported that both the viral transforming gene "mos" and the "C-onc" gene "C-mos" of moloney murine sarcoma virus can induce neoplastic transformation only when their respective sequences are linked to the LTR sequences (i.e., as chimeric DNA). These sequences were also able to induce neoplastic transformation in gibroblasts. Vande Woude (see references 15, 16) has found that the DNA sequence is also transcribed in their transformed recipient cells. Therefore, like ALV of which the entire viral genetic information is not required for transformation, apparently only LTR + “C-mos" 131 sequences are the key elements for transformation. Using related methods, similar results have been reported from Oskarsson EE-El' (169) who cloned the murine cellular Src gene (C-Src) obtained from normal mouse cells and failed to transform recipient cells unless this sequences was attached to the sequence located at the 5' end of the murine leukemia virus (i.e., containing the ”strong" viral promoter), then transformation took place at a high effi- ciency. Furthermore, very recent evidence from Cooper 25 3;. (306) indicated that the efficiency of transformation of 3T3 cells by subgenomic fragments of ASV DNAs containing only Src (i.e., without LTR) is 100 - 1,000 fold lower than the efficiency of transformation when complete virus (in- cluding LTR) DNA is used. According to the promoter insertion model the specific integration of viral DNA is required for lymphomagenesis. Such specific integration should be a rare event and prob- ably occurs only once and very few times among the entire population of infected target cells. Therefore, this model is consistent with the clonal origin of the tumors, and also can explain why viruses such as ALV and CSV with the appar- ent lack of a unique transforming gene can induce tumors EE 3329. It also provides a molecular basis for the multi- potence of the virus by which different host genes possibly are responsible for different types of neoplastic diseases. Despite much information in favour of this model, some of which has been discussed above, there are a few reports 132 which are consistent with the model. For example, re- cently Cooper and Neiman (307) observed that following transfection, the DNA obtained from LL tumors induced ifl yiggg transformation at high frequency in NIH/3T3 mouse cells. However, they found that proviral DNA sequences in NIH/3T3 cells (including the LTRI), apparently was not required for such transformation. The nature of this in- consistency is, at present,rufl:clear. However, as quoted in Hayward E; El: (15) very recently Cooper's group has found that the lymphomas from which the DNA was derived do contain ALV proviruses integrated next to the "C-myc" gene. It is possible that the Cooper transfection assay is detect- ing some event after the initiation event, i.e., secondary event (i.e., tumor progression) in a multi-step process which ultimately leads to lymphomagenesis (also see below). Also, Payne g; 3;. (15) and Cooper and Neiman (quoted in Ref. No. 15) observed that in some cases, surprisingly, the ALV provirus integrates downstream instead as in the usual cases - upstream, of the "C-myc" gene. The question of how the viral promoters activate the "C—myc" expression when it is downstream is not clear, at present. To resolve these apparently contradictory observations, further inves- tigations are needed. The detailed mechanisms regarding the general validity of "promoter-insertion" model by these viruses (especially CSV) remains to be elucidated. However, it is possible to assume that more than one "C-onc" gene (including "C-myc) 133 may be responsible for leukemogenicity and we need more probes to detect them. Furthermore, the "C-myc" plays the "initiator" role in tumor formation, so that it sets the subsequent possible event(s) which ultimately lead to a tumor. All possible steps in the multi-step process of tumorogenesis could logically be linked to one another in an organic linkage relationship. Finally, I would like to finish this chapter with some general data, as well as some speculation regarding cancer-causing agents. The question of what is the real cause of cancer is still not clear. There are a variety of cancers and they need not have a single cause. In recent years several hypo- thetiCall models about cancer have been reported. Some in- vestigators, for example Cairns (308) , believe that cancers are the product of a physical or chemical disruption of a cell's genetic material. A vast majority of known carcino- gens (i.e., chemical carcinogens) can damage the DNA of some somatic cells (i.e., cause point mutations such as frame-shifts, a basic change in the composition or structure of a gene, etc.) and if this damage is placed appropriately, it causes the cell to divide in an uncontrolled fashion, giving rise to a clone of cancerous cells. For example, several tumor malignancies can be generated by mutation at specific loci (309). Although such a simplified model can accommodate very different carcinogens including chemicals, radiation, viruses, etc., it is not complete. It may be 134 true that a vast majority of carcinogens (about 90%) can produce genetic mutations but the rest (i.e., about 10%) may just somehow disrupt the control mechanisms of the cell. In addition, if damage to DNA is sufficient to cause cancer, why is the length of the induction period induced by different carcinogens, different? Assuming that the retrovirus transforming genes are derived from cellular genes ("C-ones") as hypothesized by Bishop (310), and as several studies have shown, the func- tions of many retrovirus oncogenes are also present and highly conserved in normal vertebrate cells, their functions may be required for normal growth and development (1, 2, 5, 6, 315). Only when such "C-onc" sequences are placed under the influence of viral regulatory control elements (i.e., "strong" viral promoters) in proper infected cells does the neoplastic transformation occur. The neoplastic transforma- tion may be a consequence of dosage of the oncogene product and the virus may simply disturb the control cell division and differentiation properties by overloading cells with normal gene product. If the constitutive eXpression of one or more potentially oncogenic cellular genes ("C-onc”) which are normally expressed at low levels with no harm to the cells is the key factor in cancer induction, then what one needs are the means to activate such a gene. A huge number of different agents, e.g., viruses, chemical, irradiation, etc., can be used for such purposes. 135 Hayward 2; 3E. (15), speculating on the role of trans- forming retroviruses in neoplasia, suggested tha both "slow" and "acute" viruses may induce neoplastic transfor- mation in slightly different ways. The acute viruses were very likely originally derived by insertion of a cellular gene into the viral genome via a process of recombination in such a way that the latter gene was placed under the control of a viral promoter. In the "slow" transforming viruses (i.e., CSV, ALV, etc.), the viral promoter is inte- grated into (or adjacent to) the "C-onc" gene, as has been extensively discussed in this thesis. Both mechanisms, however, are similar with respect to the activation of a normal cellular gene in the process of tumorogenesis. So far all the information about transformation provides direct support for the hypothesis that potential neoplastic trans- formation genes are encoded in the genomes of normal avian and mammalian cells: the transforming capacity and their possible mode of action of these viruses (i.e., the rela- tively fast tumorogenesis by these viruses); the fact that a great number of malignancies can be generated by mutations; and that neoplastic transformation can be induced by DNAs of both chemically transformed cells (i.e., methyl-chloathrene- transformed quail cells), (262, 311, 316) as well as normal cells (i.e., normal uninfected CEFs) (307, 312, 316). In all of these and other different carcinogenesis (viruses, chemicals, some drugs, irradiation etc.) it is possible to speculate that perhaps the "C-onc" genes may be the main 136 targets or common key factors in initiation events leading to tumorogenesis. This tumorogenesis can be activated by viruses and mutations, chromosomal rearrangement etc. or induced by the vast majority of carcinogens. The observa- tion that in ALV apparently no viral genetic information is needed for the maintenance of transformation in lymphoma tumors is also consistent with this possibility. The availability of molecular hybridization probes for more than ten known "onc" genes can be very useful in testing the validity of these speculations. They can be used to identify the origin of a tumor. At present with such a limited number of "C-onc" genes at hand, using them for screening may not be of great advantage in the diagnosis of human cancer - until oncogenes for human cancer are identified. All in all, despite the advanced knowledge obtained in cancer research in recent years and despite the improvement in human cancer treatment, i.e., surgery, radiation therapy, chemotherapy, etc., and furthermore, despite reasons for optimism; and finally despite my personal wishes; I think that it would be unrealistic to expect a cure for cancer, the mankind's most dreaded disease, in upcoming years. How- ever, it is my sincere desire that the present study shed some light toward our understanding of the molecular mechan— ism of carcinogenesis and help pave the way for better strategies in designing therapeutic methods for human canc er . 137 IX. §EEEE£X 1) The structure of the unintegrated CSV DNA was analyzed. 2) The CSV proviruses of the primary bursal and meta- static tumors characterized by EcoRI enzyme indicated that for the majority of the tumors the "TS" bands fall into six size classes, which suggest that CSV integration in at least six different sites of the chromosome can lead to development of tumors. 3) Each tumor contains from one up to four proviruses and many tumors carry a single provirus, suggesting that the presence of a single provirus is sufficient for lympho- magenesis. 4) Tumors in infected males appear to have relatively fewer numbers of TS bands than those of the females. In addition, deletions or other structural alterations appear to occur more frequently in the female birds. 5) All tumors examined display at least one distinct "TS" band, which strongly implicates CSV proviruses in the induction of lymphoma tumors. 6) In the vast majority of tumors metastatic tumors appeared to have proviral DNA patterns identical to the primary bursal target; this is consistent with the clonal origin of tumors. 138 7) While in a few cases the complete structure of the provirus apparently may not be needed for neoplastic trans- formation, in many tumors the presence of apparently com— plete viral genomes seems to indicate that extensive dele- tion is not a requirement of transformation. 8) Analysis of the DNA of several infected birds at the preleukotic stage suggests random integration of CSV viral genes into the host genome. . 9) In nearly all the CSV induced - LL tumors analyzed, an apparent linkage between CSV DNA and "C-myc" in the chromosome of the tumor cell is demonstrated. This data, together with the specific integration of LLV DNAs near the same chromosomal site in the LLV-induced tumors, strongly implicate that the "C-myc" is the specific target gene for bursal lymphomagenesis. 10) Amplification of the chromosomal region encom- passing CSV-DNA and the "C-myc" gene has occurred in certain tumors. 11) Deletions or structural alterations involving the host sequences are observed in certain tumors. 12) Endogenous sequences weakly hybridizable to REV-A DNA are present in the chromosomes of 71, lSst72 and 6.3 birds. APPENDIX APPENDIX Avian Retrovirus (I) Transforming (1Q vitro) oncogene neoglasm Rous sarcoma virus (RSV) .553 sarcoma Fujinami sarcoma virus (FSV) fps sarcoma etc. MyeIocytomatosis virus (MC-29) ‘mgg myelocytomatosis Erythroblastosis virus (AEV) .259 erythroblastosis etc. L172 one 10”? c:r-;;;r-I2222h;;;c:n (II) Non-transforming(ig vitro) Avian leukosis virus (ALV) N0 Lymphoid leukosis Reticuloendotheliosis virus (REV) Endogenous virus (RAV-O) non-oncogenic LTR rm E:}--1F§F----C:3 .139 140 _IE Vitro E vivo Fibrcblast Latency Neoplaan Trans- Period Indiced fornaticn Avian sarccna virus 7-14 days f ibrosarccua + (PIA, B77 0 o 0) Slow leukosis virus 14-30 weeks lymphoid - (RAV-l , RAV—z) leikosis Acute lalkemia virus 1-3 weeks erythroblastosis + (AEV, M::-29 . . .) nyelocytatatosis Endogenous virus (RAV-O) 141 PARENTAL RNA W'RNA l-‘IYBRID (7) Lucas DUPLEX DNA OPEN W DUPLEX DNA (7) CLDSD CIRCLIjR “FLEX DNA mas... MlPTION VIRAL K332”!!! WA. 5' 3‘ Win N l:— 4 (‘I 3m 1 de sum WA /\ Cbeed Cinder DNAe 0 Integrated , , :fl_-————_ ue ===-: :fl‘: Sell-none waving of the Ineer and dealer unintegrated DNAe of celle recently hinted by a retrovirus. The brewed provlrue. aleo morn. ie m in chronically Infected cells. The immediate precmor of the Negro“ genome is “flown (Weiltem. Am Rev. Biocnern. l980). 142 ON THE COMPARISON OF REVS AND ALSVS SOME MAJOR SIMILARITIES: Characteristic structure Mode of replication Similarity in tumor induction (i.e., in respect to latent period, organ distribution and surface IgM production) SOME MAJOR DIFFERENCES: Genetically & serologically unrelated Difference in their R.Tr. (i.e., REV has a preference for Mn2+ insteand M92+ for ALSV etc.) Extent to which their viral nucl. acid hyb. with host cell DNA Bouyant density Morphology Limitation of phenotypic mixing & physiological compl. only for Egy_glypr. (thus far). 143 SOME MEMBERS OF REVS: REV-T - (Twiehaus, I958, & Sevoian, 1964, isol. f. turkeys), a mixture of REV-T and REV-A. CSV - (Cook, 1969, isol. f. chickens) SNV - (Trager, l959, isol. f. ducks) DIAV - (Conuin & McGhee, l969, isol. f. ducks) SOME MAJOR SIMILARITIES: Relatedness in their genomic seqs (3 80% seq. homology) Structural proteins Antigenicity of their DNA pol. gs antigene Morphology 144 Extraction of i M.N. DNA from chicken tissues iv Dig. with R. Ends ("mini" to test the extent of completion and main samples) 0.8% Agarose gel elect. and staining with EtBr visualized by I: u.v. light induced fluorescence Denat. and neut. of the DNA I; DNA transfer to nitrocellulose filter papers (using a mod. of Southern method, 1975) I Baking (in vacuo at 80°C = 2-3 hr.) the filters . Rehyb. of 4' Preanml. 4———-— filters Using progerm probe (. 1 x 10 cme Hybridization + [2X55C, 0.IXSSC 4' 0.1% SDS and Washing 0.IXSSC (IXSSC = 0.15 M NaCl - 410C 0.015 "3 Citrat6)] 50°C Autoradiography 4: Develop. the x-ray films 145 GROWTH, AMPLIFICATION, TRANSFORMATION AND ISOLATION OF RECOMBINANT DNA CLONES: P SNV-saII 608 PMc-pst (plyyg"-pst) P-flggg" EcoRI (ASV) P-"gggfl-puvll (AEV) fmy_fl-¢ 4A EcoRI subcloned DNA (AMV) 4r CsCl-EtBr G. cent. ass/or HAP purification Further purification of DNA (i.e. RNAase + proteanase K + Ext. of DNA) ~I 6-50 I purification of inserts from PBR. or A using low melting agarose (sea-plaque) Labeling of the DNA with P32 mainly by N.Tr. (occas. CDNA rep. (for ASV & CSV): purified virus (end. RX), purified 60-705 (exog. RX). 146 Stage I (0-8 wks) Viral infection Integrations of viral genes are "random“. Competent viruses are released. Stage II (8-12 wks) Target-cell transformation Integration of viral genes at a specific site(s) (e.g. near a cellular leuk gene) triggers the transformation process. WIRE FR: LEUK . DNA I ‘ —MRNA Stage III (12-16 wks) Tumor growth Due to host immunity, only those transformed cells which harbor defective viral genomes selectively grow up into tumors. Stage IV (2 16 wks) Metastasis Primary tumor cells migrate out of the bursa and are arrested on the secondary site. I47 yams monster, ‘3' metal. frADSf. Pfizer: fig Lfi'lak”‘i PLenka“: ; Mfrs—ST: 148 the structu wotei envel e (v-core pro.tein) n Glycogatein I (Mn \‘ \xxxxxixim "\\\‘ 5 \ . \ I LTR gag .'0": l I . j #1 r T Cell adenine Cell The promoter insertion model at oncogenesis. The ALV provirus is shown schematically with the long terminal repeat (LTR) enlarged. lu’tiation could occur from either the left or the right LTR of the provirus. 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