1‘2 . .3113; .213}: . I. .33. :3. . :9? v t g u. {xii . 1.)... 3 y 9&5»... firm: . Illllllllllllllllll’lllllllllllll 3 1293 01044 5272 This is to certify that the dissertation entitled ENHANCER AND MIDDLE T CONTROL OF AGE- AND ORGAN SPECIFIC POLYOMAVIRUS REPLICATION IN THE MOUSE presented by LARRY GREGORY MARTIN has been accepted towards fulfillment of the requirements for Ph . D. degree in MICROBIOLOGY mm 1m, Major professor Date 10-14-94 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY l Mlchigan Statel Unlverslty PLACE DI RETURN BOXto roman Ihb chockoutfrom your mood. TO AVOID FINES Mom on or baton dd. duo. DATE DUE DATE DUE DATE DUE MSU In An Mun-1M WOMEN Opportunity Indium 1 _. .._.__—__—-~ 4— —-. ENHANCER AND MIDDLE T CONTROL OF AGE- AND ORGAN-SPECIFIC POLYOMAVIRUS REPLICATION IN THE MOUSE BY Larry Gregory Martin A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 1994 ABSTRACT ENHANCER AND MIDDLE T CONTROL OF AGE- AND ORGAN-SPECIFIC POLYOMAVIRUS REPLICATION IN THE MOUSE BY Larry Gregory Martin Polyomavirus infects numerous organs in both neonate (less than 1 day old) and adult (6 weeks old) mice. The polyoma enhancer directs both tissue specific viral transcription and replication in the mouse. The viral early protein middle T antigen also has an essential role in viral DNA replication. In this thesis, I have examined the relationship between the enhancer and middle T antigen on age- and organ- specific viral replication in the mouse. In order to establish a more complete Organ specific pattern of viral replication, normal Balb/c mice were intraperitoneally infected for a period of 3 days when less than 24 hours old, at 3, 6, 9, 12, 15, 21, 28, 35, and 42 days of age. From our analysis the mammary gland, skin and bone possessed a greater capacity to replicate polyomavirus and produce infectious viral particles than all other organs tested. In addition, I performed finer mapping on the enhancer. Enhancer mutants were constructed which removed the A domain middle T responsive sites PEAl and PEA3, the complete B domain and the distal half of the B domain. The middle T mutant 18-5 used in this study contains a 50 base pair deletion in the middle T gene. Here, I looked at the levels of replicated viral genomes and live virus production in neonate and adult mice at 3 and 7 days postinfection. A strong requirement for either the 8 domain or the PEAl and PEAB sites was observed in all organs of neonatal mice. Elimination of the PEAl and PEA3 sites diminished replication in bone of adult mice. Deletion of the B element caused a decrease in viral replication in the mammary gland, skin and bone. Additionally, a loss in virus production was seen when both of the above elements were removed. Neonatal and adult mice displayed a strong requirement for both middle T antigen for viral replication and virus production. Overall, these results demonstrate that middle T has a more important function in viral DNA replication and live virus production than enhancer elements PEAl and PEA3 as well as the B domain. To my parents, Isaac and Sylvia and my sister and brother, Connie and Terry iv ACKNOWLEDGEMENTS I have been able to accomplish this milestone in my academic career with the guidance, understanding, support and unending friendship of my major professor Dr. Michele M. Fluck. For this, I celebrate her. I would like to thank the members of my guidance committee, Dr. Susan Conrad, Dr. Richard Schwartz, Dr. Ronald Patterson, and Dr. Sandra Haslam for their interest and for sharing their collective thoughts and invaluable expertise with me throughout the course of my studies. Finally, although too numerous to mention, I would like to thank all of my friends, specifically Dr. Michael J. Gonzalez, Enid Bauza-Gonzalez, Michael John Gonzalez-Bauza, Ray A. Moreno, Houman Dehghani, Natalie Moore-Brown, Diane Rendenius, Dr. Casandra Simmons, Karen Van Atta, Dr. Ben Cathey, Vanessa Wickliff, Dr. Ronald Buckley and the support staff in the Department of Microbiology. TABLE OF CONTENTS PAGE List Of Tables...0....O0.00000000000000000000..0... Vii List Of FigureSOOOO00....OOOOOOOOOOOOOOOOOOOOOO0.0. Viii Chapter 1 Chapter 2 Chapter 3 Literature Review Introduction.......................... 1 Discovery and pathogenesis of polyomavirus....................... 4 Middle T antigen of polyomavirus...... 9 The non-coding, regulatory region of polyomavirus....................... 27 Summary............................... 53 References ...... ...................... 55 Age dependent DNA replication of polyomavirus in the mouse Abstract.............................. 72 Introduction............... ........... 73 Materials and Methods................. 75 Results............................... 78 Discussion............................ 87 References............................ 94 Enhancer and middle T control of age- and organ-specific polyomavirus replication in the mouse Abstract................... ............ 98 Introduction............. ...... ........ 100 Materials and Methods.................. 103 Results................................ 115 Discussion............................. 139 References.... ......................... 150 Table Table Table Table IA. IB. IIA. IIB. LIST OF TABLES PAGE Fold decrease in replication relative to WT (neonatal mice, 7 DPI)..................... 131 Fold decrease in replication relative to WT (adultmice’ 7DPI)......OOOOOOOOOOOOOOOOOO 132 Neonatal organ viral titers (7DPI) ...... ... 137 Adult organ viral titers (7DPI).. ...... .... 138 vfi Chapter 1: Chapter 2: Chapter 3: Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES 1. 2. Physical map of the polyoma virus genome................. A schematic representation of the 421 amino acid middle T antigen ..... . .............. Diagram of three hypothetical signal transduction pathways mediated by middle T antigen. Physical map of the enhancer region of the polyomavirus A2 genome ..... ............... Physical map of the enhancer subdomains of polyomavirus required for transcription and replication.............. Physical map of the enhancer regions of natural variants and host range mutants of polyomavirus ............. .... Analysis of polyomavirus genome levels in mouse organs at various ages of development.................. Titers of polyoma virus in organs of mice infected at different ages A. Organ viral titers (skin and bone)... ........... B. Organ viral titers (mammary gland).............. C. Organ viral titers (kidney, liver and lung)..... Physical map of the polyoma virus enhancer and mutants... A schematic representation of the 421 amino acid middle T antigen.................... Levels of viral genomes of enhancer and middle T mutants in comparison to the wild type A2 strain....... ........ vm PAGE 3 13 29 31 36 45 80 84 85 86 107 111 117 Figure Figure Figure Figure Levels of viral protein expression in NIH3T3 and BMK cells........................ Replication analysis of enhancer and middle T mutants by in situ hybridization A. Neonates.................. B. Adults.................... Analysis of polyomavirus genomes extracted from specific organs of mice infected as neonates and adults A. Neonatal mice (3 and 7 DPI).. ..... ...... B. Adult mice (3 and 7 DPI)............. Diagram of the distribution of polyoma enhancer and middle T mutants in organs of neonate and adult mice.... ix 120 123 124 128 129 148 Chapter 1 Literature Review Introduction Polyoma is a small DNA virus. Its double stranded circular genome of 5297 base pairs is packaged into an icosahedral capsid. The genome contains an early region, a late region and a non-coding regulatory region (see Figure 1). The regulatory region comprises 244 nucleotides and regulates the activation of transcription and replication. The early region expresses a precursor transcript which is differentially spliced into three transcripts coding for the viral proteins large T, middle T and small T. Large T is a multifunctional nuclear viral protein of 100 kDa which is important for ‘transcription. and replication and. is also capable of immortalizing cells. Middle T antigen is localized on the cytoplasmic face of the cellular membrane. This protein of 55 ZkDa is responsible for transforming cells. The small T protein of 22 kDa has been found in the nuclear and cytoplasmic compartments of cells but its function is not fully understood (2, 3). Encapsidation of viral genomes requires three viral proteins synthesized from a differentially spliced precursor transcript from the late region. These capsid proteins are VPl (45 kDa), VP2 (35kDa) and VP3 (23kDa). Figure 1. Physical map of the polyomavirus genome The inner circle represents the restriction endonuclease sites of MspI/HpaII on the polyoma genome. The early and late coding regions are depicted as the outer circles, each of which express a precursor message that is differentially spliced into the T antigens (Large T, middle T and small T), or capsid proteins (VP1, VP2, VP3) (jagged lines represent introns) . The non-coding enhancer/origin region (nucleotides (nts.) 5021-5265) separate the two coding regions, and is located on the late side of the origin of replication. This figure is taken from Soeda et al. (1b). Figure 1. A lytic infection by polyomavirus culminates in the production of thousands of progeny viruses. Following adsorption of the virus to the cell surface, the virus finds its way to the nucleus where uncoating, gene expression and replication occurs. The early viral proteins appear about 10-15 hours after infection depending on conditions (4). Viral and cellular DNA synthesis begins approximately 12-15 hours post infection. late viral gene expression begins after the start of DNA synthesis. Infectious viral particles begin to appear by 20-25 hours postinfection. Virus maturation continues until cells begin to die (4). This thesis ‘will focus on several parameters which influence the replicative capacity of polyomavirus in the mouse. The parameters discussed are the enhancer and middle T as regulators of polyomavirus replication. Therefore, this literature review will address the following topics: 1. Discovery and pathogenesis of polyomavirus, 2. Middle T antigen of polyomavirus and its role in viral replication, 3. The non-coding, regulatory region of polyomavirus and its role in tissue specific viral replication. Discovery and pathogenesis of polyomavirus In an attempt to transmit leukemia to newborn mice by inoculation with filtrates of leukemic tissues, Gross (5) discovered that a certain proportion of mice developed solid tumors which he found were due to a virus. This virus was recovered from different sources of AKR mice with spontaneous leukemias. In an independent investigation, Stewart and Eddy (7) propagated and isolated a virus in mouse embryo cultures, observing that this virus was able to retain its oncogenic activity after serial passages. They showed that newborn mice infected with viral culture fluid developed numerous neoplasms. Vogt and Dulbecco were the first to show that polyomavirus could transform rodent embryo cells in vitro (2, 24). Studies of polyomavirus were facilitated by the advent of plaque assays to quantitate virus which provided a new tool by which to study viral infections (22, 23). Plaque techniques applied to animal viruses provided a method for producing genetically homogeneous lines of virus (8). The pathogenesis of polyomavirus was studied further following inoculation in newborn mice (9, 10). Neonatal mice undergo a generalized infection by polyomavirus. The virus replicates in different organs at high titers when measured by plaque assay (9). Virus infections were analyzed by measuring titers at various intervals after inoculation. The highest infectivity titers were attained between the 7th and 14th day following inoculation and declined thereafter (9, 12) . Highest titers were observed in the kidneys, salivary glands, liver, lungs and spleen and lower titers were seen in the thymus, brain and blood. Growth curves in newborn mice showed that these mice are very sensitive to infection but much less sensititve when infected as adults (12, 132). Inoculation of neonatal mice by different routes (subcutaneously, perirenally or intranasally) established a disseminated infection replicating to high titer in the kidney during the first two weeks. A recombinant virus, PTA (site directed mutation in VP1 of the viral strain PTA) showed that VP1 specificity is critical for the induction of high level viral genome amplification in the kidney. A direct correlation .between the ability of the virus to replicate efficiently and to spread within the animal was observed. Although the kidney showed the highest levels of replication, the skin, bones and lung showed levels to varying degrees. Tissues which are targets for tumor induction were also shown to be sites for a productive infection (143). In newborn immunocompetent mice the clearing phase which leads to the disappearance of the virus follows the systemic phase of infection. Virus was found persistent in many of the same epithelial (skin, mammary and salivary glands) and mesenchymal (bone) tissues in which tumors developed. These data began to document the influence of age and tissue specificity of the host on the pattern of viral infection (138). Further studies on the pattern of polyomavirus replication wasstudied from the time of infection until tumor formation. These mice (adult nude six week old male and female) were injected subcutaneously with the A2 strain of polyomavirus and the sites of viral DNA replication were determined by whole mouse section hybridization. Viral DNA was detected mainly in the bones and mammary glands of both males and females. The spleen, salivary glands, lymph nodes and skin were also viral targets and no signal was observed in the kidneys, lungs or liver, which were shown to be sites of replication in newborn immunocompetent BALB/c mice. By comparing levels of viral replication in mice infected as neonates with those infected as adults replication patterns in groups of organs were classsified based on the level of viral replication in different organs of the mouse. The observed patterns included: class I organs (mammary gland, skin and bone) which supported high levels of replication in neonatal mice and moderate levels in adults and class II organs (kidney, liver and lung) which supported high levels in neonates and very low or no replication at the adult stage. These results suggested that the viral replication potential declines significantly with the age of the mouse and shows organ specificity. It appears that the majority of polyomavirus induced tumors in mice are derived in organs which support high levels of viral replication» JDifferences in organ specific 'viral replication may represent the regulation of organ specific transcription factors (162). The induction of tumors in newborn mice by polyomavirus occurs in a variety of organs and tissues. Some tumors affect mesenchymal (bone connective tissue and renal medulla) and epithelial (salivary, mammary gland, skin and thymus) tissues (7, 8, 18, 19, 20). Visible tumors do not appear until several weeks after the drop of peak virus titer (12, 19). As many as 30 different cell types contain viral genomes after inoculation of newborn mice with polyomavirus and approximately one dozen give rise to tumors (16). Studies exploring polyomavirus infections have shown that tumor induction and/or cell selectivity can be influenced by virus dose, route of infection and age of the host (17). It was observed that athymic (nu/nu) homozygous animals are sensitive to tumor induction by polyomavirus showing a shorter latency period and a higher incidence for tumors than newborn (nu/+) heterozygotes. It has been reported that athymic (nu/nu) mice when injected subcutaneously at six weeks of age with polyomavirus A2 strain or mutants which contain mutations in their enhancer (able to infect only undifferentiated embryonal carcinoma cells) induced only a very narrow set of tumors (mainly mammary adenocarcinomas and osteosarcomas) . Mutations which affected the enhancers did not seem to effect the histotype of tumors but did affect the latency period and the frequency of tumor induction. Therefore, tumor induction in adult nude mice by polyomavirus represented a new experimental model system in which to study viral infections in comparison to the neonatal system (136). Polyomavirus serves as a excellent model system for studying mechanisms of host cell deregulation, proliferation and cellular transformation. Regions of the polyomavirus genome involved in organ specific ‘viral replication. are being characterized by site specific mutagenesis. This thesis will focus on the role of the enhancer and middle T antigen as agents of tissue tropic viral replication. Middle T antigen of polyomavirus Early studies delineated the region of the polyomavirus genome required for transformation of mammalian cells. Middle T antigen is the transforming protein of polyomavirus (see Figure 1). This is based upon properties of hr-t mutants. Host range transformation defective (hr-t) mutants were isolated in Tom Benjamin's lab that contained a mutated middle T and small T protein (30). These viral mutants lost most or all of their ability to grow in the untransformed parent cell line (normal 3T3 cells) but retained the ability to grow on polyoma-transformed cell lines. A variety of cell types such as phenotypic revertants of polyoma transfomed 3T3 cells, primary baby mouse kidney epithelial cells and primary mouse embryo fibroblast were found permissive for growth of hr-t mutants [i.e. NG18 (18-5 in this dissertation]. Several cell types were found poor for growth of hr-t mutants such as NIH3T3 cell lines, 10 radiation and chemical carcinogen transformed NIH3T3 cells and late passage mouse embryo fibroblasts to name a few (30, 31). Host range and the ability to transform cells coincided with the viral coding region later shown to encode middle T. These hr-t mutants were also shown to promote only a single round of cell division, while the wild type virus promoted multiple rounds (33, 35, 44). The growth. defect. of 1hr-t. mutants in. NIH3T3 cells became apparent only late in the virus growth cycle, after viral DNA replication and synthesis of the structural proteins (54). Studies of growth properties with site- directed mutants that either eliminated or altered middle T without affecting small T were conducted in order to define the effects of middle T on virus growth from those of transformation. variants of polyoma that completely eliminated middle T (i.e. 808-A) resulted in poor growth of the virus in NIH3T3 cells (viral DNA levels equaled that of wild type but virion assembly was deficient). Variants with an altered middle T (mutants, 1387-T and 1178T) maintained wild type levels of viral DNA and efficient viral DNA encapsidation. These results supported the fact that middle T is the viral protein that plays a major role in the induction of a productive viral infection (wild type levels of encapsidated viral genomes) in NIH3T3 cells (47). Utilizing antisera raised in rats against the early tumor antigens of polyoma virus (Large T, Middle T and Small T) permitted the identification of viral specific plasma 11 membrane proteins from infected or transformed mouse cells. The main component was identified as a 55kDa protein. This protein was absent from the plasma membrane of cells infected with transformation defective mutants (i.e. NG-18). This defined a role and connection between viral specific plasma membrane proteins and cell transformation (1, 32). Other polyoma middle T antigen variants contain different mutations such the substitution of Leucine for Proline at amino acid 248 (i.e. NPTY). Mutations in this region in polyoma middle T antigen also abolished transformation (51). The middle T 'mutant (1387-T) causes premature termination of the middle T protein which destroys membrane localization. This mutant lacks the carboxy terminus of middle T, thus anchorage of the protein into the plasma membrane was found to be defective. This mutation also abolished cellular transformation (39) (see Figure 2). Other mutations were generated around the middle T mutation of the transformation defective mutant NG59 (contains an in-phase insertion substitution (Asp to Ile- Asn) at amino acid 179). This region of middle T antigen is important for both neoplastic cellular transformation and binding to pp5oc-src. This single point mutation represented the smallest change in the middle T protein that abolishes transformation. Mutants of middle T that were unable to associate stably with the cellular protein pp6oc' src lacked kinase activity (as determined by in vitro 12 Figure 2. A schematic representation of the 421 amino acid middle T antigen. Depicted above the line are the names of various mutants, and below the line the amino acids affected by different mutations. Below these mutational sites are the binding’ domains of c-src, Shc, PI3 kinase binding, the hydrophobic membrane insertion domain, the region in common with small t antigen, and the position of the intron. Bottom line depicts the position of the deletion in the middle T mutant 18-5 used in this study. l3 . m endows 2852 73:3 8 03¢ a m 6:3 «to sowmoofioga 86%. 23 86:3 26 § 8 £T+mm xod H55. 2.. 96cm. 2m « 59:8 96:3 0.8.0 . a L .3 a a: «mm «an 2m 8m 8.. s as :78 a on: 02:: cm... F [L F 0. fl « e R: 32 $9. 98 mhzsbz buxom“ 14 kinase assays). Mutants which mapped in this region and showed kinase activity were transformation competent, while those which lacked kinase activity were transformation defective. Therefore, ppsoc-src association is important to polyomavirus middle T transformation (43, 59). The relationship between middle T antigen and ppsoc-src and signal transduction is discussed later in this chapter in further detail. Cloning and individual expression of middle T, small T and large T described the biological significance of each T antigen. ‘Viable jpolyoma mutants defective in 'middle. T showed that (1). the persistence of large T antigen was not required for the maintenance of the transformation phenotype, (2). small T antigen alone was not sufficient for inducing the full expression of the transformation phenotype and (3). middle T antigen was necessary and sufficient for transformation by polyoma (36, 37). The investigation of polyoma oncogenesis in mice was also studied in transgenic mice. Middle T antigen cDNA was placed under the control of the polyoma regulatory sequences and used to generate transgenic mice. Inoculation of newborn mice with high titers of recombinant virus resulted in tumor formation in many different tissues. A broad spectrum of tumors were observed when neonatal mice were infected by wild type virus (laboratory reference strain) and a limited number of tumors were induced in transgenic mice in the presence of middle T antigen. Middle T 15 transgenics developed hemangiomas (a tumor of endothelial cell origin) (135, 137). A transgenic mouse strain which expresses the middle T antigen of polyomavirus under the control of an enhancer that is known to be active only in adult mouse tissues [immunoglobulin heavy chain (IgE) enhancer] induced tumor formation in several adult organs. Tumors of the salivary and thyroid glands, along with mammary tumors, liver haemangiomas and adenocarcinomas were found (141). Another line of transgenic mice which carries the polyomavirus middle T antigen cDNA linked to the herpes simplex thymidine kinase promoter resulted in mice that developed neuroblastomas at a high frequency. Multiple neuroblastomas developed in mice between 2 and 3 months of age. Expression of this transgene was restricted to the neurons of the central and peripheral nervous tissue, thus giving credit to the tissue specificity of promoters and enhancers (142). When transgenic mice carry the entire polyomavirus early region it was found that they devleoped both vascular and bone tumors. This set of tumors are a subset of the spectrum of tumors in mice infected by the wild type strain, with an expansion of tumors of vascular origin. Mice of individual lineages developed lymphangiomas and fibrosarcomas. All tumors were of mesenchymal origin and expressed the polyoma transgene transcripts. Tissues (testes) which express the transgene at higher ratios were refractory to tumorigenesis while other tissues that 16 expressed the transgene at much lower levels were susceptible to tumor formation (145). Mammary gland specific expression of the polyomavirus middle T antigen was examined by establishing transgenic mice that carried the middle T protein under the control of the mouse mammary tumor virus promoter/enhancer. The expression of middle T antigen resulted in an overall transformation of the mammary gland, resulting in mammary adenocarcinomas. These tumor bearing mice also developed secondary metastatic tumors in the lung. These observations showed that middle T protein is a potent oncogene in the mammary epithelium as well as other organs in the mouse (153). As stated earlier, the region of the polyomavirus genome which is identified with cellular transformation was localized to the early region. Further analysis of middle T antigen began to uncover other regions of this protein which affect transformation (see Figure 2). Continued dissection of polyomavirus middle T antigen has lead to a better understanding of its role in both cell growth and transformation. Although middle T has no known enzyme activities, there are associated enzyme activities. The major effects of middle T appear to come from its regulation of phosphorylation events in cells. An important association of middle T is with the cellular tyrosine kinase c-src. Early experiments indicated that a fraction of middle T 17 protein formed stable complexes ‘with. pp60¢"src in. cells productively infected with or transformed by polyomavirus. This complex could be isolated by immunoprecipitation with antibodies to either middle T or pp6oc'5rc. The associated in vitro tyrosine kinase acitvity was not that of middle T but a property of the associated pp60°"src (55). Other than the carboxy terminal hydrophobic domain of the middle T protein there are other sequences of amino acids at the amino terminal half which are highly sensitive to changes. N-terminal deletion mutants are transformation defective and deficient in their ability to associate with ppeoc-src. A large portion of the exon 1 (amino acid 92-191) encoded region of middle T is important for association with pp6oc‘ src and polyoma transformation (56, 61, 69). Further analysis of middle T antigen and cellular c-src showed that the associated tyrosyl kinase activity of c-src was enhanced following polyomavirus infection or transformation when compared to uninfected mouse cells or mouse cells infected with transformation deficient polyomaviruses (53, 57). In order to determine whether the interaction of middle T antigen and the protein tyrosine kinase pp6oc'5rc was essential for middle T transformation, cells which lacked endogenous Src (disruption of both Src alleles) were infected with a retrovirus which encoded middle T. Retroviral infection of cells caused an induction of foci (transformed. cell colonies) on cell monolayers and cell l8 colony formation in soft agar. Animal studies showed that Src negative mice (newborn and 2 weeks old) developed visceral hemangiomas, which were indistinguishable from the tumors induced by the wild type 'virus with respect to morphology, frequency and latency. These studies suggested that Src may not be essential for middle T induced transformation, however, middle T induced cellular transformation may be compensated for by other cellular kinase proteins such as Yes, Fyn and phosphatidylinositol kinase whose activity was found associated with middle T antigen in Src negative cells (157). In retrospect, transgenic mice created with middle T antigen under the control of a MMTV promoter were totally deficient in tumor induction. The src-family of protein kinases includes a number of protein tyrosine kinases (c-src, c-yes, c-fyn, c-fgr, c-lck, c-hck, c-lyn and c-tkl). Middle T antigen has been found to co-precipitate with another tyrosine protein kinase. Polyomavirus middle T was found associated with and phosphorylated by the c-yes oncogene product. Middle-T was found to activate the kinase activity of ppéoc-src and was also shown to modulate the kinase activity of p62-C‘Yes. Since the association between p62°"Yes and middle T exist, this association was also involved in the complex transfomation event by polyomavirus (62). The c-src region involved in complex formation with middle T antigen is highly conserved among the 19 tyrosine kinases mentioned above. It is conceivable that other members of this family may be equally capable of associating with and being activated middle T antigen. Antibodies against synthetic peptides to c-fyn revealed that pp59¢"fYn is able to form a stable complex with middle T. Mutants defective in transformation (i.e. N659) do not associate with pp59c‘fyn. In contrast to ppéoc-src middle T's association with pp59¢"fYn does not lead to a significant increase in tyrosine kinase activity of pp59c" fyn. Deregulation of tyrosine kinase activity of more than one member of the src-family of tyrosine protein kinases following polyomavirus transformation was found to occur (65). Middle T antign also causes the activation of another cellular protein called protein kinase C. Protein kinase C (PRO) is a Ca2+ -activated phospholipid-dependent protein kinase. Protein kinase C becomes activated by diacyl glycerol generated by the turnover of phospholipid in membranes and prefers membrane associated proteins as substrates. Tumor promoters such as the phorbol esters have been found to bind and activate protein kinase C. In order to investigate the possible role of protein kinase C on middle T phosphorylation in vivo, 12-O-tetradecanoylphorbol- 13-acetate (TPA) was used. TPA rapidly stimulated phosphorylation of a 58 kDA middle T species (phosphorylation in vivo on serine and tyrosine in immune complexes) when it was added to cultures infected by wild 20 type polyomavirus or transformed NIH3T3 cells. The 58 kDA species was not phosphorylated by transformation deficient viruses such as NG59 or Py1387T. This indicated that the 58 kDa form of middle T is phosphorylated through the action of protein kinase C (60). The mediation of cellular signals from the membrane to the transcriptional apparatus has been recognized by the activation of promoter regions on phorbol ester inducible genes such as collagenase and stromelysin. A conserved 9 base pair motiff is recognized by a common cellular protein described as transcriptional factor' AP-l. Treatment of cultured cells with TPA results in a rapid increase in the binding activity of AP—l to the TPA responsive element. This implied a possible receiving end to a complex pathway, which is responsible for transmitting the signal by phorbol ester tumor promoters (64). Phosphatidylinositol 3-kinase (PI3-K) is an enzyme which catalyzes formation of a minor lipid, phosphatidylinositol 3-phosphate (74). PI kinase activity was detected in immunoprecipitates made with middle T or pp60c-src specific antisera from cells infected with polyoma virus. 'This detection indicated, an association. between middle T/c-src and PI3-K. Studies with middle-T defective mutants demonstrated a correlation between PI3-K activity and transformation. Thus, middle T immunoprecipitates prepared from cells infected by transformation defective 21 viruses were less active in phosphatidylinositol (PI) phosphorylation than wild type immunoprecipitates (58, 74). The removal of tyrosine 315 (substitution of phenylalanine for tyrosine 315) results in the inability of mutant middle T to bind phosphatidylinositol 3 kinase but middle T continues to bind and activate pp5oc-src. Therefore, the binding and phosphorylation of middle T (on tyrosine 315) by ppGO-C'Src is very important for binding phosphatidylinositol 3-kinase (66). Cells productively infected or transformed with wild type or a mutant virus (Py1178T whose middle T antigen fails to promote binding of phosphatidylinositol 3 kinase but binds and activates pp6oc‘5rc) exhibit an increase in inositol tri-phosphate levels. The data suggested that phosphoinositide turnover in normal and transformed cells occurs by phospholipase C, which appears to be the rate limiting enzyme in the formation of inositol tri-phosphate, and that middle T causes an increase in these levels by accelerating the cleavage of phosphatidylinositol 4,5- bisphosphate by phospholipase C (68, 76). Another tyrosine phosphorylation site on middle T antigen (Tyrosine 250) acts as a binding region for the SH2 domain of the transforming protein Shc (see Figure 2). Shc becomes phosphorylated on tyrosine and binds the SH2 domain of Grb2. This stimulates p21ras activity through the mammalian homologue of the Drosophilia guanine-nucleotide- exchange factor 803 (164). 22 A variety of other cellular proteins have been implicated in the signal mediated transduction pathway that may lead to transformation by polyomavirus middle T antigen. Studies of revertants of ras transformed mouse cells showed that pp60V‘5rC acts upstream of p21c-ras in the same signaling pathway. These revertants were resistant to transformation by pp60V'SrC and was confirmed by micro- injection of anti-p21‘3'ras antibodies into pp60v’src transformed cells which resulted in a transient morphological reversion. If the activation of ppsoc-src by middle T antigen :mimicked. the effects of the activated pp60V‘src then it may be possible that p21c-ras could also play a role in middle T antigen induced transformation. The dominant. suppressor' of IK-ras 'transformation (Krev-l) (encodes p21rap-1A, which is thought to block p21c-ras function by competing with p21c-ras for ras-GAP (GTPase activating protein), which stimulates the hydrolysis of p21c-ras bound GTP without affecting the GTPase activity of Krev-l) could. revert. the ‘transformed. phenotype of .Rat-2 cells in a dose dependent manner when co-transfected with plasmids that expressed middle T antigen and the human Krev- 1. Thus, transformation by middle T antigen required p21°' ras function and implied that middle T antigen lies upstream of p21c-ras in the same signal pathway (77). Ras has been shown to transactivate PEAl which binds within the polyoma virus enhancer. This function was studied by transcription from a reporter gene 23 [chloramphenicol acetyl transferase (CAT)]. Oncogenic Ras (Ha-Ras-Val) increased the rate of transcription from this element which contained four head-to-tail copies of the PEAl motif. There was a correlation between the capacity of various oncogenes to activate transcription from PEAl and to transform cells, and the oncogenic Ras also induced binding of nuclear proteins to the PEAl motif (129). Ras proteins are plasma membrane bound and have been found to transduce signals from the outside of cells to internal targets within cells. The identification of transcription factors which are activated by the ras oncogene has been studied by site directed mutagenesis of the alpha domain of the polyomavirus enhancer. This specific region of the enhancer has been found to bind cellular proteins (PEAl, PEA3 and PEA2). This short sequence (ras responsive element) to which these factors bind has been found to mediate Ha-ras activation. It also mediates activation by TPA and serum. PEAl binds specifically to this domain. Analysis in LMTK' cells demonstrate that the RRE mediates transcription by serum, TPA, middle T antigen and ras and that the ras protein, middle T and TPA can substitute for serum in the activation of transcription. In the presence of high serum (10%) cells cannot be further stimulated by TPA or ras expression. A. mutation of the RRE prevents binding of PEAl which suggest that transcription factor PEAl activity is regulated by ras, middle T antigen, TPA and serum factors. The polyoma 24 enhancer is very similar to the TPA inducible element found in the SV40 enhancer, the metallothionein MTIIA gene and upstream of the collagenase, interleukin-2 and transin genes (114, 115, 117). Raf-1 has important connections to growth factor receptor activation at the cell membrane to transcriptional events in the nucleus. It has been shown by using Raf-1 dominant negative mutants that Raf-1 is required for serum, TPA and Ras induced expression from the oncogene responsive element (A core) of the polyomavirus enhancer. This mutant appears to titrate out a Raf-1 activating factor which is induced by Ras expression following serum or TPA treatment of NIH3T3 cells. Thus, the dependence of Raf-1 on serum and TPA induced transcriptional activation of genes appeared to be driven by AP-l and c-ets binding motifs. Therefore, Raf- 1 appears to transactivate promoters containing AP-l and c- ets binding sites following its activation by phosphorylation (128). The stimulation of API and c-ets mediated transcription has been shown following transformation with the raf oncogene. It has also been shown that the oncogenic activation of raf increases its ability to activate PEA1 (API). The raf protein kinase is one of the later elements in the cascade of events of mitogens found to activate PEAl and transduce a signal (118). Other studies indicate that a variety of mitogenic signals increase c-raf—l phosphorylation and its associated protein kinase activity 25 (118a). The phosphorylation by raf may be directly or indirectly important for the activation of transcription, since mutations in the ATP binding site of v-raf prevents trans-activation of PEAl. The activation of PEAl may be an important event in transformation because its activity is up-regulated by several oncogenes and various derivatives of raf and ras has been shown to activate PEAl, and transform cells (118). Two purified cellular proteins (36 and 63 kDa) have been shown to associate with polyomavirus middle and small T antigens. Analysis of these two proteins reveals that the 36 kDa unit is the catalytic subunit of protein phosphatase 2A (PP2A) and the 63 unit is the regulatory subunit of the PP2A holoenzyme. PPZA-like phosphatase activity was detected in immunoprecipitates of wild type polyoma middle T antigen. In the middle T complex both the 63 kDa and 36 kDa protein were found in an equimolar ratio. The 36 kDa catalytic subunit could be immunoprecipitated by anti-pp60c‘ src only from cells which expressed wild type middle T antigen. Moreover, there may be a relationship between the formation with PP2A and middle T antigen and transformation (70). It was found that middle T antigen is associated largely with only PP2A in transformed cells and middle T antigen exists in two other complexes, each containing a Src family kinase and PP2A. These membrane associated complexes exhibit both phosphoseryl protein phosphatase and tyrosine protein kinase activities. 'This suggest that middle T antigen is in complex with PP2A and c-src but the failure to detect PP2A association with pp60‘3‘src in normal cells suggest that middle T antigen is directly involved in the formation of this complex in transformed cells (75). Polyomavirus middle T antigen contains a cluster of cysteine residues (amino acids 120-153) which is also present in small T antigen. A mutation at cysteine 120 to a tryptophan has been found to abolish the ability of middle T to associate with PP2A, pp60c-src and PI-3 kinase rendering the mutant deficient in transformation. The results indicate that the residue at position 120 which is present in middle T and small T but not large T is important in complex formation. PP2As' role in complex with middle T is less well defined but the transformation defect could result from the failure of the middle T mutant to associate with one or a combination of other cellular proteins isolated thus far in complex with middle T (78, 79). Small T antigen of SV40 has been shown to cause constitutive stimulation of both ERKl (mitogen activated protein kinase), and MEKI (mitogen activated protein kinase kinase), without affecting the upstream activation of Raf, thus inducing cell growth. This may also provide a functional role for both middle T and small T of polyomavirus which have been found to bind the 36kd and 63 kd subunits.of PP2A (80). In summary, middle T binds c-src and activates the c- src associated tyrosine kinase activity, phosphorylating 27 middle T antigen on tyrosine 315. This activated complex binds PI-3 kinase at tyrosine 315 on middle T antigen. Transduction of the signal via another possible pathway to ras and further to raf occurs by the binding of middle T - c-src complex to shc via tyrosine 250 on middle T. The binding of middle T - c-src complex to phospholipase C gamma on the other hand has not been demonstrated. Evidence shows an increase in the turnover of phosphatidyl-inositide, increase cellular concentrations of Diacyl glycerol, activation of protein kinase C and an increase in phospholipase C gamma activity. The middle T downstream targets appear to be PEA1(AP1) and PEA3 (c-ets) cellular transcription factors. Middle T has been shown to activate PEAl and PEA3 mediated transcription (115, 117). Recent studies on middle T antigen of polyomavirus, suggest that middle T activates a mitogenic pathway that is similar’ to ‘the ligand/receptor' activated. tyrosine Ikinase signal transduction pathway of PDGF (80a). To date there are three ways in which middle T may activate three branches in this pathway (see Figure 3). The noncoding. regulatory region of polyomavirus The origin region of polyomavirus contains several genetic elements required for DNA replication (see Figures 1 and ‘4). The :minimal cis-acting sequences required for polyomavirus DNA replication were determined by constructing 28 Figure 3. Diagram of three hypothetical signal transduction pathways mediated by middle T antigen. The association of middle T antigen with a variety of cellular proteins induce a cascade of signalling events from the outside of the cell to the nucleus, that may result in neoplastic transformation. Middle T binds the cellular membrane protein c-src, and activates its tyrosine kinase which causes the autophosphorylation of c-src and the phosphorylation of middle T on tyrosine 315 (pathway III), or 250 (pathway II). This activated complex binds the SHZ region of Phosphatidylinositol-3 kinase (PI3-K), which phosphorylates a minor lipid (phosphatidylinositol (PI)), and catalyzes the formation of phosphatidylinositol-3 phosphate (PI3-P), to which no known phospholipase has been found to cleave, and whose physiological role is unknown. A number of cellular proteins (Shc, Sos, Gap, Ras Raf, MEK, and MAP) were found to transduce a signal via phosphorylation events, culminating in the activation of the transcriptional activators APl and c-ets. The middle T / c- src complex can also cause the activation of phospholipase C-gamma (PLCG) resulting in the activation of protein kinase C (PKC) . The effect of these interactions have not been completely determined, but appear to induce the expression of cellular genes, and may account for the role of middle T antigen in neoplastic transformation. 29 . n museums“ gel 3. 9w... :5: Suzi » .62: 3.53.8 3 .883 mm E< 8§_§3%o_§£ 30 Figure 4. Physical map of the enhancer region of the polyomavirus A2 genome. The 244 base pair enhancer region (nucleotides (nts). 5023-5267)) has been divided into two basic elements, A and B (nts. 5023-5130, 5131-5267 respectively). The middle T responsive elements, which bind PEAl, the mouse homologue to AP1, and PEA3, the mouse homologue to c-ets have sites in the A and B elements. Other cellular transcription factors (PEA2, EFC, PEBl, EBP20, and UVRE (APl-like site)) are also shown to bind at multiple sites in both domains. L = late start site and E = early start site. BC] I 3 EH [fjflflémfl l l BZAQB la 193d OdB EVSd CV36 1V3d EVEd 380 Figure 4 . 32 viral-plasmid recombinants. Their replicative capacity was assessed in permissive mouse cell lines (MOP cells). Deletion analysis defined separate genetic elements. The origin contains an adenine-thymine rich segment, a 32 base pair guanine-cytosine rich palidrome, large T antigen binding sites, the initiation site of bidirectional replication and a non-coding regulatory region upstream (85). DNA sequences necessary for transcription of eukaryotic genes by RNA polymerase II in vivo are located upstream of the transcription initiation site and are referred to as enhancers. Enhancers are operationally defined as having the ability to function over very large distances and usually in either orientation. Early studies of the enhancer region of polyoma involved linking the hemoglobin Beta-1 gene of rabbit to a 244 base pair fragment from the enhancer of polyoma virus (A2 derivative), not including the origin of replication, in order to assess the level of stimulation of transcription. Following transfection of the recombinant DNAs into mouse 3T6 and human HeLa cells, the polyoma sequences strongly enhanced the level of the Beta- globin gene transcripts over a distance of 1400 base pairs (82). Sequences of the polyomavirus genome shown to enhance transcription were interrupted by deletion mutations around the PvuII site (nucleotide (nt.) 5130). Within this region, the late region transciptional promoter elements, the late mRNA leader sequence and a DNase I hypersensitive domain 33 were found. Other deletion mutants within the non-coding region of the polyoma genome helped to define other essential and. non-essential regulatory' elements (84, 92, 93). Upstream activating sequences are essential for the expression of the early genes of polyoma as well as SV40. These elements are known as transcriptional enhancers. Enhancers have been shown to possess tissue specificity in different cell types and are best represented by the immunoglobulin enhancers that are specific for' cells of lymphoid origin. The polyomavirus enhancer has another unique function which involves cis-acting sequences essential for DNA replication. It was found in SV40 that enhancer sequences could function as either transcriptional or replicatory activators. The finding that viral enhancer sequences could activate DNA replication in an orientation and position independent. manner suggested, that the polyomavirus transcriptional enhancer and replication activator are one in the same (86, 87) (see Figure 4). Investigation into the structure of the polyoma enhancer proceeded by insertng different fragment regions of the polyoma enhancer in both orientations under a reporter gene (5' or 3' of a hybrid test gene to the chicken collagen type I promoter fused to the CAT gene). Activation of the collagen promoter defined a major enhancer element called A (nucleotides (nts.) 5021-5130) and a second minor element called B (PvuII to PvuII fragment: nts. 5130-5267). It was 34 shown that at least two enhancer units, generally called the A and B domain, could be found within the enhancer region of polyomavirus. Certain regions of the A and B domains are highly homologous to very important sequences in other enhancers. The domains activated promoters very poorly when alone. For full efficiency of activation surrounding sequences were needed. Each of these enhancer units showed different relative efficiencies of promoter activation in different cell types (i.e. fibroblasts and embryonal carcinoma undifferentiated cells) (87). In order to further define and localize the sequence elements important for the activation of viral DNA replication and enhancer activity, another extensive set of deletion :mutants ‘was constructed. Of the 244 base pair enhancer region, four elements were found to be involved in activation of replication. These functionally redundant elements were called A, B, C, and D (nts. 5108-5130, 5179- 5214, 5148-5179 and 5020-5098 respectively). In many cases a combination of two or three elements was sufficient for efficient replication. Transfection assays of recombinant plasmids also showed that the "replication activator" has one of the characteristics of an enhancer in that it can function independently of its orientation. Thus, the polyomavirus enhancer was found to be required in cis to activate both viral replication and gene expression (89) (see Figure 5). 35 Figure 5. Physical map of the enhancer subdomains of polyomavirus required for transcription and replication. This figure shows transcriptional and replicational elements and their boundaries in relation to a restriction map of the polyoma A2 enhancer. I. represents transcriptional domains and II. represents the overlapping replicational domains whose domain endpoints are defined in the text. . m madman .56 9.00 .x30 0.50 .x30 28 28 58 was 2% m o < o .28. .226. mezmzwom mmoz z zml I I m I I I .1321 I I .< I I I 108 S nc ana ' enhance ta ts Hirt extracted, gel purified (USBioclean kit, United States Biochemical) DNA from viral stock lysates served as templates for PCR amplification of the enhancer region (thermal cycler by MJ Research Inc.). Enhancer templates of both WTA2 and mutant viruses were created using primers MF9 and MF10. Amplified enhancer products (gel purified using Magic Preps DNA. Purification System (Promega) served as templates for sequencing (7, 8, 9, 10, 11). Initial sequencing reactions were carried out using the fmol DNA Sequencing System (Promega). This system allowed a maximum of 100 bases to be read. Therefore, all sequences were confirmed by dye terminator sequencing (Applied Biosystems, model 373A) at Michigan State University's sequencing facility. This system allowed a maximum sequence reading of approximately 400-500 bases from a PCR product. All sequencing results confirmed precisely each enhancer deletion. All sequence data were subjected to a transcription sequence-specific recognition site computer program to detect possible newly formed transcription factor binding sites created by the mutagenic procedure. The GCG (Genetics Computer Group, Inc.) programs were utilized on an operating system at the Biochemistry Department, Michigan State University. 109 Cells and viruses Mouse NIH3T3 cells were grown in Dulbecco's modified Eagle's medium. (DMEM) supplemented ‘with 5 or 10% heat- inactivated calf serum. and antibiotics, maintained. in a humidified incubator at 37°C with 5% C02. Baby mouse kidney cells (BMK) were prepared by asceptically removing the kidneys from 6 day old neonatal mice (ICR, Balb/c mice, Harlan Industries). Kidneys were minced and trypsinized by a standard laboratory procedure. Packed tubule cells were seeded on 60mm plates in DMEM supplemented with 10% horse serum and antibiotics. The following polyoma strains were used: wild type A2 (standard reference laboratory strain) (69): 18-5 (an hr-t mutant constructed by site directed mutagenesis from wild type A2 which contains approximately a 50 base pair deletion in the Msp I fragment #4 (see Figure 2) (16, 20)). Assay of viral DNA and proteins Viral genome levels were measured at 4 and 60 hours post-infection. Viral DNA samples were collected by the method of Hirt (6). 5% of the total viral DNA samples were digested with EcoRl, electrophoresed on 1% agarose gels, blotted and hybridized with a polyoma (WTA2) specific probe, and exposed to x-ray film. Blots were quantitated using a beta analyzer (AMBIS). 110 Figure 2. A schematic representation of the 421 amino acid middle T antigen. Depicted above the line are the names of various mutants, and below the line the amino acids affected by different mutations. Indicated below these mutational sites are the binding domains of c-src, Shc and PI3 kinase as well as the hydrophobic membrane insertion domain, the region in common with small T antigen, and the position of the intron. The bottom line depicts the position of the deletion in the middle T mutant, 18-5. 111 . m mun-roam 6103525.; ._. mg: +1.33“ 1:) 368 E 358 5:600 ecu «2 an 23.8 2&8... 8s... ... 3.05 @3103 he Eggéwg J/Em an want as 45W. 2. - 8 t 904. 02?? WNT « + b 8 9.2....an b. 8n. 2: $2 ....8 zonHz< .r wJODHz <5>Jom 112 Early protein (Large T, Middle T and Small T) levels were measured at 60 hours post-infection. Total protein was collected from cells by lysis in SDS reducing buffer (0.5 M Tris-HCl, pH 6.8, 10% glycerol, 10% (w/v) SDS, 5% B- mercaptoethanol, 0.05% (w/v) bromophenol blue). Samples were boiled at 100°C for 5 minutes and stored at -70°C. Protein amounts were quantitated using the Bio-Rad Protein Assay in SDS concentrations less than 0.1% at 595 nm. 1 ug protein samples along with low range prestained SDS-PAGE Standards (Bio-Rad) were separated on a 10% SDS polyacrlyamide gel (Mini-Protean II Dual Slab Cell, Bio- Rad). Proteins were transferred approximately 24 hours at 50 volts (4°C) onto membrane (PVDF, Bio-Rad). Blots were washed and blocked in 5% nonfat dry milk and blot buffer for 30 minutes, rinsed and probed with anti-T antibody (polyoma polyclonal serum collected from the acites fluid of Brown Norwegian rats, which carried a polyoma-induced tumor of the peritoneum cavity). Serum was diluted 1:500 in 5% nonfat dry milk blot buffer and applied to membranes with slow shaking for 2 hours at room temperature. Blots were washed and probed with secondary antibody (sheep anti-rat HRP) at a dilution of 1:3000 in blot buffer and 5% nonfat dry milk. Blots were incubated for 45 minutes, washed and immediately subjected to Enhanced Chemiluminescence (ECL, Amersham), and then exposed. to ‘x-ray film (Hyperfilm-MP, Amersham) for various time intervals. 113 Transformation Analysis FR3T3 cells were plated at a density of 2.0 x 105 cells per 35 mm tissue culture dish. Cells were grown to confluency and serum starved (0.5% serum) for two days. Cells were infected at an MOI of 5. Following infection, cells were maintained in growth medium (DMEM, 5% calf serum and antibiotic supplementation) and re-fed approximately every 5 days. Plates were counted at 15 days post-infection for foci (neoplastically transformed cell colonies) development. Mice infections Balb/c mice were purchased from Harlan Industries. Neonatal (less than 24 hours old) and adult nude (6 weeks old) mice were infected intraperitoneally with 1.5 x 106 PFUs of WTA2 and each enhancer mutant in a 50 ul total volume. All animals were sacrificed at 3 and 7 days post- infection. In situ hybridization Whole mouse bodies were embedded in 3% carboxymethylcellulose and frozen above the vapors of liquid nitrogen. Sagittal 40 um sections were collected on tape, fixed in 95% ethanol/acetic acid, dehydrated in 95% ethanol and air dried. Sections were denatured in 95% formamide at 65°C for 30 minutes and pre-hybridized (50% deionized formamide, 5x SSPE, 2.5x Denhardts, 100ug/ml non-homologous DNA) for 18-24 hours at 37°C. Sections were hybridized 114 using fresh pre-hybridization solution with a multiprimed, column purified polyoma whole genome probe (106 cpm/section) for 72 hours at 37°C. Sections were washed in several changes of SSPE, stained using Gills Hematoxylin, washed in 1% lithium carbonate and air dried. Sections were exposed to Amersham Hyperfilm-MP with intensifier screens for various times at -70°C. Regions positive for polyoma specific genomes were compared to serially stained sections in order to identify tissues positive for DNA replication. Extraction of nucleic acids from organs Organs ‘were removed from.:mouse. cadavers immediately after sacrificing and stored in liquid nitrogen or processed. Organs were weighed and homogenized in 5 ml of digestion buffer (100mM NaCl, 10mM Tris pH8, 25mM EDTA pH8) at 30,000 rpm for 10 seconds with a Tekmar Tissumizer. 2 ml of the homogenate was removed and further processed for live virus analysis. The remaining 3 ml was brought to 0.5% sodium dodecyl sulfate (SDS) and 0.1 mg of proteinase K per ml. Tissue was digested at 50°C for 12 to 18 hours with shaking. Samples were extracted by standard techniques. Total DNA was precipitated by addition of 1/10 volume of 3M sodium acetate and 2.5 volumes of cold 95% ethanol followed by incubation overnight or longer at -20°C. DNA was collected by centrifugation at 10,000 rpm for 30 minutes at 4°C and resuspended in sterile double distilled water. DNA concentrations ‘were determined. at 260 nm and equivalent 115 amounts of DNA were digested with Eco RI. DNA samples were electrophoresed on 1% agarose gels and blotted. Blots were hybridized to a 32F polyoma specific probe (WTA2) and quantitated using a beta imager (AMBIS). Live virus analysis Mouse organs were aseptically removed and homogenized in digestion buffer. 2ml samples removed from organ lysates mentioned above were sonicated (Raytheon, Sonic oscillator, DF101) for 1 minute and heated to 45°C for 15 minutes. Viral titers were determined by the plaque assay method on monolayers of NIH3T3 cells (1.5 x 105 cells per 35mm plate) (16, 17a). RESULTS Growth properties of the viruses: viral DNA, prpteip and transfppmation To examine the level of viral genomes in a lytic infection, NIH3T3 and baby mouse kidney (BMK) cells were used. BMK cells are important because they are permissive for middle T mutants (18-5 in this study). Samples were harvested at 4 and 60 hours postinfection (Figure 3) (4 hour data are not shown). A fraction of each viral DNA sample was linearized with EcoRI. A 5.3 kb fragment corresponding to the viral genome was observed. Mutants A2 (dl 2B) (data not shown), A2(dl B) and A2(dl P3,1) grew just as well in most samples as the wild type A2 strain in 3T3 cells and BMK 116 Figure 3. Levels of viral genomes of the enhancer and middle T mutants in comparison to the wild type A2 strain. NIH3T3 and BMK (Baby mouse kidney) cells were infected with each virus at an MOI of 2. At 60 hours postinfection duplicate viral DNA samples were isolated by Hirt extraction. 5% of the total viral DNA samples were digested with EcoRI and electrophoresed through a 1% agarose gel, blotted, and hybridized to a polyoma specific probe that represented the entire A2 genome. Middle T mutant (MT’) = 18-5. 117 AI2 A2(dll B) A2(lle3,l) MIT" l I l l I I l l Figure 3. 118 cells. The middle T mutant 18-5 failed to grow in 3T3 cells, but replicated well in BMK cells. Viral early proteins from each mutant were measured . Equal aliquots of extracts of infected cells were separated on polyacrylamide denaturing gels, transferred to membranes and incubated with a rat polyclonal antibody (1:500 dilution). The membranes were then incubated with a secondary antibody (sheep anti-rat HRP, 1:3000 dilution). Viral proteins were detected by Enhanced Chemiluminescence (Figure 4). An abundance of large T antigen was detected at 100 kDa in NIH3T3 and BMK cells from wild type A2, A2(dl B), A2(dl P3,1), and 18-5 infected cells. The middLe T protein (55 kDa), and the small T protein (23 kDa) were synthesized by A2(dl B), and A2(dl P3,1) at levels less than wild type in 3T3 cells but at comparable levels to wild type in BMK cells. 18-5 failed to synthesize middle T and small t antigen (16). Studies using viruses with a mutation only in the middle T protein have shown that in the absence of middle T, small T retains an important function in viral growth (13, 16, 25). Cell transformation by the mutants mentioned above was analyzed in a rat cell line (FR3T3). In comparison to the wild. type .A2 laboratory reference strain, each enhancer mutant (A2(dl B), A2(dl 28) and A2(dl P3,1)) was able to neoplastically transform rat cells in a period of 15 days (data not shown). The hr-t mutant 18-5 was completely defective in transformation 119 Figure 4. Levels of viral protein expression in NIH3T3 and BMK cells. Total cellular protein was extracted 60 hours postinfection. One ug of each protein sample was separated by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide) . Proteins were transferred onto membranes and probed with anti-T antibody (serum from Brown Norwegian rats, which carried a polyoma induced tumor) at a dilution of 1:500. Blots were probed with a secondary antibody (sheep anti-rat HRP) at a dilution of 1:3000. Viral early proteins were detected following enhanced chemiluminescence of membranes. M = molecular weight marker, u = uninfected, a = protein expression in BMK cells and a' = protein expression in NIH3T3 cells. a & a' = WTA2, b & b' = A2(dl P3,1), c s. c' = A2(dl B), and d s. d' = 18-5 (middle '1' mutant). LT = large T antigen, MT = middle T antigen, and st = small t antigen. Note the absence of middle T and small t antigen in cells infected with the viral middle T mutant (18-5). 120 N1 0 a b c d d' c. U . €- 106 - LT» 80 - MT! 50 - 32 - 27- 18- BAAK 313 Figure 4. 121 which supports its failure to produce the viral transforming protein middle T antigen as mentioned above (16, 20). Therefore, all enhancer mutants are transformation competent except the middle T mutant 18-5 which is transformation defective. Analysis of viral peplicapion in mice i. In situ hybridization sppdigs. To study the role of the polyomavirus enhancer and middle T antigen on viral replication in the whole mouse, the technique of in situ hybridization of whole-body mouse sections was employed (12, 19, 13). Neonates were infected within 24 hours of birth and adults at 6 weeks of age. Serial 40um saggital sections allowed for the visualization and identification of a number of target organs of viral replication. Figure 5(A) shows sections of neonatally infected mice sacrificed at 7 days postinfection. This time-point preceded the onset of the viral induced immune response (17a, 19). As previously observed, the wild type A2 strain (demonstrated. the Ihighest levels of replicated viral DNA (13). In this experiment, shorter exposures to x- ray film showed high levels of replicated viral genomes in kidney, skin, salivary glands and bone. Sites of lower levels of ‘viral genomes include the stomach, intestinal linings and liver. Viral genomes were absent in the brain and stomach. The capacity to produce a systemic infection was not affected by deleting the second half of the B 122 Figure 5. Replication analysis of enhancer and middle T mutants by in situ hybridization. (A). Neonatal mice 'were infected as described in Material and Methods and sacrificed 7 days postinfection. 40 um sagittal sections were prepared from two individual mice from the same litter, processed, and probed with a 32P labeled polyoma whole genome probe. Note the lack of viral replication in mice infected with the middle T mutant. (B). Viral replication in adult mice following infection with enhancer and middle T mutants 7 days postinfection. Sections from two individual mice are shown for each viral strain. Note the lack of viral dissemination in mice infected with the MT' strain. A2 = wild type strain, A2(dl ZB) = deletion of the distal half of the B domain (nts. 5204-5264), A2(dl B) = deletion of the complete B element (nts. 5141-5264), A2(dl P3,1) = deletion of the PEA1 and PEA3 sites in the A element (nts. 5109-5122) and MT“ = middle T mutant 18-5. 123 A ‘0 Q 6 ° 4" 5 . ... A It"- . l O ' O o ...-r A2 A2(d|28) A2(dl a) ...}...- ‘ A2(c||P3.1) MT" Figure 5 A. ad I . 0 o o . .. .0 'r- s- . 5 ... O ' e A2(dl P3,1) 124 a ." . .- I" , t. 9 ' ‘ .9 .. . -' " ’ 8' rig-'- .5A2(dlB1 J O . 0 .‘. K .0 ‘ ri- MT" Figure 5 B. 125 enhancer (A2(dl 2B, nts. 5204-5264)), but was significantly affected by removing the complete B element (A2 (dl B)) or the PEA1 and PEA3 sites in the A element (A2 (dl P3,1)) (Figure 5A). The pattern of organs infected by these mutants resembles the same pattern of wild type virus, but with a lower level of viral genomes. The skin, salivary glands, and bone contain stronger viral DNA signals while the kidney, stomach, and intestinal linings show less intense signals of viral replication. At both short and long term exposures, low intensity focal signals (smaller, single sites of replicated viral genomes), which probably represent targeted regions within these organs, are found in the skin, bone, and kidney. Stronger signals are seen in the salivary glands and muscular regions. Replication of the middle T mutant (18-5) was not detectable. Long term exposure reveals minute foci at the site of inoculation which may represent low level site specific viral replication without dissemination. To examine replication in adult tissues we infected adult female athymic (nu/nu) mice with the various viral strains. Figure 5(B) displays sections from mice infected for 7 days. Systemic infections did not occur for any of the viruses. As previously observed, wild type virus is restricted in growth showing replication only in skin, bone, salivary glands and spleen (mammary glands are not observable in these sections). However, in subsequent DNA extractions of the mammary gland, viral replication was 126 observed. Long term exposures to x-ray film do not reveal viral genomes the kidney, liver, or lung. Enhancer mutants A2(dl P3,1), and A2(dl B) reveal the same generalized pattern of infection. In contrast to enhancer mutations, a middle T defective mutant (MT', 18-5) diminishes the ability of the virus to replicate in adult mouse tissues. Long term exposure shows ‘minimal signal in. glandular organs (i.e. salivary glands). These results suggest that the spectrum of tissues involved in mutant middle T infections in adult nu/nu mice is smaller than that of wild type A2 infections. In addition, the level of viral replication of the mutants is much reduced. Deletions of the B element or PEA1 and PEA3 do not significantly change the viral replication pattern in comparison to wild type A2 infections except for an increase in the amount of replication of these deletion mutants in the skin. The middle T mutant 18-5 replicated poorly in adult mice (Figure 5B). (ii) Viral replication in mouse organsA To further evaluate and better quantitate viral replication in specific organs, DNA was extracted from neonatal organs, linearized, electrophoresed, blot hybridized, and viral genome levels quantitated. Figure 6(A) and Table I A demonstrate the level of viral genomes in organs of neonatal mice at 3 and 7 days postinfection. All enhancer deletion mutants tested showed low levels of viral genomes in neonatal mammary gland, skin, bone and kidney by 3 days 127 Figure 6. Analysis of polyomavirus genomes extracted from specific organs of mice infected as neonates and adults. Organs were removed from cadavers at 3 and 7 days postinfection (dpi) and processed as pooled samples of two mice per virus per time point. Total DNA was extracted and 2ug samples were digested with EcoRI, electrophoresed in 1% agarose, transferred to nylon membranes and hybridized to a polyomavirus specific probe containing the complete viral genome. The membranes were scanned with a beta imager (AMBIS) for each organ. Sample loading was corrected by hybridization to a mouse gene and polyoma genome signal was normalized as the ratio of these two signals. (A). Level of viral genomes in neonatal mice. (B). Level of viral genomes in adult mice. The numbers at 3 dpi and 7 dpi reflect the level of viral genomes. A2(dl 2B) = deletion of the 2nd half of the B element (nts. 5204-5264). 128 4 6 9.33m 529v. ng 29w D250 Egg 0 or 5' Eu 5 oz... 9 mos ._<.Ezom_z mmEOsz .._ '50 5 E500 NVSHO / 13Aa'l 3WON39 'IVHIA < 129 m 6 333m uZOo zEm ozSo >532: w a t 2 1 8 6. I v «a 2. 8 Iron _ a A ... _ v o 2 ... m .... 8 5.3... w m w m a m m. P P M m 1m, w w w m m w w m m. w w .m m w w 'E' weeps...” ”3...... H O ........................................................................................................................... 1m CON ........................................................................................................................................................................................... aim 8m ......................................................................................................................................... 8.. ........................................................................................................................................................................................... \1 8m in k oz< 9 mo: :39. mmEOZMG ._ m NV'SUO / 'IBIG'I BWONS'E) ‘IVUIA 130 Table I. Fold decrease in replicated genome levels of enhancer mutants relative to wild type. Quantitative values of neonatal (A) and adult (B) organ viral genome levels were measured for each organ by scanning membranes with a beta scanner (AMBIS). The relative level of viral genomes for a given mutant was computed relative to that of the wild type A2 strain (WT) and is presented as a ratio of WT/mutant. 131 fl H OHQMB gnu-3.. .56 En e mos. 1.5.20sz BF 8 2 R .253 on m m k $.91 .325) 2 9 mm m. a _em¢<_2_2> O._. m_>_._.<1_m_m ZO_._.mfi5wm ._.>> O... w>_._.<._wm ZO_._.%._‘,_2<_M_‘wz Ea e wme ._ z z is greater. 148 . N. gunman 848:8 3.288 g ane. ..mmswm . 3339 t. 6 83..me 6:639. 653 g S 336.. 1. Erik E “tangime .5 83958 : 5:539 852...? 6:839. E .o 85.-WV a. a 3 o 8.358 3:815: 385 .6 Swim... .865 85039 -E 3.98 8 3.86.. _ fl .6. .E .0... .n 3 him: 3.2.0.. ‘1 OF! OE OE IE Fl 5! E g s a a... g i la! in” build g Id! Uri I gs 149 did not in many cases inhibit viral replication in the organs studied. This reflects the redundancy of the enhancer. The mutants used in this study did permit us to further’ elucidate the role of the polyoma enhancer and middle T antigen in tissue specific viral replication. Additional analysis on tissue specific viral DNA replication is ongoing with mutants in which all known middle T responsive sites or non-middle T responsive sites are removed. 10. 11. 150 REFERENCES Twigg, A.J., and Sherratt, D. (1980). Trans- complementable copy-number mutants of plasmid ColEl. Nature. 283:216-218. Higuchi, R. (1990). Recombinant PCR. p. 177-183. Innis, M.A., Gelfand, D.R., Sninsky, J.J., and White, T.J. (ed.), PCR Protocols. A guide to methods and application. Academic Press, Inc., San Diego, Calfornia. Higuchi, R., Krummel, B., and Saiki, R.K. (1988). A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16:7351-7367. 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Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell. 39:653-662. ICHIGQN STQT “WW! E UNIV. LIaRnRIEs I WWMWWWMWWW 0445272 13112 23031