Identification of a Packaged Cellular mRNA in Virions of Rous Sarcoma Virus

A novel messenger activity has been identified by in vitro translation of the 70S virion RNAs of a variety of avian leukosis and avian sarcoma viruses. When the 70S virion RNA complex was heat dissociated and the polyadenylated RNA was fractionated on neutral sucrose gradients, a polypeptide of 34,000 daltons (34K) was observed in the translation products of 18S polyadenylic acid-containing virion RNA. Aside from the p60(src)-related subgenomic messenger activities, this was the only prominent messenger activity that sedimented at <20S. It was determined that the 34K protein was not virally coded because (i) messenger activity for the 34K protein was not generated by mild alkaline hydrolysis of 35S genomic RNA, (ii) the 34K proteins synthesized in response to different virion RNAs had identical tryptic peptide maps, and (iii) the tryptic peptide map of the 34K protein coded for by virion RNA was identical to that of a major in vitro translation product of 34,000 daltons made from 18S uninfected chick cell polyadenylated RNA. The 18S RNA was shown to be contained within virion particles, rather than part of a cellular structure copurifying with virus preparations, by demonstrating the presence of 34K messenger activity in virion cores made from detergent-disrupted virus. This cellular mRNA, however, was not observed in the virion RNAs of Rous-associated virus types 0 and 2 avian leukosis viruses and therefore is not packaged by all avian retroviruses. Since no other cellular message has been detected by this assay, it seems likely that the 34K mRNA found in 70S virion RNA is the result of selective packaging of an abundant host cell mRNA by certain avian retroviruses.     

Relationship Between Moloney Murine Leukemia and Sarcoma Virus RNAs: Purification and Hybridization Map of Complementary DNAs from Defined Regions of Moloney Murine Sarcoma Virus 124

Complementary DNAs (cDNA's) specific for various regions of the Moloney murine sarcoma virus (MSV) 124 RNA genome were prepared by cross-hybridization techniques. A cDNA specific for the first 1,000 nucleotides adjacent to the RNA 3' end (cDNA 3') was prepared and shown to also be complementary to the 3'-terminal 1,000 nucleotides of a related Moloney murine leukemia virus (MLV) genome. A cDNA complementary to the "MSV-specific" portion of the MSV 124 genome was prepared. This cDNA was shown not to anneal to Moloney MLV RNA and to anneal to a portion of the viral RNA of about 1,500 to 1,800 nucleotides in length, located 1,000 nucleotides from the 3' end of MSV RNA. A cDNA common to the genome of MSV and MLV was also obtained and shown to anneal to the 5'-terminal two-thirds, as well as to the 3'-terminal 1,000 nucleotides, of the MSV RNA genome. This cDNA also annealed to the RNA from MLV and mainly to the 5'-terminal half of the MLV genome. It is concluded that the 6-kilobase Moloney MSV 124 RNA genome has a sequence arrangement that includes (i) a 3' portion of about 1,000 nucleotides, which is also present at the 3' terminus of MLV; (ii) an MSV-specific region, not shared with MLV, which extends between 1,000 and 2,500 nucleotides from the 3' terminus; and (iii) a second "common" region, again shared with MLV, which extends from 2,500 nucleotides to the 5' terminus. This second common region appears to be located in the 5' half of the 10-kilobase MLV genome as well. Experiments in which a large excess of cold MLV cDNA was annealed to (3)H-labeled polyadenylic acid-containing fragments of MSV RNA gave results consistent with this arrangement of the MSV genome.     

Identification of proteins encoded by the Gazdar murine sarcoma virus genome by in vitro translation and comparison with Moloney murine sarcoma virus 124.

The gene products of Gazdar murine sarcoma virus (Gz-MuSV) were identified by in vitro translation of Gz-MuSV virion RNA. An overlapping set of proteins with approximate molecular weights of 37,000 (37K), 33K, 24K, and 18K were synthesized from the transforming gene of Gz-MuSV, v-mosGz. In addition, Gz-MuSV-specific RNA directed the in vitro synthesis of a 62K gag gene protein and a 37.5K env gene-related product. The Gz-MuSV-specific in vitro translation products were compared with the in vitro translation products of M-MuSV 124, an independent isolate with a similar v-mos gene. This analysis showed that the 62K Gz-MuSV gag gene protein and the 37K, 33K, 24K, and 18K v-mosGz proteins were almost identical to the M-MuSV 124 62K (gag) and 37K, 33K, 24K, and 18K (v-mosMo) proteins that we previously identified and characterized. The 37.5K env gene product from Gz-MuSV does not have a correlate in the M-MuSV 124 translation products. These results were analyzed in the context of expectations based on similarities and differences in genetic organization of these two viral genomes.     

Isolation of a Transformation-Defective Deletion Mutant of Moloney Murine Sarcoma Virus

A transformation-defective (td) deletion mutant of Moloney murine sarcoma virus (td Mo-MSV) and a transforming component termed Mo-MSV 3 were cloned from a stock of clone 3 Mo-MSV. To define the defect of the transforming function, the RNA of td Mo-MSV was compared with those of Mo-MSV 3 and of another transforming variant termed Mo-MSV 124 and with helper Moloney murine leukemia virus (Mo-MuLV). The RNA monomers of td Mo-MSV and Mo-MSV 3 comigrated on polyacrylamide gels and were estimated to be 4.8 kilobases (kb) in length. In agreement with previous analyses, the RNA of Mo-MSV 124 measured 5.5 kb and that of Mo-MuLV measured 8.5 kb. The interrelationships among the viral RNAs were studied by fingerprinting and mapping of RNase T₁-resistant oligonucleotides (T₁-oligonucleotides) and by identification of T₁-oligonucleotides present in hybrids formed by a given viral RNA with cDNA's made from another virus. The nontransforming td Mo-MSV RNA lacked most of the Mo-MSV-specific sequence, i.e., the four 3′-proximal T₁-oligonucleotides of the six T₁-oligonucleotides that are shared by the Mo-MSV-specific sequences of Mo-MSV 3 and Mo-MSV 124. The remaining two Mo-MSV-specific oligonucleotides identified td Mo-MSV as a deletion mutant of MSV rather than a deletion mutant of Mo-MuLV. td Mo-MSV and Mo-MSV 124 exhibited similar deletions of gag, pol, and env sequences which were less extensive than those of Mo-MSV 3. Hence, td Mo-MSV is not simply a deletion mutant of Mo-MSV 3. In addition to their MSV-specific sequences, all three MSV variants, including td Mo-MSV, shared the terminal sequences probably encoding the proviral long terminal repeat, which differed from their counterpart in Mo-MuLV. This may indirectly contribute to the oncogenic potential of MSV. A comparison of td Mo-MSV sequences with either Mo-MSV 124 or Mo-MSV 3 indicated directly, in a fashion similar to the deletion analyses which defined the src gene of avian sarcoma viruses, that Mo-MuLV-unrelated sequences of Mo-MSV are necessary for transformation. A definition of transformation-specific sequences of Mo-MSV by deletion analysis confirmed and extended previous analyses which have identified Mo-MuLV-unrelated sequences in Mo-MSV RNA and other studies which have described transformation of mouse 3T3 fibroblasts upon transfection with DNAs containing the Mo-MSV-specific sequence.     

Biological and Serological Properties of Viral Particles from a Nonproducer Rat Neoplasm Induced by Murine Sarcoma Virus (Moloney)

The viral particles present in a nonproducer rat neoplasm induced by murine sarcoma virus (MSV) Moloney isolate, as detected by electron microscopy, were found to be biologically active on normal kidney cells of random-bred Osborne-Mendel rats. The virus is designated here as MSV (0). MSV (0) differs from other pseudotypes of MSV in its host range, antigenicity, and interference pattern.     

Rescue of Murine Sarcoma Virus from a Sarcoma-Positive Leukemia-Negative Cell Line: Requirement for Replicating Leukemia Virus

The nature of murine sarcoma virus (MSV) "defectiveness" was investigated by employing an MSV-transformed mouse 3T3 cell line which releases noninfectious virus-like particles. Rescue kinetics of MSV, observed after murine leukemia virus (MuLV) superinfection of these "sarcoma-positive leukemia-negative (S + L -)" mouse 3T3 cells, consisted of a 9- to 12-hr eclipse period followed by simultaneous release of both MSV and MuLV with no evidence for release of infectious MSV prior to the production of progeny MuLV. Addition of thymidine to the growth medium of MuLV-superinfected S + L - cells at a concentration suppressing deoxyribonucleic acid synthesis inhibited the replication of MuLV and the rescue of MSV. MSV production closely paralleled MuLV replication under a variety of experimental conditions. These results suggest that replication of MuLV is required for the rescue of infectious MSV from S + L - cells and that one (or more) factor, produced late in the MuLV replicative cycle, is utilized by both viruses during virion assembly. During the course of these experiments, virus stocks were recovered which contained infectious MSV in apparent excess over MuLV. These stocks were used for generating new S + L - cell lines by simple end point dilution procedures.     

Immunological Studies on Viral Polypeptide Synthesis in Cells Replicating Murine Sarcoma-Leukemia Virus

Antibodies to disrupted murine sarcoma-leukemia virus (MSV[MLV]) were used to study the synthesis of viral polypeptides in the transformed, virus-producing rat cell line 78A1. When cultures were labeled for 10 min with radioactive amino acids, about 9% of the total labeled proteins were precipitated with antiserum against purified MSV(MLV), and 3 to 4% were precipitated with the same antiserum after it had been absorbed with an extract from uninfected rat cells. The difference is due to the presence in the unabsorbed antiserum of antibodies to cellular proteins that are present in purified virus preparations. Intracellular viral proteins labeled with radioactive amino acids were isolated by immunoprecipitation and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The mobilities of intracellular viral polypeptides were identical to those of the purified virion. However, labeled polypeptides having electrophoretic mobilities lower than that of the major virion polypeptide, the group-specific antigen of molecular weight 31,000, were present in higher proportion in the total cell extract and in the membrane fraction than in the virion. These polypeptides appear to be of cellular origin for they were present only in minute amounts in the immunoprecipitates obtained with the absorbed serum. After a 10-min labeling period, radioactive proteins were assembled into extracellular virions rapidly for the first 4 hr followed by a slower rate. More than 2% of the total proteins of the cell labeled in a 10-min pulse were assembled into virions at the completion of a 24-hr chase. The high-molecular-weight polypeptides with the same mobilities as those detected in the immunoprecipitate of intracellular proteins were found in virions released from cells after a 10-min pulse. A larger proportion of these high-molecular-weight proteins was detected in virions released after short chase periods (30-120 min) than after longer chase periods (6-24 hr). Two possible interpretations of these data are that the high-molecular-weight cell-derived polypeptides (i) have a turnover rate higher than that of the major virion polypeptides or (ii) are cleaved proteolytically from the virions during long incubation in the culture media.     

Sequence and translation of the murine coronavirus 5''-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.     

Detection of a non-structural protein of M r 11 000 encoded by the virion DNA of maize streak virus

A polypeptide of approximately 11 000 daltons (11 kDa protein) encoded by an open reading frame (10.9 ORF) from the virion sense of maize streak virus (MSV) DNA has been detected among the products of in vitro translation reactions programmed with RNA from infected maize plants and also in total protein extracts from infected leaves. The 11 kDa protein has not been detected in virions and is therefore proposed to have a nonstructural role.Viral DNA with an additional in-frame translation stop codon in the 10.9 ORF was not infectious when transmitted to maize plants via Agrobacterium tumefaciens agroinfection, suggesting that the 10.9 ORF may be essential for virus function. Computer comparison data show that equivalent ORFs in wheat dwarf virus (WDV) and digitaria streak virus (DSV) have some sequences in common with the 10.9 ORF of MSV. Further-more, the absence of similar sequences in geminiviruses which infect dicotyledonous plants suggests that the 11 kDa protein and its putative homologs in WDV and DSV have a function necessary only for those geminiviruses which infect the Gramineae.The significance of the 11 kDa protein in relation to expression of the virion sense DNA of MSV is discussed.     

Gene and mRNA for precursor polypeptide VI from adenovirus type 2.

We present a 1,040-base-pair-long sequences of adenoviruses type 2 DNA which encodes the complete gene for precursor polypeptide VI (pVI). pVI consists of 250 amino acids amounting to a molecular weight of 26,990. The proteolytic cleavage maturing pVI to virion polypeptide VI removes 33 amino acids from the amino-terminal end of the polypeptide, thus giving the mature polypeptide VI a molecular weight of 23,400. The UAA stop codon terminating pVI translation is separated by 84 nucleotides from the initiator triplet for the hexon gene. Both polypeptides are encoded by the same translational reading frame, suggesting the evolution of pVI and hexon as separate proteins by the introduction of a termination codon and selection of a new splice acceptor site in an ancestral fused polypeptide chain. The splice site where the common tripartite leader is attached to the pVI mRNA precedes the initiator codon for pVI translation by one nucleotide and forms, together with other late splice acceptor sites, a late adenovirus consensus acceptor site. We also demonstrate that the 3' end of the mRNA's belonging to the L2 3'-cotermination family is located only 31 nucleotides upstream from the splice junction of the pVI mRNA. Furthermore, we show that four novel polypeptides of molecular weights 80,000, 39,000, 36,000, and 10,500 are encoded by region L2.     

Proteins of Yaba Monkey Tumor Virus I. Structural Proteins

Yaba virus proteins were characterized by polyacrylamide gel electrophoresis. Electrophoresis of Yaba virion (proteins) dissociated by sodium dodecyl sulfate and 2-mercaptoethanol in continuous and discontinuous buffer systems yielded 37 polypeptide species by staining and by counting bands of radioactively labeled polypeptides. The molecular weights of the viral polypeptide species were found to range from 10,000 to 220,000 by comparing the relative distance of migration of viral proteins with proteins of known molecular weights. Two polypeptides were removed from purified virions by nonionic detergent nonidet P -40 treatment, and the amount of one polypeptide was reduced. Purified cores yielded 21 polypeptide species, none of which was labeled with radioactive glucosamine.     

Synthesis of murine mammary tumor viral proteins in vitro

The coding potential of murine mammary tumor viral genomic RNA was investigated by in vitro translation of various size classes of RNAs isolated from the virions. The major products of translation of full-size 35S polyadenylylated virion RNA were gag-related polyproteins of 75,000, 105,000, and 180,000 daltons (P75, P105, and P180, respectively). Studies on the kinetics of translation of these three proteins established that they were synthesized independently and that the smaller proteins were not post-translational cleavage products of the larger proteins. Tryptic peptide mapping showed that almost all of the P75 sequences were contained within P105 and almost all of the P105 sequences were contained within P180. The syntheses of all three proteins were inhibited by m7GTP, indicating that they were translated from capped mRNA's. Although a 24S polyadenylylated RNA had been identified as the intracellular mRNA for env precursor polyprotein, no such protein could be translated from the 24S polyadenylylated RNA isolated from the virions. However, translation of a 14S size class of polyadenylylated virion yielded four prominent proteins of about 36,000, 23,000, 21,000, and 20,000 daltons. These proteins were unrelated to murine mammary tumor viral structural proteins, as suggested from tryptic peptide mapping and immunoprecipitation data. They might be the products of an as-yet-unidentified gene located near the 3' terminus of the murine mammary tumor viral genomic RNA.     

Intracellular Viral RNA Species in Mouse Cells Nonproductively Transformed by the Murine Sarcoma Virus

The size and quantity of virus-specific RNA in five non-virus-producing mouse cells transformed by the Moloney isolate of murine sarcoma virus (MSV) was determined. Hybridization of RNA from transformed cells with the [(3)H]DNA product of the RNA-directed DNA polymerase of the murine sarcoma-leukemia virus was used to detect and quantitate virus-specific RNA. The amount of virus-specific RNA in non-virus-producing cells was less than one-sixth of that found in virus-producing cells. A striking correlation was found between the amount of intracellular virus-specific RNA and the degree of agglutination by conconavalin A previously reported for the four non-virus-producing NIH/3T3 cell lines (Salzberg and Green, 1974). A major RNA subunit sedimenting at 26 to 28S was detected in all five MSV-transformed non-virus-producing cells. This could represent the RNA genome of defective MSV.     

Both VP2 and VP3 are synthesized from each of the alternative spliced late 19S RNA species of simian virus 40.

The late 19S RNAs of simian virus 40 consist of a family of alternatively spliced RNAs, each of which contains open reading frames corresponding to all three of the virion proteins. Two approaches were used to test the hypothesis that each alternatively spliced 19S RNA species is translated to synthesize preferentially only one of the virion proteins. First, we analyzed the synthesis of virion proteins in simian virus 40 mutant-infected monkey cells that accumulate predominantly either only one spliced 19S RNA species or only the 19S RNAs. Second, we determined the virion proteins synthesized in a rabbit reticulocyte lysate programmed with specific, in vitro-transcribed 19S RNA species. These results indicated that VP2 and VP3, but not VP1, are synthesized from all 19S RNA species. Quantitative analysis of these data indicated that individual 19S RNA species containing a translation initiation signal upstream of the VP2 AUG codon were translated in a cell extract three- to fivefold less efficiently than were 19S RNA species lacking this signal and that the precise rate of synthesis of VP2 relative to VP3 varied somewhat with the sequence of the leader region. These data are consistent with the synthesis of VP2 and VP3 occurring by a leaky scanning mechanism in which initiation of translation at a specific AUG codon is affected by both (i) the intrinsic efficiency of ribosomes recognizing the sequences surrounding the AUG codon as an initiation signal and (ii) partial interference from 5'-proximal initiation signals and their corresponding open reading frames.     

Translation of bovine leukemia virus virion RNAs in heterologous protein-synthesizing systems.

Bovine leukemia virus 60 to 70S RNA was heat denatured, the polyadenylic acid-containing species were separated by velocity sedimentation, and several size classes were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The major RNA species sedimented at 38S and migrated as a single component of molecular weight 2.95 x 10(6) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The predominant polypeptides of the in vitro translation of bovine leukemia virus 38S RNA were products with molecular weights of 70,000 and 45,000; minor components with molecular weights of 145,000 and 18,000 were also observed. Two lines of evidence indicate that the 70,000- and 45,000-molecular weight polypeptides represent translation products of the gag gene of the bovine leukemia virus genome (Pr70gag and Pr45gag). First, they are specifically precipitated by a monospecific antiserum to the major internal protein, p24, and second, they are synthesized and correctly processed into virion proteins p24, p15, and p10 in Xenopus laevis oocytes microinjected with bovine leukemia virus 38S RNA. The 145,000-molecular weight polypeptide was immunoprecipitated by the anti-p24 serum and not by an antiserum to the major envelope glycoprotein, gp60. It contained all the tryptic peptides of Pr70gag and additional peptides unique to it, and thus represents in elongation product of Pr70gag in an adjacent gene, presumably the pol gene. The 18,000-molecular weight product was antigenically unrelated to p24 and gp60 and shared no peptides in common with Pr70gag, Pr45gag, or the 145,000-molecular weight polypeptide. It was maximally synthesized on a polyadenylic acid-containing virion 16 to 18S RNA, and we present evidence that this RNA is a 3' end-derived subgenomic fragment of the bovine leukemia virus genome rather than a contaminating cellular RNA.     

Polymorphism of avian sarcoma virus src proteins.

The src gene products of seven different avian sarcoma viruses were compared. In vitro translation of virion RNA yielded products identified unambiguously as p60src in the case of two stocks of the Schmidt-Ruppin strain, three stocks of the Prague strain, the Bryan strain, and the Bratislava 77 strain of avian sarcoma virus. Differences in the electrophoretic mobility of these seven p60src proteins in sodium dodecyl sulfate-polyacrylamide gels, corresponding to variation in the apparent molecular weights ranging from 56,000 to 60,500, were observed. Antigenic variability was also found; only three of the seven viruses tested encoded a p60src, which was precipitated by antisera derived from rabbits bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Examination of the methionine-containing tryptic peptides of the seven ;60src proteins by two-dimensional mapping revealed four common peptides but marked variability in the five to eight other peptides in each protein. Clear differences in the peptide maps of p60src were observed, both between different strains of virus and within strains. In the three cases examined, p60src synthesized in transformed cells was found to be essentially identical to that synthesized in vitro. We conclude that there is significant polymorphism in the p60src proteins of the avian sarcoma viruses.     

Characterization of Rous sarcoma virus src gene products synthesized in vitro.

The cell-free synthesis of three major proteins from virion RNA of nondefective Rous sarcoma virus (RSV), but not from RNA of transformation-defective deletion mutants, has been observed. The apparent molecular weights of these transformation-specific proteins are approximately 60,000 (60K), 25K, and 17K. Tryptic maps of methionine-containing peptides revealed the 17K, 25K, and 60K proteins to be overlapping in sequence. However, only partial homology was observed between the 17K, 25K and 60K proteins synthesized from Schmidt-Ruppin strain, subgroup D, RSV RNA and those synthesized from Prague strain, subgroup B, RSV, RNA. About half of the methionine peptides in the Schmidt-Ruppin strain, subgroup D, 60K protein were shared with the Prague strain, subgroup D, 60K protein, and the rest were distinct to each. The virion RNAs coding for the 60K, 25K, and 17K proteins were found to be polyadenylated and to sediment with maximal mRNA activity at about 23, 19 to 20, and 18S, respectively. In addition, transformation-specific proteins with molecular weights of 39K and 33K were observed by in vitro synthesis. These proteins are also related to the 60K, 25K, and 17K proteins and were synthesized from polyadenylated RSV RNA of approximately 21 to 22S. RNase T1-resistant oligonucleotides were analyzed in parallel, and the src-specific oligonucleotides were found to be first present in equimolar amounts in those gradient fractions sedimenting at 21 to 22S. Our data suggest that synthesis of the 60K protein is initiated near the 5' terminus of the src gene, whereas the 39K, 33K, 25K, and 17K proteins are initiated internally in the src gene. All of these proteins appear to be initiated independently, but they may have a common termination site.     

Alternative Translation Initiation of Theiler’s Murine Encephalomyelitis Virus

DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) produce a chronic demyelinating disease in which the virus persists but has a restricted expression. We previously reported that TO subgroup strains, in addition to synthesizing the picornaviral polyprotein, use an alternative initiation codon just downstream from the polyprotein's AUG to translate an 18-kDa protein called L* that is out of frame with the polyprotein (H. H. Chen et al., Nat. Med. 1:927-931, 1995; W. P. Kong and R. P. Roos, J. Virol. 65:3395-3399, 1991). L* is critically important for virus persistence and the induction of the demyelinating disease (Chen et al., 1995; G. D. Ghadge et al. J. Virol. 72:8605-8612, 1998). We have proposed that variations in the amount of translation initiation from the L* AUG versus the polyprotein AUG may occur in different cell types and therefore affect the degree of expression of viral capsid proteins. We now demonstrate that ribosomal translation initiation at the polyprotein's initiation codon affects initiation at the L* AUG, suggesting that ribosomes land at the polyprotein's initiation codon before scanning downstream and initiating at the L* AUG. We also find that the viral 5' untranslated region affects utilization of the L* AUG. Surprisingly, mutant DA cDNAs were found to be infectious despite the presence of mutations of the polyprotein initiation codon or placement of a stop codon upstream of the L* AUG in the polyprotein's reading frame. Sequencing studies showed that these viruses had a second site mutation, converting the reading frame of L* into the polyprotein's reading frame; the results suggest that translation of the polyprotein during infection of these mutant viruses can be initiated at the L* AUG. These data are important in our understanding of translation initiation of TMEV and other RNAs that contain an internal ribosome entry site.     

Transformation of Rat Liver Cells with Chicken Sarcoma Virus B77 and Murine Sarcoma Virus

Rat liver cells in vitro were transformed with chicken sarcoma virus B77, giving RL(B77) cells, and with murine sarcoma virus (Harvey), giving RL(MSV) cells. Rat liver cells transformed spontaneously in vitro were designated RL cells. In addition, the RL(MSV) cell line was adapted for growth in culture fluid containing 25 mug of 5-bromodeoxyuridine per ml. All cell lines were tumorigenic in 1-wk-old rats. The number of cells needed for induction of tumor growth was 1,000-fold higher in the case of RL(B77) cells in comparison with RL(MSV) cells and RL cells. No production of viral particles from any of the cell lines investigated was detected by plating concentrated supernatant fluid of the cultures on different secondary embryo cells with and without fusion by Sendai virus, by labeling with uridine-5-(3)H, or by assay for deoxyribonucleic acid polymerase activity. The viral genome was rescued by fusion of RL(B77) cells with chicken cells. Chicken sarcoma virus rescued from (RL(B77) cells differed in plating efficiency on duck cells from B77 virus rescued from transformed rat embryo cells. No virus was rescued after fusion of RL(MSV) and RL cells with mouse, rat, or chicken embryo cells. Infectious murine sarcoma virus can be induced by 5-bromodeoxyuridine from RL(MSV) cells.