wank H .‘.' y _ "M. ““x- . "P“. ““““ v.71: I'I’ifr‘ hi§,t.‘. .‘k'v.\‘ ANALYSIS OF POLYRIBOSOMES AND INT RACELLULAR I RNA FROM FELINE LEUKEMIA VIRUS INFECTED'CELLS ' Dissertation for the Degree 'of Ph. 7 D. ’- MICHIGAN STATE UNIVERSITY ANTHONY JOSEPH VCONLEY 1977 I MICHIGAN STATE UNIVERSITY Il IIIIIIII lIlIIIIlIIIjIII * 3 1293 10580 TL"? ._:‘ A". I" "9/ £13. I‘ E1; A.’ III 'I‘ 1.; __ .,. ”It, .q., Mic; 21231.; q as :8 U fum University v1 éfimfim - - - . -~ This is to certify that the thesis entitled ANALYSIS OF POLYRIBOSOMES AND INTRACELLULAR RNA FROM FELINE LEUKEMIA VIRUS INFECTED CELLS presented by Anthony Joseph Conley has been accepted towards fulfillment of the requirements for film—degree in my and Public Health .A A Major professor 0—7639 I I I I I ...,,_. ,fl’ ,4 I an mas I MAGICZ " k "e g WQ’ESB E I I The chron the Rickard st: virus contains submits which The intr analyzed by us hybridization ribonucleopmt and 1-952 virI Size cl Valocity sedi and hydridiza nuClear and c Class, which fralct10118 al specifiC 368 the I"31y(A)+ ABSTRACT ANALYSIS OF POLYRIBOSOMES AND INTRACELLULAR RNA FROM FELINE LEUKEMIA VIRUS INFECTED CELLS By Anthony Joseph Conley The chronically infected thymus tumor cell line, F-422, produces the Rickard strain of feline leukemia virus (FeLV—R). This oncorna— virus contains 50 to 608 genomic RNA, which can be denatured to yield subunits which sediment at 285, molecular weight of 1.8 X 106. The intracellular virus-specific RNA from this cell line was analyzed by using in_yi££g synthesized complementary FeLV-R DNA. By hybridization kinetics analysis cytoplasmic, polyribosomal, and nuclear ribonucleoprotein (nRNP) particle RNAs were found to be 2.09%, 2.63%, and 1.95% virus-specific, respectively. Size classes within subcellular fractions were determined by velocity sedimentation in the presence of 99% dimethylsulfoxide (DMSO) and hydridization of the fractionated RNA to the FeLV—R DNA. The nuclear and cytoplasmic extract fractions contained a 28 to 308 size class, which corresponds to the size of the genomic subunit. Both fractions also contained 363, 238, and 12 to 188 RNA species. Virus— specific 368, 238, and 188 species were present in both the total and the poly(A)+ polyribosomal fraction. The nRNP particle fraction contained 365 classes were presence in I smaller than observed in t ing of FeLV—I To furt the POlyribo: 1mm and r nascent prott P01yribosome, throuShout tI FeLV IgG hour to 0,402_ TI tions of ant FeLv 18G Was Virus‘SPe-cif the Polyribo Specific ant absorbed IBG The binding reduced by t The Fe approximate l Anthony Joseph Conley contained 368, 285, 23S, and 188 virus—specific RNA. The four size classes were also present in the nRNP particle poly(A)+ RNA. The presence in the total nuclear and the nRNP particle fractions of smaller than genomic subunit size RNA, which corresponds to sizes observed in the cytoplasm and polyribosomes, suggests nuclear process- ing of FeLV—R RNA in these chronically infected cells. To further understand the mode of oncornavirus RNA translation, the polyribosomes from F—422 cells were examined by using both immuno— logical and nucleic acid hybridization techniques. Virus—specific nascent proteins were detected by binding 125I—labeled anti-FeLV IgG to polyribosomes. Normal rabbit serum (NRS) IgG bound at a level of 0.02% throughout the polyribosome region of the gradient. In contrast anti— FeLV IgG bound to rapidly sedimenting polyribosomes at a level of 0.25 to 0.40%. The peak binding was at 4008 polyribosomes. Two prepara— tions of anti—FeLV p30 had little or no binding. The binding of anti— FeLV IgG was further studied to determine its specificity for nascent virus—specific protein. NRS IgG did not compete with anti—FeLV IgG for the polyribosomal binding sites. Total viral protein and p30 absorbed specific antibody from the IgG preparation and the binding of the absorbed IgG was reduced in relation to the amount of protein used. The binding of anti-FeLV IgG to puromycin treated polyribosomes was reduced by the same proportion that nascent proteins were released. The FeLV—R DNA probe hybridized to two polyribosomal regions, approximately 400 to 4508 and 2508, and to a slower sedimenting region, approximatel treatment, 1 regions, 1e: size classes as described major peaks tained only obtained wit to the 343, Specific Rm is not virus 228, and 185 VimS‘Specii FeLV‘R‘Speci Anthony Joseph Conley approximately 808, within the polyribosomal gradients. After EDTA treatment, the DNA still hybridized to RNA from slower sedimenting regions, less than 808, but not to the two polyribosome regions. The size classes of virus-specific RNA within these regions was determined as described above. The 400 to 4508 polyribosomes contained three major peaks at 335, 228, and 188; whereas, the 2508 polyribosomes con— tained only 348 and 188 RNA. RNA from the approximately 808 regions obtained with and without EDTA treatment contained 288 RNA in addition to the 348, 228, and 188 virus-specific RNA. The absence of 288 virus— specific RNA in polyribosome regions suggests that FeLV—R subunit RNA is not virus—specific mRNA in infected cells. The presence of 348, 228, and 18S RNA within polyribosome regions which also contain nascent virus—specific proteins, suggests that there are three species of FeLV—R—specific mRNA in the F—422 feline thymus tumor cell line. ANALYSIS OF POLYRIBOSOMES AND INTRACELLULAR RNA FROM FELINE LEUKEMIA VIRUS INFECTED CELLS By Anthony Joseph Conley A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1977 III [III III III ;I .I DEDICATION To Jane ii (I >V‘ <:/ I we for guidan to acknowl Dr. Loren I I acl Health and my aPPreci; travel ass; ACKNOWLEDGMENTS I would like to express my appreciation to Dr. Leland F. Velicer for guidance and support during my dissertation research. I also wish to acknowledge the guidance received from Dr. Ronald J. Patterson, Dr. Loren R. Snyder, and Dr. Fritz M. Rottman. I acknowledge financial support from the National Institutes of Health and especially from Jane C. Conley. I would also like to express my appreciation to the Department of Microbiology and Public Health for travel assistance to attend scientific meetings. iii LIST OF TA} LIST OF FIC INTRODUCTK LITERATURE l. Oncoi TABLE OF CONTENTS ................................. vi LIST OF TABLES.................. LIST OF FIGURES.................................................. vii INTRODUCTION..................................................... 1 LITERATURE REVIEW................................................ 3 l. Oncornavirus RNA........................................... 3 Viral Genome............................................ 3 Viral Genomic Subunit................................... 4 Chemical Characteristics of the Subunit................. 4 2. Oncornavirus Proteins and their Biosynthesis............... 5 Low Molecular Weight Proteins........................... 6 Oncornavirus Glycoproteins.............................. 7 RNA-Directed DNA Polymerase............................. 8 3. Integration of Oncornavirus DNA-~the Provirus.............. 8 4. Organization of the Oncornavirus Genome.................... 9 5. Intracellular Oncornavirus-Specific RNA.................... 12 13 Virus—Specific RNA in Infected Avian Cells.............. Detection of Intracellular Avian Oncornavirus—Specific RNA with Probes of Specific Sequence........... 16 ago... Virus-Specific RNA in Infected Mammalian Cells.......... 17 Endogenous Oncornavirus—Specific RNA in Mammalian Cells. 20 21 Mammalian Sarcoma Virus—Specific Intracellular RNA...... iv TABLE OF CONT 6. Translz Trar Pol} 1 REFERENCES. . . ARTICLE I. I ARTICLE II . I TABLE OF CONTENTS—-continued Page 6. Translation of Oncornavirus RNA........ ..... ......... ..... 25 Transcriptional Control and Intranuclear Processing.... 25 Polyribosomal Virus—Specific RNA and Cell Free Trans— lation...................... .. ........ ............. 28 REFERENCESIO000.00.00.00it.cpl-COIIOOIOIOOIOOOOOOOIlotiono'OICO 31 ARTICLE I. Detection and Analysis of Intracellular Feline Leukemia Virus—Specific RNA...................... 47 ARTICLE II. Analysis of Polyribosomes from Cells Infected with Feline Leukemia Virus........... ............. .... 93 v TABLE 1. Virus-S} 2- Virus-S] LIST OF TABLES TABLE Page 1. Virus—Specific RNA in Infected Avian Cells. . . . . . . . . . . . . . . . . 14 2. Virus-Specific RNA in Infected Murine Cells. . . . . . . . . . . . . . . . 18 vi ' .iduifil-V iSIIIIIIIIliaafizr FIGURE ARTICL l. Polyac: N a w A LI! 0\ \l ,_4 N La) FeLV e1 Hybrid: afl-labv Relatii cellul; - Siles‘ by ana: - Sucrosv excludv plasmiv . DMSO 3] fr< ' Sizes ( ARTICL] Sedimer ' Sedimei mixture Sed imer FeLVIPC ' The Par tiOn of LIST OF FIGURES FIGURE ARTICLE I — Detection and Analysis of Intracellular Feline Leukemia Virus-Specific RNA . Polyacrylamide gel electrophoresis of the product of the FeLV endogenous RNA—directed DNA polymerase reaction...... Hybridization of 32P-labeled FeLV 28$ subunit A with 3H-labeled FELV DN Iotoooo-oo-nooooctoo-counoobvo-oooooooo 3. Relative concentrations of virus—specific RNA in sub— cellular fractions from F-422 cells....................... 4. Sizes of intracellular virus-specific RNA, as determined by analysis under denaturing conditions.. ....... .......... 5. Sucrose gradient sedimentation analysis of sepharose ZB excluded polyribosomes and unfractionated total cyto- plasmic extract.................................... ..... .. 6. DMSO gradient sedimentation analysis of rapidly labeled RNA from polyribosomes and nuclear RNP particles... ...... . 7. Sizes of intracellular virus~specif1c poly(A)+ RNA........ ARTICLE II-Ana1ysis of Polyribosomes from Cells Infected with Feline Leukemia Virus 1. Sedimentation analysis of 125I-Ingpolyribosome mixtures.. 2. Sedimentation analysis of 125I-anti FeLV:p01yribosome mixture preincubated with an excess of unlabeled NRS and sedimentation analysis of disrupted 14C—amino acid labeled FeLV:polyribosome mixture .......... ............ ...... ..... . The partial release of nascent protein and Partial reduc— tion of anti—FeLV binding by puromycin treatment ........ .. vii Page 60 63 65 68 71 74 77 107 110 6. Sizes LIST OF FIGURES-continued FIGURE 4. Sedimentation analysis of absorbed 125I-anti FeLV: polyribosome mixture.......................... ........... 5. Polyribosomal location of virus-specific RNA.............. 6. Sizes of FeLV—R-specific polyribosomal RNA ....... ......... viii Page 113 116 118 culture, can copy of its R. considerable 1 The fel: oncornawiruse: species. Thus systems for: tb An inter leukemia virus which do not body to a tram feline oncorna the origin and gene products which occur in The pres major unique c The viral pol cm of the vi l _ _w: ~11”?ng INTRODUCTION The oncornaviruses occupy an unusual position in the field of This class of viruses can infect and transform cells in virology. culture, can cause neoplasia in vertebrates, and can synthesize a DNA copy of its RNA genome. Consequently, the oncornaviruses have received considerable research attention in recent years. The feline leukemia viruses comprise the one group of mammalian oncornaviruses known to be horizontally transmitted in an outbred Thus, the feline leukemia virus systems are excellent model species. systems for the study of possible virus-induced neoplasia in humans. An interesting immunological phenomenon is seen in the feline Cats which have had an apparent infection but leukemia Virus system. which do not have feline leukemia virus—induced neoplasia, have anti- The nature of this antigen, bOdy to a transformed cell surface antigen. feline oncornavirus membrane antigen, is not known. Thus, the study of the origin and molecular nature of feline leukemia virus components or gene Products can aid in our understanding of the mechanisms and events which occur in neoplastic diseases. The presence of an RNA—dependent DNA polymerase is probably the major unique characteristic of the molecular biology of oncornaviruses. Ike Viral polymerase directs the synthesis of a double Stranded DNA COPY of the Viral RNA genome which can be integrated in the hOSt C311 apparently of precurso lational fez thymocyte ce (1) de vi (ii) dc (iii) an Thus, there are a number of questions about replication, genome. production, and control of production of the viral macromolecules. A particularly interesting problem concerns control of the amounts of the various viral gene products synthesized. These products are apparently synthesized in nonequimolar quantities, and some in the form of precursors; a situation which raises questions about the virus— specific messenger RNA and the translational mode of this RNA. The overall aim of this research was to study the intracellular feline leukemia virus-specific RNA and to determine some of the trans- lational features of this RNA in the chronically infected F—422 Among the specific aims of this research were to thymocyte cell line. (1) determine the amount of feline leukemia virus—specific RNA within subcellular fractions. (ii) determine the sizes of these intracellular RNA species. (iii) examine virus-specific protein synthesis on F—422 polyribo— somes by using anti—feline leukemia virus serum as an immuno- logical probe for nascent viral protein, and in vitro synthe— sized feline leukemia virus DNA as a nucleic acid probe for virus-specific RNA. (iv) determine the major size class or polyribosomes synthesizing viral proteins. (V) specifically determine the size of the polyribosomal- associated presumptive, Virus—specific mRNA. The 11 specific RN specific m3 replication brief disgu be Presente RNA, Protei viral genom l. W Viral which has a tions (58,1 RNA, Can be genous Smal, achieVed by SUIfoxide o: struCtux-e (. native Statt 1'0in near Probably fr: which are 31 LITERATURE REVIEW The major topic of this review is intracellular oncornavirus- specific RNA and the possible translational mode(s) for oncornavirus- specific mRNA. Since other aspects of oncornavirus structure and replication are important in a development of this central theme, a brief discussion of the important properties of these viruses will also These reviews include short discussions of oncornavirus be presented. RNA, proteins, the viral replication cycle, and organization of the viral genome. l. Oncornavirus RNA The genome of oncornaviruses consists of RNA, Viral Genome: which has a sedimentation coefficient of 50 to 708 under native condi— This RNA, referred to as genomic high molecular weight tions (58,148). RNA, can be dissociated to 30 to 40$ subunits and various minor hetero- Dissociation of the genome has been genous smaller species (58,143). achieved by the use of heat or denaturing agents, such as dimethyl— sulfoxide or formamide, which disrupt hydrogen bonding and secondary Electron microscopic studies have shown that, in the structure (7,38). native state, the genome consists of subunit dimers noncovalently joined near the subunit 5'—termini (30,80,81). The 3'—termini are Probably free and can be detected by electron microscopic techniques which are specific for polyadenylation (12). 3 11113 oncornavir subunit mo tation in mentation acrylamide the electn size among 408 genomn 28S for the genous felj Virus (Msv- Virus (R‘Mu 175), Sedim for the mol Subunits. lar weight A spe. electroPhorl subunit is i and is the ( (35). The! size Subunit % unit RNA has Viral Genomic Subunit: The genomic subunits from most known oncornaviruses have been analyzed. Four techniques have been used for subunit molecular Weight determinations. These are: velocity sedimen- tation in neutral sucrose gradients after denaturation, velocity sedi— mentation in denaturing sucrose gradients, electrophoresis in poly— acrylamide gels (PAGE) after denaturation, and length measurement in the electron microscope. Notwithstanding the considerable variation in size among the oncornaviruses, the subunit is referred to as the 30 to 40$ genomic subunit RNA. Subunit sedimentation coefficients range from 288 for the Rickard strain of feline leukemia virus (FeLV—R), the endo— genous feline RD—ll4 virus, and the Soehner-Dmochowski murine sarcoma virus (MSV-SD) (21,36,37), to 35 to 44$ for Rauscher murine leukemia . virus (R-MuLV) (7). There are comparative electron microscopic (23,88, 175), sedimentation (22), and electrophoretic (31,35) data available for the molecular weight determination of Rous sarcoma virus (RSV) RNA subunits. These data are in agreement and the RSV subunit has a molecu- lar weight in the range of 2.7 to 3.5 x 106. A special case exists for some avian sarcoma viruses, where two electrophoretically separable subunits have been found (34). The a subunit is approximately 10 to 15% larger than the b_subunit (34,35), and is the only subunit present within recloned avian sarcoma viruses (35). The b_subunit is electrophoretically identical to the single size subunit present in the avian leukosis viruses (35). Chemical Characteristics of the Subunit: The oncornavirus sub— unit RNA has properties which are similar to eukaryotic mRNA. Polyadenylatit leukemia-sarc< avian myelobla from R-MuLV m2 of its specifi respectively 1 these sequence The gene is a charactez In early EXPEI 0f RSV subunit harvested virn short intewa] ments were peI detected in an; RNA (20,112). RNA (147). SL‘ methylated nuc dominant m6th5 2 Onco - ' rua , VlrL The Oncc considerable s of sodiUm dOde guanidine hydr cal techniqueS Polyadenylation of subunit 3‘—termini has been detected for the murine leukemia-sarcoma viruses (70,82,l97,1ll), RSV (78,82,109,169), the avian myeloblastosis virus (AMV) (135), and FeLV (21). Subunit RNA from R-MuLV may also contain poly (C) and poly (G) sequences by virtue of its specific binding to poly (G) and poly (C) agarose columns, respectively (103). However, not all oncornaviruse subunits contain these sequences (55). "cap”, The general structure 7mG(5')ppp(5')Nmpr, referred to as a is a characteristic of the 5'-terminus of most eukaryotic mRNA (115). In early experiments, free phosphorus was not found at the 5'-termini of RSV subunit RNA (128). Further studies on RNA from long—term harvested virus and virus harvested from cell cultures at extremely short intervals confirmed this observation (78). From this work experi— ments were performed to demonstrate that the cap structures could be detected in avian oncornavirus RNA (77,48,136) and murine oncornavirus RNA (20,112). However, no cap structures could be detected in FeLV—R RNA (147). Subunit RNA from RSV, MuLV, and FeLV-R contain internally methylated nucleotides (20,136,147). N6-methyladenosine is the pre- dominant methylated species. 2. Oncornavirus Proteins and their Biosynthesis The oncornavirus protein components have been the subject of considerable study. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) and agarose gel filtration in 6! guanidine hydrochloride (GuHCl) have been the most widely used analyti- cal techniques (45,59,98). It is now assumed that the leukemia viruses contain thr4 weight stru< (iii) the R1 viruses cent is involved component 01 are in agree cases have t Proteins obs be host cell Low mc molecular we Proteins the M four low 11101 “5,46,64,19 beefl dEtermi unstable 76, cursor can b fected Call been °°nfinn 0f the Precu The mu molecular We these protei contain three major classes of proteins, i.e. (i) the low molecular weight structural proteins, (ii) the envelope glycoproteins, and (iii) the RNA-dependent DNA polymerase. The nondefective avian sarcoma viruses contain these three classes of proteins plus a component which is involved in cell transformation (32). The nature of this additional component or its presence in virions is not known. These assumptions are in agreement with the genetic capacity of the viruses and in some cases have been verified (see section 4). Most of the remaining minor proteins observed in analytical SDS-PAGE of virions are considered to be host cell or serum contaminants (137). Low molecular weight oncornavirus proteins are designated by their molecular weight in thousands, prefixed by a lower case p, glyco— proteins the same, prefixed by the lower case gp_(5). Low Molecular Weight Proteins: The avian oncornaviruses contain four low molecular weight structural proteins—“p27, p19, p15, and p12 (45,46,64,19). Their structural positions within virions have also been determined (108,138,139,l40). They are synthesized by way of an unstable 76,000 dalton, intracellular precursor (166,167). The pre— cursor can be detected by pulse-label and immunoprecipitation of in- fected cell proteins (166). The precursor-product relationship has been confirmed by pulse~chase experiments and tryptic peptide analysis of the precursor and the viral proteins. The murine and feline leukemia viruses contain analogous low molecular weight structural proteins (57,59,98,119,137). Synthesis Of these proteins also proceeds by intracellular cleavage of comparable size precur further con extremely 1 similar exp QM avian, mun 92,177,178) components Avian (18,45) whi Plex (83). CiPitation a n°n81ycos ti” is ava s)’Tlthesis 0 Among designated “ins (33,6 rePorted (6 an apparent Position an be Confirme‘ and tryptic cellular pr. size precursors (4,72,100,101,124,158). R—MuLV infected cells may further contain a 200,000 dalton precursor (4,95,96,124). This extremely large precursor was not observed by other investigators in similar experiments with the same virus (116,158). Oncornavirus Glycoproteins: The glycoprotein components from avian, marine, and feline oncornaviruses have been studied (45,59,68, 92,177,178). Morphologically, the glycoproteins are the virus—specific components of the envelope. Avian oncornaviruses contain two glycoproteins, gp85 and gp37 (18,45) which are linked by disulfide bonds in the envelope as a com— plex (83). Pulse—chase labeling experiments combined with immunopre— cipitation of intracellular proteins suggest that gp85 is produced from a nonglycosylated 70,000 dalton precursor (60). However, no informa— tion is available about the relationship of this precursor to the synthesis of both glycoproteins. Among the variety of murine oncornavirus strains, glycoproteins designated gp70 or gp69/71 appear to be the major envelope glycopro— teins (33,68,90,92,93,102). Other minor envelope components have been reported (69,71,93,118). These have been designated gp45 and p15 (E); an apparently nonglycosylated envelope constituent. The structural position and viral specificity of the two smaller proteins has yet to be confirmed (90). R-MuLV gp69/71 and p15 (E) antigenic determinants and tryptic peptides are present in an unstable 90,000 dalton intra- cellular protein (4,39,124). The unstable precursor is partially glycosylated (96,124) and may be an intermediate in the cleavage of a very high1 has been d4 535g to contain and Mizutar directed t< properties, These topic discussed 1 Of these er The a Weights of unit Can be aPPears to Unlik COnsist of (2,50,113,1 DNA Polymer Product (16 iflsn Early formation 0 that Ontorn very high molecular weight precursor——260,000 to 300,000 daltons, which has been detected intracellularly (124). RNA—Directed DNA Polymerase: The oncornaviruses were first shown to contain an RNA—directed DNA polymerase by Baltimore (8) and Temin and Mizutani (146). Since that time numerous investigations have been directed toward the characterization of this enzyme, its catalytic properties, and involvement in oncornavirus infection and replication. These topics have been amply reviewed (58,91,145,l48) and will not be discussed here. However, a brief presentation of some characteristics of these enzymes follows. The avian polymerase consists of two subunits with molecular weights of approximately 65,000 and 105,000 (44,76). The smaller sub— unit can be generated from the larger by proteolytic cleavage (92), and appears to be the major enzymatically active subunit (56). Unlike the avian enzyme, the mammalian oncornavirus polymerases consist of one protein with an estimated molecular weight of 70,000 (2,50,113,l49). Although the mode of biosynthesis of the RNA—directed DNA polymerase is not known, the avian polymerase is a virus—specific product (164, and section 4). 3. Integration of Oncornavirus DNA*—the Provirus Early observations suggested that productive infection and trans- formation of cells with oncornaviruses required DNA synthesis immedi— ately after infection (6,142). These observations led to the hypothesis that oncornaviral RNA is copied into DNA, the provirus (144), which can .,_R be incorpc directed E "reversely has been r and has be genome (89 the infect of forms r RSV and M-1 daltons fo; copies of , which are , is conside1 viral RNA , 4. E33113 Since Problems ha cOTIStructec' i.e., is as Unique, 3 17 In av was °bServe dePendent 13 Suggest rea be incorporated into the host genome (143). The discovery of the RNA- directed DNA polymerase (8,146) confirmed that genomic RNA can be "reversely transcribed" into DNA. Since then, infectious proviral DNA has been recovered from RSV and Moloney—MuLV infected cells (65,130), and has been demonstrated to be covalently integrated into the host genome (89,162). The DNA intermediate appears to be synthesized in the infected cell cytoplasm (51,161), where it can exist in a variety of forms ranging from linear molecules of 2.5 to 3.0 X 106 daltons for RSV and M—MuLV (87,160), to closed circular molecules of 5 to 6 X 106 daltons for M-MuLV (51,130). This suggests that double-stranded DNA copies of the viral genomic subunit are the major proviral molecules which are eventually integrated (89,127,159,162). The integrated DNA is considered the transcriptional template for the production of progeny viral RNA and virus—specific mRNA (58,145). 4. Organization of the Oncornavirus Genome Since the oncornavirus genome consists of subunits, a number of problems had to be resolved before genetic and physical maps could be constructed. The major obstacle concerned the complexity of the genome, i.e., is each subunit identical, a polyploid genome; or is each subunit unique, a haploid genome? In avian oncornavirus systems, high frequency of recombination was observed for the viral markers—host range, transformation, and RNA- dependent DNA polymerase (32,76,165,l76). These observations would suggest reassortment of genomic subunits. However, analysis of the RNA 0f specific recombinants suggested that this was not the case. Nondefectiv host range could not b is the prob indirect su Two d oncornavira reassociati analysis wh An em 0f annealin RN4- In va tional to t RSV RNA was all’l’miimate melecular w are Suggest Same tYPe o Purificatio aPProximate °PPosite th The s measUrement The general determinati lO Nondefective sarcoma—positive recombinants, containing a leukosis virus host range marker, retains the g_subunit RNA (11,32). This result could not be obtained from reassortment and indicated that crossing—over is the probable mechanism of recombination. These analyses lend indirect support to polyploidy. Two direct approaches have been used to relate the size of the oncornaviral genome to the complexity of its RNA. In the first, reassociation kinetics analysis is used, in the second a chemical analysis which determines an apparent complexity (11,14) is used. An empirical relationship has been established between the rate of annealing of RNA to a complementary probe and the complexity of that RNA. In vast RNA excess, the rate of annealing is inversely propor— tional to the complexity (15,17). By this method, the complexity of RSV RNA was determined to be 9.3 X 106 daltons (141) and that of M—MuLV, approximately 9.0 X 106 daltons (43). These values are close to the molecular weight determinations of l X 107 for the 708 genomic RNA, and are suggestive of unique subunits. Baluda et a1. (10) performed the same type of analysis on AMV RNA, employing strict conditions for the purification of RNA standards. Their results indicate a complexity of approximately 3 X 106 daltons, suggesting identical subunits, and are opposite the results obtained in the previous two investigations. The second, chemical method has consistently yielded a complexity measurement of approximately 3.5 X 106 daltons for RSV RNA (11,14,32). The general approach for this method is the isolation and molar yield determination of a series of unique oligonucleotides from T1 RNase digests o complexit; of approx: and is nor viruses e1 The of RSV get T1 RNase-d ably sligh gene order leukemia v In t 3"Polyade from theSe binant RNA 1°Cat€d sat the 3'rterx 3"terminus oligonucla 3"terminus In"ether 0118 2800 to 50C the Subunit Son of n0nd ll digests of subunit RNA. This data can then be used to calculate the complexity of the RNA (11,14). Since these data support a complexity of approximately 3.5 X 106 daltons, a polyploid genome is most likely and is now the accepted complexity. This further implies that oncorna- viruses encode approximately 300,000 daltons of virus—specific protein(9). The construction of genomic physical maps and the identification of RSV gene order has been achieved by using the same technique of T1 RNase-derived oligonucleotide isolation. Although there are prob— ably slight differences among the oncornaviruses, the general deduced gene order can be considered valid for both avian and mammalian sarcoma— leukemia viruses. In these experiments, the oligonucleotides were oriented to the 3'—polyadenylated end of the genomic subunits. Maps were constructed from these orientations by comparison of deletion mutant and recom— binant RNA oligonucleotides (24,73,170,171,l72,173). Wang et al. (170) located sarcoma marker specific oligonucleotides, designatedjgagg, near the 3'—termini. They also observed conserved sequences at the 3'—terminus, 5' to the polyadenylation (170). The location of the sarg oligonucleotides and the presence of conserved sequences at the 3'—terminus were confirmed in a similar fashion (24,73). Host range marker oligonucleotides, designated—egg, were found in the range of 2800 to 5000 nucleotides from the 3'~terminus, i.e., near the middle of the subunit, in nondefective RNA (171). This analysis employed compari— son of nondefective RNA with RNA from defective deletion mutants (171). The same map location for env was determined by correlation of recombine et al. (1' 22;, betvn tions were marker p27 terminus ( are probab 5'-terminu using vira observed ( The sidered es 5"868-p01 Viruses do marker has Viruses . 5. 32353252 The I sYnthesis 1 Probes were mAmmalian c Suggested t and Coald b selected by found that l2 recombinant and parental oligonucleotides (73,172). Finally, Wang et al. (173) mapped RNA~dependent DNA polymerase ts markers, designated— 22;, between 6000 and 8000 nucleotides from the 3'—terminus. Correla— tions were also made in these recombinants between the parental protein marker p27, and the origin of oligonucleotides at or near the 5'— terminus (173). The viral structural protein sequences, designatedfgag, are probably located at the 5'—terminus. Evidence for gag at the ~5'—terminus is also found from in_xi££g protein synthesis experiments using viral RNA. Structural protein precursors are the major products observed (54,79,105,l68). The gene order consistent with all these data and which is con— sidered essentially correct for nondefective avian sarcoma viruses is: 5'—gag—pol-env—sarc—50 conserved N-poly(A)-3' (9,173). Avian leukosis viruses do not have the sagg sequences (170), and a virus—specific sagg marker has not yet been identified for the murine and feline oncorna— viruses. 5. Intracellular Oncornavirus—Specific RNA The RNAedependent DNA polymerase can be used in an in_yit£g_DNA synthesis reaction to prepare DNA copies of the oncornaviral RNA. DNA probes were first used to detect virus—specific RNA in both avian and mammalian oncornavirus infected cells (26,155). The initial experiments suggested that intracellular RNA was of the same polarity as viral RNA and could be best detected using preparations of complementary DNA pre— selected by hybridization with viral RNA (26). Garapin et al. (49) found that when the reaction was performed in the presence of actinomyci: also found methods ini (27,74,110, RNA usuall} mycin D or Rease the standa: specific R1 exPeriments from varior been develc P°1Y(I) se; hound comp] techlliques RNA' In th teChniques Virus has been fc CharaCter is studieS Wit cells have In all th es tated relat for virUS~s l3 actinomycin D, single stranded DNA was the major product. This DNA was also found to be a relatively uniform copy of the genome (49). The methods involved in complementary DNA preparation are well documented (27,74,110,ll4). The complementary DNA used for analyzing intracellular RNA usually has been prepared by synthesis in the presence of actino— mycin D or preselected by hybridization with viral RNA. Reassociation kinetics analysis, in RNA excess, with viral RNA as the standard has been generally used to quantitate intracellular virus— specific RNA under steady state conditions. Hybridization saturation experiments have been used for determinations of homology of RNA species from various cell lines to the DNA probes. Recently, techniques have been developed for specific binding of RNA:DNA—poly(dC) hybrids to poly(I) sephadex columns (25) and specific binding of RNA to matrix bound complementary DNA (16). Besides use in quantitation, these techniques allow for the analysis of newly synthesized virus—specific RNA. In the presentation that follows these types of hybridization techniques have been widely used. Virus—Specific RNA in Infected Avian Cells: Virus—specific RNA has been found in cells infected with avian oncornaviruses. The major characteristics of these RNAs are summarized in Table I. In most studies with the avian oncornaviruses, chicken embryo fibroblast (CEF) cells have been the usual host cell and RSV has been used for infection. In all these studies, the amount of virus-specific RNA has been quanti- tated relative to the total amount of intracellular RNA. The values for virus-specific RNA range from approximately 0.1% (120) to 2.0% (16). l4 A «3 In- men + mmm NA. :8 138 use :2 +3.38 ma + mum Nu. 33318.8 emu o mammumémm A 2V +A$2oa I- NN S332 Ea Rmu>mm A n: in n} um. 3 no. :8 H38. emu Saguuflafiwsmm ill III Noe. uwwaodz III nut new. ofismmHmouzo A3: I- 3- non. :8 H33 has casebfiaeumémm III III NN. cu NH. ummHosz u..- i- no. 3 Mn”. 03313.6 A a: l... mo... 3 3 maonowouuuun 5. 3 um. :8 38. H8 caaébfisfimsmm In: men cu oA «mnm In: Hmsomoafinzaom A83 E- u». "3.. :8 A38 emu o wuwfimlsmm hwoaoson A a: 398m» A: -u- 2 unanimous emu Elma mononowom 302 Saw hug—3:0 oouumuoq .300 9.5.; Ausowowwuoou nowumucoawvomv nofiwfioonmtmans NV uaazaawonsm 5:8 :33 3835 5 <5 udfloamlmfig 5 mafia In general : of RNA unc ; It t i labeling a i tion. Par ' specific 5 L DNA probe, ( Specific R I] stable. c | early time k In a examined 1 ‘ RSV (120). E determined ‘ infection, I Sh°rtly th t| In c0ntras ‘ Rece ‘ applied to | Specific n than genom longfl lab‘ SynthP-Size‘ (16), 15 In general, these values constitute the accepted range for measurement of RNA under steady state conditions. It has also been possible to measure newly synthesized RNA by labeling and specific selection or by rapid infection and transforma— tion. Parsons et al. (104) measured newly synthesized RNA for virus— specific sequences by labeling, annealing the RNA to a heterologous DNA probe, and selecting virus—specific RNA:DNA hybrids. Nuclear virus- specific RNA was detected within 15 minutes and its level remained stable. Cytoplasmic virus—specific sequences were also present at early time points, however the level continued to increase. In another experiment, virus—specific intracellular RNA was examined immediately after infection and transformation of cells by RSV (120). The amount of RNA at various times postinfection was determined by hybridization kinetics analyses. Within 12 hours post- infection, virus-specific sequences were detected intranuclearly and shortly thereafter reached a stable level of approximately 0.03%. In contrast, cytoplasmic virus—specific RNA levels continued to increase. Recently, the pulse labeling and selection techniques have been applied to an examination of the size of newly synthesized virus— specific nuclear RNA (16). An RNA species, approximately 15% larger than genomic subunits was detected. This species was not detected in longer labeling periods, suggesting that the viral RNA species are synthesized as a precursor which must undergo intranuclear processing (16). The been exam whole celi RNA (16,61 consist of classes we virus-spec (120). % for prepar synthesize. f°rm3tion 1 tive of th¢ hybrids. 1 DNAenv ’ WEI DNA se Mann and c “115 (16,2 Virus~produ suggests th 363 and 24s DNA err 243 intrace Populatim ‘ within One 1 16 The sizes of intracellular avian oncornavirus-specific RNA have been examined (16,63,88,120). When analyzed under native conditions, whole cell virus-specific RNA is quite heterogenous (88). Denatured RNA (16,63) or specific subcellular fraction RNA (120), were found to consist of more discrete species. Specifically, 36S and 248 size classes were found (16,63). In another study, polyribosomal associated virus—specific RNA was found to consist of 358 and 10 to 308 species (120). Detection of Intracellular Avian Oncornavirus—Specific RNA with Probes of Specific Sequence: Stehelin et a1. (132,133) devised methods for preparation of specific RSV gene sequence DNA probes. Briefly, DNA synthesized from a nondefective RSV template was hybridized to trans— formation defective RNA. The nonhybridizing DNA sequences, representa— tive of the sarc gene, designated DNA , were separated from the sarc hybrids. In an analogous manner env gene DNA sequences, designated DNAenv’ were prepared (62). DNABare was found to hybridize to 24$ RNA present in the cyto- plasm and on polyribosomes of Schmitt—Rupin—RSV transformed hamster cells (16,28). Since the 24S RNA is also present in SR—RSV infected virus-producing and transformed CEF cells, the DNAsarc hybridization suggests that the RSV sage gene is transcribed (or processed) into both 363 and 248 intracellular virus—specific RNA (28). DNAenv as well as representative DNA, hybridized to both 368 and 248 intracellular RNA (63). Either the 248 RNA is a heterogenous population of transcripts, or both §a£g_and g§g_sequences are present within one transcript. P_\,_._ F.._. ,_‘ PA Vi: detectiox reported mentary 1 402 of tl part of P 0.00052, be involv Vi; teristics ized in T used for Specific locatiOu, Gre Specific j in total . Cients of 0‘72 of U RNA (163), (150), an< Polyribos,‘ the membrg The 3508 s ity of Vir 17 Viral RNA has been used as a specific hybridization probe for the detection of intracellular virus—specific RNA. Stavnezer et al. (131) reported the presence of negative strand sequences, i.e., RNA comple— mentary to viral RNA. These sequences are homologous to approximately 40% of the viral RNA. They exist in nuclei and in the cytoplasm as part of RNA duplex structures at relative concentrations of 0.005% and 0.00052, respectively. Their function is not known. They may however, be involved in virus-specific mRNA processing (131). Virus-Specific RNA in Infected Mammalian Cells: The major charac— teristics of virus—specific RNA in infected mammalian cells are summar- ized in Table II. M~MuLV and R—MuLV infected cells have been widely used for these analyses. The characteristics of intracellular virus— specific RNA which have been studied and determined include subcellular location, quantity of RNA, and sedimentation values. Green and colleagues (126,150,153,155,163) examined M—MuLV specific RNA in infected cells. Virus-specific sequences were present in total cellular RNA as two distinct sizes with sedimentation coeffi— cients of 358 and 208 (155). These sequences comprised approximately 0.7% of the total RNA and approximately 0.6% of polyribosomal—associated RNA (163). Both species appear to contain 3'-termina1 polyadenylation (150), and these sizes were found to be unevenly distributed on cellular polyribosomes. Free polyribosomes only contained the 355 RNA whereas the membrane-bound polyribosomes contained both the 353 and 205 (126). The 3508 sedimenting polyribosomes of both classes contained the major- ity of virus—specific sequences in the 78Al M—MuLV infected cells (163). IIINIII..C (CVLGKCA nuuuuuauuooo nevusucufiuvumvAufluflUOQMImgwb NV thJHHoonam ocwq HNoU moat. .UHHOU flannel: VQUUUNEH EH <2“ UHNWUOQMIODHHx» uNH NASH 1" (‘lllII‘[‘1|.‘1[/‘lil\[lillll\[l. l1lIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Hmafinfiz mQH mHN + mom Ill +Aumsn uoauwame->a=z nvnmv :owumuwvwnnzm oz wwm no. 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Amoav cauauwsm-msna> some: .I. new. mmaomoasusaom Hq=z moawnouum ouoz mnam . suauemao coaumuon mess Hams sagas Auuuuuewuwoo uoaumueaEAeamvAuawwooawamsua> NV umassauonpm .maaoo means: souuuwaw ea 42m oswwuuamumauss "HM usmflumz AAom mnu mo “undone new we mwmmuosmouuoofio How upwamakuomwaom H ouswam N. w amber—0.2 20 .v N. l 1:: we as mm. mm i . r l r .m we as l 1 mm. mmN .< N '0 3-0: x was H., and the hybr. in Figure 2, DNA/RNA rati to RNase dig tions on the plate protec Since the ma the data fur mm formed to de RNA fraction can be perfo concentratic concentratic determined h fraction wit exPP—riments With essentj Stilts of the DNA hybridi: t0 the viraj Plasmic RNA ribosomal R] L952 virus. uninfected : 61 and the hybrids formed were assayed for RNase resistance (14). As seen in Figure 2, the DNA product protected 32.5% of the viral genome at a DNA/RNA ratio of one. At a ratio of l7.5/l.77% of the RNA was resistant to RNase digestion. This was the greatest ratio tested due to limita— tions on the amount of DNA product which could be prepared. The incom— plete protection indicated that the entire genome was not transcribed. Since the maximum protection observed was at a DNA/RNA ratio of l7.5/l, the data further indicates non—uniform transcription. Amount of Intracellular Virus-Specific RNA: Experiments were per— formed to determine the rates of hybridization of various intracellular RNA fractions with the FeLV DNA. RNA—DNA hybridization in RNA excess can be performed to determine the value of Crt(%); the product of the concentration of RNA and the time at half saturation (3). The relative concentration of virus-specific RNA in those fractions tested, can be determined by comparison of the Crt(8) obtained for the RNA from each fraction with that Crt(%) obtained for the viral RNA (11,43). These experiments were performed with either varied time or concentration, with essentially identical results obtained by both methods. The re— sults of these experiments are seen in Figure 3. The amount of viral DNA hybridized is plotted as a function of Log[104Crt]. By comparison to the viral Crt(%) for 288 FeLV subunit RNA, unfractionated F-422 cyto— plasmic RNA was found to contain 2.09% virus—specific RNA. Total poly- ribosomal RNA contained 2.63% virus-specific RNA, and nRNP particle RNA, 1.95% virus—specific RNA. Under these conditions, cytoplasmic RNA from uninfected feline embryo lung fibroblasts (FLF—3) did not hybridize to l i 00. 62 [([.l .No no venouwxomn unnuwAmou ommzm so now wouoounou mum mosam> .oHumn Aomlmmm mo usoo nude say .Aqu ponfiuomou mm < ummzm oeumouoame Sues woummmwu onus moamamm .moaumnsonfl Houwm .oufluooHoss Hofia\aeo wHN was <29 onu mo umnu was .okuooaosn Hofie\amu me mos 42m wen mo xufi>fluom UHMHoomm unH .mH ousmflm you wonfluomow mm nonmeunn <29 >Aom poaonmalmm a»? $2 £553 mum 50m conceding no couumuaeflfim .N 3am: 63 IOO O (D VNH lNViSlSBZ-J BSVNH °/o 20 Figure 2 DNA / RNA RATIO VN / I I «may . n+l+liuel+l+lal+levx o .{lli‘l‘rl ll. .AoMImm :uH3 macaumuuaoodoo wdflkum> um Aom mam mnowuomuw umHSHHoundm msowum> onu .mHHmo qulm .m ousmmm Scum wumaon osa Scum muofiuomuw HmHSHHoonsm CH 42M QHMHoommlmsuH> mo mfloaumuunoosoo o>fiumamm 65 n madman mmtiommdmmoz - Tao so; 03 m a m N _ -I q - VNCI lNVlSlSBE-J HSVB'IOFIN lS °/o the FeLV D] and polyri] a maximum 1 the sequem particle R] S_iz_e_ extracted < Materials 4 gradient, 4 and analyz¢ in Figure 4 value of 2: Present fa: Weight est: Parallel mg resoIVed P‘ aniounts of were also 1 cl’rOplasmn RNA detect. partiCles I 66 the FeLV DNA probe. Maximum hybridization for the viral, cytoplasmic, and polyribosomal RNA ranges from 76 to 84%. The nRNP particle RNA had a maximum at slightly more than 60%. This may indicate that not all the sequences present in the other fractions were present in nRNP particle RNA. Size of Virus-Specific RNA in F-422 Cytoplasmic Extracts: RNA was extracted directly from F-422 cytoplasmic extracts as described in Materials and Methods. After sedimentation in a 5% to 20% (w/v) sucrose gradient, containing 99% DMSO, the RNA was precipitated from fractions and analyzed for virus—specific RNA using 3H-FeLV DNA probe. As seen in Figure 4A, the major cytoplasmic species present sedimented with a value of 288 (molecular weight estimate of 1.8 X 106). There was also present faster sedimenting material with a value of 368 (molecular weight estimate of 2.8 X 106). In an earlier analysis using only a parallel marker gradient, this RNA was present as a more completely resolved peak sedimenting at approximately 363 (data not shown). Minor amounts of material sedimenting at approximately 233 and less than 183 were also present. Due to the denaturing conditions of the analysis, cytoplasmic RNA of genomic size (SO-60$), would not be detected. The RNA detected would include genomic subunits from virion precursor particles present in the cytoplasm as well as non-virion virus-specific RNA. Size of Total Nuclear Virus—Specific RNA: The results of nuclear RNA size analysis are shown in Figure 4B. Peaks with values of 308 6 (mol wt N 2 X 106), 398 (mol wt W 3.5 X 106) and 243 (mol wt m 1.2 X 10 ) . \l \4(‘||[l ll 67 Figure 4. Sizes of intracellular virus-specific RNA, as determined by analysis under denaturing conditions. RNA extracted from the various subcellular fractions were analyzed on sucrose gradients containing 99% DMSO by the method previously described (5). Parallel ribosomal RNA gradients were run with each analysis. Fractions (A), (C), and (D) also contained internal ribosomal RNA markers. Arrows indicate internal marker position in panels (A), (C), (D), and parallel marker in panel (B). After centrifugation, gradients were fractionated, processed, and assayed for virus— specific RNA as described in the text. The amount of RNA was plotted as relative virus—specific RNA, using the relationship described by Fan and Baltimore (11), where 50% hybridization has a relative value of one. (a) 8.1 ug total cytoplasmic RNA. (B) 14.5 pg total nuclear RNA. (C) 4.3 pg polyribosomal RNA. (D) 9.1 pg nuclear RNP particle RNA. RELATIVE VIRUS-SPECIFIC RNA '0 .0 01 9“ CD L003 h. P9 C) 68 A. Cytoplasmic Q, Polysomal nined 28$ '88 285 '88 me 2.0 - l l "2.0 ts I .5 r «L5 RNA a Nuclear 1; Nuclear RNP Method 11 1 nuclear g 30 __ 28$ IfS 4,. [5 288 '88 particle E ‘ .1 w or eat + LO I .0 - +0.5 5 lb I5 50 2'5 516 s éo és FRACTION NUMBER Figure 4 were seen. 7.0 x 105). were employ ing S value within the nuclear spe species obs V3132 from F-422 l described (, ribosomes i: cl’tOplasmic material cox monomers anc This I Specific RNA is seen in F material Wit of 2.3 X 106 Sadimenting ent of the C when Sepharo before Extra nOtable that 288 in this 1 69 were seen. There is also a shoulder in the range of 12-188 (3.2 to 7.0 X 105). In analysis of nuclear RNA, only parallel gradient markers were employed. We have found up to one fraction variations in determin— ing S values when using only parallel gradient markers. Consequently within the experimental limits of the method, the size of the three nuclear species was comparable to the three largest cytoplasmic RNA Species observed when internal markers were used (Figure 4A). Virus—Specific RNA from Cellular Polyribosomes: Polyribosomes from F—422 cells were purified by the Sepharose 2B exclusion method as described (10). A 20% to 45% (w/v) sucrose gradient analysis of poly— ribosomes isolated by this procedure compared to an analysis of a total cytoplasmic extract is shown in Figure 5. The Sepharose 2B excluded material consisted entirely of polyribosomes, almost devoid of ribosomal monomers and sloWer sedimenting material. This polyribosome fraction was extracted and analyzed for virus— specific RNA as described for the cytoplasmic extract RNA. The analysis is seen in Figure 4C. There were three distinct peaks of hybridizing material with values of 368, 238, and 188; molecular weight estimates of 2.8 X 106, 1.1 X 106, and 0.7 X 106, respectively. The two slower sedimenting peaks present here, are more discernable than in the gradi- ent of the cytoplasmic extract RNA. The same three species were obtained when Sepharose 2B excluded material was pelleted through 2.0 M_sucrose before extraction and DMSO gradient analysis (data not shown). It is notable that there was no peak of hybridizing material at a value of 288 in this polyribosomal RNA. “i 70 Figure 5. Sucrose gradient sedimentation analysis of Sepharose 2B excluded polyribosomes and unfractionated total cytoplasmic extract. Total polyribosomes were prepared as described in the text. Analysis was performed on 4.8 ml 20% to 45% (w/v) linear sucrose gradients in the SW50.l rotor, 40,000 rpm for 1.25 hr. (A) Sepharose ZB excluded polyribosomes. (B) Unfractionated cyto— plasmic extract. ___.__—_ ______————.— A254 '45 A Crude Cytoplasmic Extract L803 T: - ----- use 23 extract. rose .3 _ cyto- .2r .lr- _B_, Sepharose 28 Excluded Polyribosomes [808 .6 - .2 Figure 5 TTDP l—_ g ,l A254 A254 .£> Eirug Many aspect presumptive present hyp mRNA in euk with protei 54)- The n Processed a analysis of Virus-speci int° Possih nuclear pro The h isolated by Figure 4D. SPECies Wit mates of 2' The two Slc total nucle PreSEnt in fractions, Pared in 0: tions. Con indicate 8 these two f Show“ in F1 Virus-Specific RNA from Intranuclear Ribonucleoprotein Particles: Many aspects of the post—transcriptional processing and maturation of presumptive mRNA in eukaryotic cells remain to be elucidated. One present hypothesis is that some aspects of processing and transport of mRNA in eukaryotic cells involves an association of those RNA species with protein in complexes designated ribonucleoprotein particles (1,27, 54). The mRNA transcripts of the integrated oncornavirus DNA may be processed and transported by the same mechanisms as cellular mRNA. An analysis of nRNP particles for virus—specific RNA, could characterize virus—specific RNA processed in this manner and give further insight into possible viral mRNA species which arise by transcription and nuclear processing. The hybridization pattern of RNA extracted from nRNP particles isolated by the method of Quinlan et a1. (38) (Method II) is shown in Figure 4D. This fraction contained four discernable virus—specific RNA species with values of 343, 283, 228, and 18S; molecular weight esti- mates of 2.6 X 106, 1.8 X 106, 1.05 X 106, and 0.7 X 106, respectively. The two slower sedimenting peaks were more discernable than in the total nuclear fraction. Since the two slower sedimenting species were present in high proportions in nRNP particle RNA and polyribosome RNA fractions, pulse—labeled total RNA, from these two fractions was pre— pared in order to compare the mean size of total RNA in these two frac— tions. Comparable sizes of total RNA from the two fractions may indicate a similar relationship between the virus—specific RNAs of these two fractions. The DMSO gradient analyses of these fractions is shown in Figure 6. The nRNP particle RNA, labeled for ten minutes with 73 sis of rapidly labeled RNA Polyribo particles ient analy somes RNP particles. 3Heuridine and nRNP RNA extracted as 5 described in performed a text. th 3 parallel marker gradient. radients wi polyribosome m nRNP parti rallel g r 20 minutes Figure 4 ) RNA from s labeled fo cles labeled for 10 symbols: (l——«O) RNA fro 3H CP M x -3 '(:> F>(:)L_‘1l 2.0 - 0.5 - 0.5 . mu. it. idicate :oplasmic 3.2 ug edin by RELATIVE VIRUS-SPECIFIC RNA 2.0 'O .0 01 .N '0: 0.5 _A_. Cytoplasmic - 28$ l8$ -- 28S _B_. Polysomal IB'S i U l l l l Q Nuclear RNP Method I 285 l8$ .- 9, Nuclear RNP Method II -- 28$ l8 - 5 no l5 ab 25 5 I0 1522) 25 FRACTION NUMBER Figure 7 Pol methods it particles and Peder. had sedim from part. (Figure 71 and 188. were also l lated by 1 for the 3: to 235, a1 78 Poly(A) containing RNA from nRNP particles isolated by both methods was analyzed. Virus-specific poly(A) containing RNA from nRNP particles isolated by the methods of Bhorjee and Pederson (2) and Kish and Pederson (26) (Method I), is shown in Figure 7C. The RNA present had sedimentation values of 358, 28S, 23S, and 17S. The poly(A) + RNA from particles isolated by the method of Quinlan et a1. (38) Method II (Figure 7D), containee major species with sedimentation values of 285 and 185. In this fraction a 33 to 35$ shoulder and a small 228 peak were also present. The molecular weight estimates for the species iso- lated by both methods were similar and were estimated at 2.5 to 2.8 X 106 for the 33 to 353, 1.8 X 106 for the 283, 1.0 to 1.1 X 106 for the 22 to 23S, and 0.6 to 0.7 X 106 for the 17 to 18S virus-specific RNA. In 1 DNA to Stl of this a; transcript describe 1 The major: ing bound been notec 0f the ter Product In: dalton mal than 283 1 molecular The us to makl analYsis a all FELV~1 this qual: Should be Sequences 79 DISCUSSION In this study we have used complementary single stranded viral DNA to study intracellular FeLV specific RNA. The major qualifications of this approach concern the uniformity and completeness of the transcript. The combination of analyses shown in Figure l and Figure 2 describe the FeLV DNA probe and indicate the level of transcription. The majority of the native DNA transcript migrates as material remain— ing bound to the high molecular weight RNA template. This result has been noted by others and is assumed to represent a faithful transcript of the template (22). As seen in Figure 1B, most of the denatured product migrates as small molecules. The small amount of 2.0 X 106 dalton material present is still approximately 1.2 X 106 daltons smaller than 288 FeLV—R genomic subunit RNA, which migrates with an estimated molecular weight of 3.2 X 106 in 2% polyacrylamide—O.5% agarose gels (6). The template protection experiment described in Figure 2 allows us to make an evaluation of the quality of the FeLV—R DNA probe. This analysis and indirectly the electrophoretic analysis suggests that not all FeLV—R RNA sequences are present in the DNA probe. DNA probes of this quality can be used in analyzing intracellular RNA (11), but it should be emphasized that certain intracellular virus—specific RNA sequences may not be detected. Recent have been 01 used here a1 (40), high I Since this ‘ form transc: (41). The v1 F-422 cytop Moloney nut to 0.9% (11 cells, 0.27 true for th ribosomes. Rauscher mu from approx oncornaviru Specific RN (18). Alth ffaction Wa WOuld be ex tiou of Vir fuIICtion as The c within nan 80 Recently, larger and more uniform transcripts of oncornavirus RNA have been obtained (8,24,40). Even though the conditions of synthesis used here are similar to those described by Rothenberg and Baltimore (40), high uniformity and complete transcription was not obtained. Since this work was completed, published results suggest that more uni- form transcription occurs under conditions of restricted magnesium ion (41). The value of approximately 2% FeLV—R-specific RNA in unfractionated F—422 cytoplasmic extracts was higher than the values reported for Moloney murine leukemia virus (M—MuLV) infected cells, 0.69% (52), 0.3 to 0.9% (11), 22% (12), 1.0 to 1.3% (23), or Rous sarcoma virus infected cells, 0.27% (43), 0.25 to 0.6% (28) and 0.2 to 2.0% (4). The same was true for the value of 2.63% FeLV—R-specific RNA from total F—422 poly- ribosomes. The virus-specific polyribosomal RNA levels reported for Rauscher murine leukemia virus (R—MuLV) (15) and M—MuLV (12.52) range from approximately 0.05% to 0.58%. The F-422 line is one of the higher oncornavirus producing cell lines and the higher levels of FeLV—R— specific RNA may be related to the apparently high viral synthetic rate (18). Although the amount of viIUS*specific RNA in the nRNP particle fraction was found to be less than the other fractions, this lower value would be expected if nRNP particle RNA contained a transient subpopula- tion of viral RNA molecules, which could be undergoing processing for function as mRNA. The consistent appearance of very reproducible sized species, within narrow ranges, allows us to use the average RNA size designations 363, 288, in subsequ The nuclear (F is compare ship is $1 51). Howe FeLV-R-spe This phenc M-MuLV ini the large: genomic m sidered a longer la tion in o the 363 s cular wei subunit. Whe denaturir found (1] PrecurSO] unfracti (11,50,5 Although 81 363, 288, 238 and 188 to identify the intracellular FeLV-R—specific RNA in subsequent discussions. The size of the major species of virus—specific RNA in total nuclear (Figure 4B) and unfractionated cytoplasmic extracts (Figure 4A) is comparable in size to the FeLV-R genomic subunit RNA. This relation- ship is similar to intracellular R—MuLV, M—MuLV, and RSV RNA (11,23,50, 51). However, both the nuclear and cytoplasmic fractions contain FeLV—R—specific RNA which sediments faster than the genomic subunit. This phenomenon has been reported for nuclear virus—specific RNA from M-MuLV infected cells (23) and RSV infected cells (4). In these cases the largest nuclear species is not more than 15% larger than the 353 genomic subunit. The largest intranuclear RSV—specific RNA is con— sidered a precursor to the genomic subunit since it is not detected in longer labeling periods and steady state RNA (4). Although the resolu— tion in our experiments does not permit an exact size determination of the 368 species found in all subcellular fractions, its estimated mole— cular Weight is approximately 50 to 70% larger than that of the genomic subunit. When cytoplasmic virus—specific RNA is analyzed under non— denaturing conditions, larger than genomic subunit species have been found (11,28). These species are considered cytoplasmic genomic RNA precursors. The presence of smaller than genomic subunit size RNA in unfractionated cytoplasm has been reported in M—MuLV infected cells (11,50,51), RSV infected cells (28,43), and RD—114 cells (33). Although the sizes of these smaller molecules vary among the virus systems at The unfrac ‘ amount of l containing . species is fraction t P01) 185) disti RNA was 1'1: ‘1 genomic St I. present it (Figure 7] l P01yribos< ' the same I ' RNA Specit \ Gielkens 1 ! showed tlu cell Pely: p°1YI-‘ibos. sP-dilneuti: l RNA» Whil f°r the 3 0“ denatu 213 and 3 (16), Th 82 systems studied, their presence appears to be a general phenomenon. The unfractionated F—422 cytoplasm (Figure 4A) contained a minimal amount of the smaller FeLV—R-specific species. However, when poly(A) containing RNA from this fraction is analyzed (Figure 7A), a 238 species is resolved, probably due to its relative enrichment in the fraction by oligo(dT) cellulose chromatography. Polyribosomal RNA from F—422 cells contains three (368, 233, and 188) distinct species (Figure 4C). It is important to note that 288 RNA was not detected in this fraction suggesting that the FeLV—R genomic subunit may not be present on polyribosomes. Three species are present in the polyribosomal poly(A) containing virus—specific RNA (Figure 7B), with approximately the same sedimentation values as total polyribosomal virus—specific RNA (Figure 4C) and probably represent the same molecules. The detection of three distinct FeLV—R—specific RNA species on F—422 polysomes is in part similar to the findings of Gielkens et al. (15) for R—MuLV polyribosomal RNA. Their analysis showed the presence of 368, 215 and 148 virus—specific RNA from infected cell polyribosomes, with the species being unequally distributed on polyribosomes. Free polyribosomes contained mostly 36S RNA, slower sedimenting membrane—bound polysomes contained the smaller 218 and 148 RNA, while the faster sedimenting membrane bound polysomes were enriched for the 36S RNA (15). These same Species were also found by analysis on denaturing, 85% formamide, gradients (16). They further showed that 218 and 365 RNA were the major poly(A) containing intracellular species (16). The 368 R—MuLV polyribosomal RNA is the same size as R—MuLV subunit RN same size Two mixing exp the isolat (2) and Ki during Met cedures. Method I, The from seve processin A w A ,__d— formed by l no eviden : by transc . analysis does indj Cleavate. evoked u poll’ribo arise by under st nucleus. A could re 83 subunit RNA; whereas, the FeLV-R 368 polyribosomal species is not the same size as the FeLV-R subunit RNA. Two methods were used to obtain nRNP particles. From control mixing experiments we found that purified viral RNA is cleaved during the isolation of nRNP particles by the method of Bhorjee and Pederson (2) and Kish and Pederson (26), Method I. Cleavage was not observed during Method II (38) isolation or any of the other isolation pro— cedures. Consequently, the integrity of nRNP particle RNA isolated by Method I, is suspect (Figure 7C). The presence of virus-specific RNA in nRNP particles is important from several aspects. Primary among these is that the transport and processing of the various virus-specific RNA size species could be per— formed by an apparently normal cellular mechanism. We presently have no evidence to eliminate the possibility that the smaller species arise by transcription separate from that of the larger species since our analysis was under steady state conditions. However, their presence does indicate a nuclear site of generation and a post—translational cleavate—recycling model as proposed for M—MuLV RNA (32), need not be evoked to account for the presence of all the smaller than subunit size polyribosomal virus—specific molecules. If the smaller species did arise by cleavage during translation, their presence in the nucleus under steady state conditions would require return transport to the nucleus. A steady state level of RNA smaller than genomic subunit size could reflect a transcriptional or nuclear post-transcriptional control mechanism gene prodt ribosomes, two separa (29,30) h: in the pr< progeny v: (T% 1.5-2 specific mRNA. Th precursor 1 l ‘ and uncle ( species. I The 1 FeLV—R in ‘ FeLV-R RN ‘ laborato: ‘ infection fibroblag polymer,E rEsults). found int F‘422 ceI Virions' prEParat' 84 mechanism for the production of non-equimolar amounts of oncornaviral gene products. These smaller RNA species, in nRNP particles and poly- ribosomes, may further indicate a physical basis for the possibility of two separate pools of oncornaviral RNA transcripts. Levin and colleagues (29,30) have found that gg_novo synthesis of M—MuLV viral protein occurs in the presence of actinomycin D, with the expected absence of new progeny virion RNA. Functionally, the half life of M4MuLV messenger RNA (T3 1.5—2 hr) (30). It could be possible that the smaller virus- specific RNA species represent the functionally stable virus—specific mRNA. The data presented here shows that some possible species of viral precursor mRNA may exist in the nuclei as smaller than subunit size RNA, and nuclear fractionation greatly increases the ability to find these species. The analyses presented here and other preliminary data concerning FeLV—R in F-422 cells, lead to some interesting observations about FeLV—R RNA metabolism in F-422 cells. Preliminary observations in this laboratory indicate that FeLV—R from F-422 cells is 2.5 logs less infectious than virus from the original FeLV—R isolate passed in feline fibroblasts, based on endpoint dilution assays of supernatant DNA polymerase activity on FLF-3 cells (Haberman and Velicer, unpublished results). Since the genomic subunit size RNA is not the largest species found intracellularly, the majority of progeny virions produced by F—422 cells may be defective in the RNA molecules incorporated into virions. Further, we have preliminary evidence that FeLV—R 50—608 RNA preparations contain a minor subunit of approximately 368 as well as the major approximal generated cleaved f larger pr which 312 If‘ specific, combined somes and 2.5 X 106 value of in all th the small 0f 34—365 porated i needed fc (a 17.55 seParate the 36s I as is trt need to I SPECies ,- intracel: 85 the major 28S subunit (Conley and Velicer, unpublished results). The approximately 368 molecule may simply contain host cell sequences generated by transcriptional readthrough, and the extra sequences are cleaved from most virus—specific molecules as is the case for the larger precursor of the 358 RSV RNA (4). However, we do not yet know which.size molecule contains the entire genomic capacity of FeLV—R.‘ If the sequences present in 36S RNA but not in 28S RNA are virus- specific, some interesting processing schemes could be devised. The combined estimated molecular weights of the 188 RNA found on polyribo— somes and in nRNP particles, and the 28S genomic subunit RNA, is 2.5 X 106. This corresponds to molecules of approximate sedimentation value of 348 which is of similar size to the largest species observed in all the subcellular fractions. It is tempting to speculate that the smallest polyribosomal species (188) is a nuclear cleavage product of 34—36S RNA, with most of the remaining RNA being eventually incor— porated into virions as a 288 species. The minimal amount of RNA needed for FeLV-R structural protein precursor Pp70 (35) is 6.3 X 105 (a 17.58 molecule). The 188 species is compatible to that needed for separate biosynthesis of structural protein precursor. In this model the 36S RNA could still function as structural protein precursor mRNA as is true of M—MuLV 36S RNA (32). However, this speculation would need to be proven by cell-free translation of individual intracellular species and by hybridization~competition experiments with the various intracellular RNA species. We Thi CA-lZlOl Public He Research Institute 86 ACKNOWLEDGMENTS We thank Alice Swanson for excellent technical assistance. This research was supported by Public Health Service grant CA212101 from the National Cancer Institute. A. J. C. was supported by Public Health Service grant CA—12101. L. F. V. is a recipient of Research Career Development Award CA-70808 from the National Cancer Institute. AW 4 ”a _ 4A H-,—~,—. l. Augenli< lab4 J.l 2. Bhorjee cul tio 3. Birnsti ple 4. Bishop, and by and mo] Nev 5. Bondura pat ‘1. 6| Brian, 87 LITERATURE CITED Augenlicht, L. H. and M. Lipkin. 1976. Appearance of rapidly labeled, high molecular weight RNA in nuclear ribonucleoprotein. 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Tsuchida, N., M. S. Robin, and M. Green. 1972. in cells transformed by RNA tumor viruses. Viral RNA subunits Science 176:1418— 1420. 52. Vecchio, G., N. Tsuchida, G. Shanmugam, and M. Green. 1973. Virus- specific messenger RNA and nascent polypeptides in polyribosomes of cells replicating murine sarcoma—leukemia viruses. Proc. Natl. Acad. Sci. U.S.A. 7_O:2064-2068. 54. Will: 92 53. Wang, L. H., P. Duesberg, K. Beemon, and P. Vogt. 1975. Mapping RNase Tl-resistant oligonucleotides of avian tumor virus RNAs: Sarcoma-specific oilgonucleotides are near the poly(A) end and oligonucleotides common to sarcoma and transformation-defective viruses are at the poly(A) end. J. Virol. ;§:1051-1o70. 54. Williamson, R. 1973. The protein moieties of animal messenger ribonucleoproteins. F.E.B.S. Letters. 21:1—6. ARTICLE II ANALYSIS OF POLYRIBOSOMES FROM CELLS INFECTED WITH FELINE LEUKEMIA VIRUS Anthony J. Conley and Leland F. Velicer J. Virol. (Submitted) 1977 93 ANALYSIS OF POLYRIBOSOMES FROM CELLS INFECTED WITH FELINE LEUKEMIA VIRUS Anthony J. Conley and Leland F. Velicer Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 94 -,__.__.._—_ _’/__— P.— * Pol cell line leukemia ‘ sized FeL‘ by bindin; (NR5) IgG of the gr; somes (pe was furth‘ Specific ' POeribos specific . absorbed The bindi‘ reduced b The (approxim (approxim hybridize two Polyr Virus-ape Sedimenta to 4503 p 95 ABSTRACT Polyribosomes from a chronically infected feline thymus tumor cell line, F-422, were analyzed by using antisera specific for feline leukemia virus (Rickard strain) (FeLV-R) proteins and ig_vi££g_synthe— sized FeLV-R DNA probe. Virus-specific nascent proteins were detected by binding 125I-anti—FeLV IgG to polyribosomes. Normal rabbit serum (NRS) IgG bound at a level of 0.02% throughout the polyribosome region of the gradient. Anti—FeLV IgG bound to rapidly sedimenting polyribo- somes (peak binding at 4008) at a level of 0.25 to 0.40%. This binding was further studied to determine its specificity for nascent virus— specific protein. NRS IgG did not compete with anti-FeLV IgG for the polyribosomal binding sites. Total viral protein and p30 absorbed specific antibody from the IgG preparation and the binding of the absorbed IgG was reduced in relation to the amount of protein used. The binding of anti-FeLV IgG to puromycin treated polyribosomes was reduced by the same proportion that nascent proteins were released. The FeLV—R DNA probe hybridized to two polyribosomal regions (approximately 400 to 4505 and 2508) and to a slower sedimenting region (approximately 808) within the polyribosomal gradients. The DNA still hybridized to RNA in slower sedimenting regions (<808) but not in the two polyribosome regions after EDTA treatment. The size classes of virus-specific RNA within these regions were determined by velocity sedimentation in the presence of 99% dimethylsulfoxide (DMSO). The 400 to 4503 polyribosomes contained three major peaks at 338, 228, and 175; whereas, from the want con RNA. Th which al are thre tumor ce 96 whereas, the 2508 polyribosomes contained only 348 and 183 RNA. RNA from the approximately 808 regions obtained with and without EDTA treat— ment contained 288 in addition to the 348, 228, and 188 virus-specific RNA. The presence of 348, 228, and 188 RNA within polyribosome regions which also contain nascent virus-specific proteins suggests that there are three species of FeLV—R-specific mRNA in the F-422 feline thymus tumor cell line. P.— ll examine: molecul: structu1 murine‘ tion of 200,000 the one The strw (FeLV—R Precurs the siz infecte I sPonds CEllula the int Since 5 35). an sis of hYPOthe of Smal ability 97 INTRODUCTION The synthesis of oncornavirus proteins has been extensively examined in recent years. By using immunological techniques, high molecular weight intracellular precursors have been detected for the structural proteins of avian myeloblastosis virus (40,41) and various murine oncornaviruses (1,19,33,38). Further, the immunological detec- tion of extremely large intracellular virus-specific proteins of 200,000 to 300,000 daltons (1,38) suggests the possibility that all the oncornavirus proteins may be synthesized as a single precursor. The structural proteins of the Rickard strain of feline leukemia virus (FeLV-R) are generated by cleavage of an intracellular 70,000 dalton precursor (26). These observations raise further questions concerning the size and number of possible virus—specific mRNA(s) in oncornavirus infected cells. In most oncornavirus infected cells a 30 to 40$ RNA, which corre- sponds in size and polarity to the viral subunit, is the major intra- cellular virus—specific species (12,32,37). It has been suggested that the intracellular viral genomic subunits function as viral mRNA (12). Since species of subunit size are present on polyribosomes (2,8,12,13,32, 35), and since RNA from these various oncornaviruses directs the synthe- sis of virus~specific products ig_!i££g (16,20,24,29,30,42), this hypothesis is probably correct. However, the presence in polyribosomes of smaller than subunit size virus—specific RNA (2,8,15,16,35), and the ability of these smaller species to direct the synthesis of j { virus-s ‘ F [ contain [ specifi plasmic ‘ polyrib . shown m tion to T1 the cell 1 A using b< ‘ These p( virus—SI A arations J ribosome 1 Sized Db \ Protein RNA pres virus—sF Most of of the r East Lan This pap RNA Tumo 98 virus-specific products in vitro (16) also suggests an mRNA function. FeLV—R genomic subunits sediment as 288 RNA in sucrose gradients containing 99% dimethylsulfoxide (DMSO) (5,9). Although a virus— specific species of this size is present intranuclearly and intracyto— plasmicly, a 288 virus—specific species could not be detected in total polyribosomes from FeLV—R infected cells (6). These polyribosomes were shown to contain an approximately 345 virus—specific species in addi— tion to 238 and 188 FeLV—R specific RNA. To further understand the mode of oncornavirus RNA translation, the cellular polyribosomes from FeLV—R infected cells were examined by using both immunological and nucleic acid hybridization techniques. These polyribosomes were analyzed to (1) determine whether nascent virus—specific determinants could be detected by using antibody prep— arations specific for FeLV—R proteins, (ii) detect the size of poly— ribosomes containing virus—specific RNA by using an in_vi££g_synthe— sized DNA probe, (iii) correlate the detection of virus-specific nascent protein and RNA, and (iv) determine the size classes of virus-specific RNA present within the major size class of polyribosomes containing virus—specific RNA and nascent proteins. Most of this work was submitted by A. J. Conley in partial fulfillment of the requirements for the Ph.D. degree, Michigan State University, East Lansing, 1977. This paper was presented in part at the Cold Spring Harbor meeting on RNA Tumor Viruses, May 25—29, 1977, Cold Spring Harbor, New York. _— ”A 931 cell line virus (Fe suspensic Egg tion in 1 Nuclear Cells la bation 1 mixture/ After on cells to Prepared Q5 F‘422 ce disrupt. (RSB) , 4 Plasmic 0-22 so heParin X 8 for tograph Centair used in 99 MATERIALS AND METHODS Cells and Virus: The chronically infected feline thymys tumor cell line (F-422) which produces the Rickard strain of feline leukemia virus (FeLV-R), was used as the source of cells. This line, grown in suspension culture, was propagated as previously described (14). Isotopic Labeling: Cells were labeled with l4C—uridine by incuba- tion in fresh medium containing 0.5 uCi 14C—uridine/ml (New England Nuclear Corp., 53 mCi/mmole) at a cell density of 2 X 106/ml for 4 hr. Cells labeled for one minute with 3H—amino acids were obtained by incu— bation in amino acid deficient medium containing 25 mCi 3H-amino acid mixture/ml (New England Nuclear Corp.) at a cell density of 50 X 106/ml. After one minute at 37°C the pulse was terminated by addition of the cells to partially frozen medium. l4C-amino acid labeled virus was prepared as described (14). Cell Fractionation: Polyribosomes were prepared from harvested F-422 cells by the method described (6). Briefly, washed cells were disrupted by dounce homogenization after swelling in hypotonic buffer (RSB), 0.01 M Tris HCl (pH 7.4), 0.01 M_NaCl, 1.5 mM MgClz. A cyto— plasmic extract was prepared and solubilized with (final concentrations) 0.2% sodium deoxycholate (w/v) and 0.2% Nonidet P-40) (w/v). Sodium heparin was added to 50 Ug/ml and the extract was centrifuged at 27,000 X g for 5 min in the $834 rotor (Sorvall). The supernatant was chroma— tographed On a column of Sepharose ZB (10). The excluded fractions containing polyribosomes were pooled, made 50 ug/ml sodium heparin and used immediately in experiments. “”1 g rupted) chromatt (NRS) WI II) was York). { 40% satl A phosphal \ on a col ‘ (IgG) f1 ( E Helmkam‘ was modi S (New Eng tion. 1 ‘ several 1 1.4 cm, g et al. ‘ albumin g; the labs ‘0 Purii of Poly: incubate 100 Preparation of Antisera: Rabbit anti—FeLV (Tween 80-ether dis— rupted), rabbit anti-FeLV p30 (prepared with guanidine HCl-agarose chromatography purified p30, designated-I), and normal rabbit serum (NRS) were prepared as described (14). Goat anti—FeLV p30 (designated- II) was obtained from F. de Noronha (Cornell University, Ithaca, New York). All sera were subjected to two successive precipitations with 40% saturated ammonium sulfate followed by extensive dialysis against phosphate buffered saline (PBS). These proteins were chromatographed on a column of Sephadex G-200 equilibrated with PBS. The gamma globulin (IgG) fractions were pooled and concentrated by diafiltration. Radioiodination of Proteins: The iodine monochloride method of Helmkamp et al. (18) was used for 1251 labeling of IgG. The procedure was modified for use in PBS and was performed at a level of 50 uCi 1251 (New England Nuclear Corp., carrier free—l7 Ci/mg) per iodination reac- tion. After iodination the proteins were extensively dialyzed against several changes of PBS. Specific activities ranged from 1.5 to 2.4 X 104 cpm/ug protein. Determination of Protein Concentrations: The method of Lowry et al. (21), was used to determine protein concentrations. Bovine serum albumin was used as the standard. Binding of 125I—IgG Preparations to Polypibosomes: Binding of the labeled IgGs was performed by direct addition of the preparations 25 to purified polyribosomes (generally 5 to 8 pg 1 I—IgG per A260 unit of polyribosomes). For direct binding experiments the mixtures were incubated at 40C for 45 min. A NRS 125I-Ingpolyribosome and an _. ,_——~i . unlabel polyril with as g 20% to at 40,C fractic eter (P g Blobel nascent from ce (final cation I—Ig for 15 incubat Puromyc A amounts “8) War for 30 1 r01:01: (; mixture lOl unlabeled NRS IgG:polyribosome mixture (control for degradation of polyribosomes under the conditions of incubation and analysis) were run withreach.analysis. Gradient analyses of Ingpolyribosome mixtures were performed on 20% to 45% (w/v) sucrose gradients in RSB. Gradients were centrifuged at 40,000 rpm for 1.25 hr at 4°C in the SW 50.1 rotor (Beckman). Equal fractions were collected and counted directly in an autogamma spectrom— eter (Packard). Puromycin Treatment of Polyribosomes: The method described by Blobel and Sabatini (3) was modified to use for partial release of nascent proteins without polyribosomal disaggregation. Polyribosomes from cells labeled for one min with 3H—amino acids, were incubated with (final concentrations) 10 mM puromycin and 50 m! KCl-total monovalent cation concentration 60 mM, at 0°C for 45 min. For the binding of 12SI—IgG under these conditions, unlabeled polyribosomes were incubated for 15 min in the mixture above, followed by addition of 125I-IgG and incubation for the additional 30 min. The mixture which did not contain puromycin was incubated only with KCl for the first 15 min. Absorption of 125I—anti FeLV IgG with Viral Proteins: Varying amounts of soluble FeLV proteins (0.4 to 32 pg) or FeLV p30 (1.0 to 50 ug) were incubated with a constant amount of 125I—anti FeLV IgG at 37°C for 30 min. The mixtures were centrifuged at 5,000 rpm in the $834 rotor (Sorvall) for 15 min. A constant amount of supernatant from each mixture was used in the binding experiments with polyribosomes. p fractim cedure ; p synthes: prepare gradiem hybridi: NaCl, 0. yeast R] Hybrid 1 102 Preparation of Intracellular RNA: RNA from polyribosomal gradient fractions was collected by ethanol precipitation in the presence of 0.2 M NaCl and 50 ug/ml carrier yeast RNA. RNA was extracted from total polyribosomes and from pooled polyribosomal gradient fractions by the TNE-9 (0.1 M_NaCl, 1 mM EDTA, 0.1 M_Tris HCl, pH 9.0) SDS—phenol pro- cedure as described (5). FeLV DNA and DNA-RNA Hybridization: Single stranded FeLV 3H—DNA, synthesized in the endogenous RNA directed DNA polymerase reaction was prepared as described (6). Hybridization of this DNA probe to RNA from gradient fractions was performed as described (6). Briefly, the hybridization conditions were 66°C in 0.01 Tris HCl (pH 7.2), 0.421 NaCl, 0.05% SDS, 0.025 mM EDTA, 0.075 mg/ml calf thymus DNA, 0.15 mg/ml yeast RNA, sample RNA, and FeLV 3H—DNA (generally 1000—1500 cpm). Hybrid formation was determined by digestion with $1 nuclease. p ments w protein these e p30 IgG tograph Also in The NRS througt bound c anti-pf approx: levell i i highes1 ‘ Peak b; ‘ somes i by the I greate was us 0f pol The bi 103 RESULTS Binding of 1251-136 to Purifigd Polyribosomes: Initial experi— ments were performed to determine if IgG preparations specific for viral proteins would bind to nascent virus—specific proteins. The results of these experiments are seen in Figure 1. Binding of rabbit anti-FeLV p30 IgG (prepared with p30 purified by guanidine HCl—agarose chroma- tography, designated anti-p30 (I) and NRS IgG are seen in Figure 1A. Also included in Figure 1A is an absorbance profile of the polyribosomes. The NRS IgG binding was at a level of approximately 0.02 to 0.03% throughout the polyribosome region of the gradient. The anti-p30 (I) bound only slightly higher, with values of 0.03 to 0.04%. In comparison, anti—p30 (II), Figure 1B, bound to polyribosomes with higher values of approximately 0.1 to 0.15%. In contrast, anti—FeLV IgG bound at a level of 0.25 to 0.32% in the polyribosome region. The binding was highest in the regions containing the fastest sedimenting polyribosomes. Peak binding in this gradient (Figure 10) was equivalent to polyribo— somes of sedimentation coefficient of approximately 4005, as calculated by the method of McEwen (22). Since this level was significantly greater than the levels obtained with the other IgGs, the anti—FeLV IgG was used in further experiments. The specificity of binding was further examined by preincubation of polyribosomes with an excess of unlabeled NRS IgG, followed by incubation with 125I—anti FeLV. The results are seen in Figure 2. The binding of anti—FeLV to polyribosomes preincubated with NRS IgG was Figure l. Sedimentation analysis of 125I-IgG:polyribosome mixtures. F-422 polyribosomes purified by sepharose 2B chromatography were incubated with the 125I-IgG preparation indicated in each panel and analyzed as described in the text. All analyses were per— formed in parallel gradients. Each fraction of the gradient was plotted as the percent of the total 12sI—IgG. (A) (O—-—O) rabbit anti—p30 I, (0--0) normal rabbit serum (NRS) IgG, ( ) A254 polyribosomes; (B) (0r-C) goat anti-p30 II, (O———O) NRS; (C) (O—-—0) total rabbit anti-FeLV, (0---0) NRS. , p ,4 4.. some mixtures. raphy were Lch panel re per- :lient was i) rabbit -) A254 total 5 (C) (H) I-IgG BOUND % l25 A, Anti-p30 (I) 0.4- 808 Absorbonce of i 0.3, total polyribosomes 0.2 O.l ——-——---_-0_. .0 A I 9 u ‘\ ‘0.--..- ~- ‘& . O.| gAnII-FeLv I I I I I l I I l I i I l' ,6 - {rd NRS ad 0 l W l 0.2 0.4 0.6 0.8 L0 Top FRACTION OF GRADIENT Figure l ‘I‘ll‘lxl‘ llAl‘ . 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