Avian Sarcoma Virus Genome Research Articles

Selective inhibition of splicing at the avian sarcoma virus src 3' splice site by direct-repeat posttranscriptional cis elements.

The direct-repeat elements (dr1) of avian sarcoma virus (ASV) and leukosis virus have the properties of constitutive transport elements (CTEs), which facilitate cytoplasmic accumulation of unspliced RNA. It is thought that these elements represent binding sites for cellular factors. Previous studies have indicated that in the context of the avian sarcoma virus genome, precise deletion of both ASV dr1 elements results in a very low level of virus replication. This is characterized by a decreased cytoplasmic accumulation of unspliced RNA and a selective increase in spliced src mRNA. Deletion of either the upstream or downstream dr1 results in a delayed-replication phenotype. To determine if the same regions of the dr1 mediate inhibition of src splicing and unspliced RNA transport, point mutations in the upstream and downstream elements were studied. In the context of viral genomes with single dr1 elements, the effects of the mutations on virus replication and increases in src splicing closely paralleled the effects of the mutations on CTE activity. For mutants strongly affecting CTE activity and splicing, unspliced RNA but not spliced RNA turned over in the nucleus more rapidly than wild-type RNA. In the context of wild-type virus containing two dr1 elements, mutations of either element that strongly affect CTE activity caused a marked delay of virus replication and a selective increase in src splicing. However, the turnover of the mutant unspliced RNA as well as the spliced mRNA species did not differ significantly from that of the wild type. These results suggest the dr1 elements in ASV act to selectively inhibit src splicing and that both elements contribute to the fitness of the wild-type virus. However, a single dr1 element is sufficient to stabilize unspliced RNA.

Tyrosine-phosphorylated epidermal growth factor receptor and cellular p130 provide high affinity binding substrates to analyze Crk-phosphotyrosine-dependent interactions in vitro.

The genome of CT10 avian sarcoma virus encodes a 47-kDa fusion protein that consists of viral gag sequences fused to a cell-derived sequence containing SH2 and SH3 domains (v-crk). Genetic and biochemical evidence suggests that v-Crk can induce transformation of chicken embryo fibroblasts by influencing the activity of cellular proteins involved in growth regulation. In this report, we have developed an in vitro microtiter assay to study the binding of bacterially expressed glutathione S-transferase-fusion proteins of v-Crk and its cellular homolog, c-Crk, to the phosphorylated epidermal growth factor receptor (EGFR). Competitive binding data are presented that compare the abilities of heterologous glutathione S-transferase-fusion proteins containing GAPSH2[N], AblSH2, SrcSH2, and PLC-gamma SH2[N] sequences to inhibit Crk binding. Results indicate that both full-length Crk and GAPSH2[N] bind the phosphorylated EGFR with high affinity and can quantitatively compete the binding of each other by competitive enzyme-linked immunosorbent assay. Binding of full-length Crk or the isolated SH2 domains of GAP or Abl resulted in a significant protection of phosphorylated EGFR against dephosphorylation by cellular phosphatase activity, but did not appear to stimulate the intrinsic tyrosine kinase activity of the EGFR. To extend these findings to p130, the major phosphotyrosine-containing protein in CT10-transformed cells, we utilized a nitrocellulose filter binding assay. Results demonstrate high affinity binding of Crk toward denatured p130 and, as is the case for phosphorylated EGFR, Crk binding can partially protect p130 from phosphatase activity. However, no apparent competition of Crk binding was noted with heterologous SH2-containing proteins including GAPSH2[N], suggesting a possible specificity of Crk-p130 binding. These data are consistent with a direct role of SH2 in the modulation of cellular phosphotyrosine status in vivo.

Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA.

The nucleotide sequences in the long terminal repeat of avian sarcoma virus that are recognized in vitro by HeLa cell RNA polymerase II have been identified. For this purpose, various 5' and 3' deletions were introduced into a cloned long terminal repeat fragment. The effects of these deletions on transcription initiation in HeLa whole-cell extracts were then studied. Three specific transcripts have been identified. The major transcript is initiated at nucleotide +1 (relative to the cap site). Deletion of the upstream sequence between -299 and -55 has no effect on the level of transcription from this start site, whereas deletion of the sequence downstream of -14 drastically reduces the levels of transcription. In contrast, deletion of the sequence downstream from the TATA box has no effect on the initiation or efficiency of synthesis of the two minor RNA species, which are initiated at around nucleotides -260 and -105. The transcription of these RNA products, however, is abolished by an upstream deletion between -299 and -55. These results suggest that HeLa cell RNA polymerase II recognizes in vitro more than one promoter site present in the long terminal repeat of the avian sarcoma virus genome and defines the sequences required for initiation of the major transcript.

Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA.

The nucleotide sequences in the long terminal repeat of avian sarcoma virus that are recognized in vitro by HeLa cell RNA polymerase II have been identified. For this purpose, various 5' and 3' deletions were introduced into a cloned long terminal repeat fragment. The effects of these deletions on transcription initiation in HeLa whole-cell extracts were then studied. Three specific transcripts have been identified. The major transcript is initiated at nucleotide +1 (relative to the cap site). Deletion of the upstream sequence between -299 and -55 has no effect on the level of transcription from this start site, whereas deletion of the sequence downstream of -14 drastically reduces the levels of transcription. In contrast, deletion of the sequence downstream from the TATA box has no effect on the initiation or efficiency of synthesis of the two minor RNA species, which are initiated at around nucleotides -260 and -105. The transcription of these RNA products, however, is abolished by an upstream deletion between -299 and -55. These results suggest that HeLa cell RNA polymerase II recognizes in vitro more than one promoter site present in the long terminal repeat of the avian sarcoma virus genome and defines the sequences required for initiation of the major transcript.

Two structurally and functionally different forms of the transforming protein of PRC II avian sarcoma virus.

The primary translation product of the PRC II avian sarcoma virus genome is a protein of 105,000 daltons (P105), and we have previously shown that approximately 50% of the P105 molecules are converted to molecules of 110,000 daltons (P110) by posttranslational modification. Fractionation of PRC II-infected cells showed that P105 was contained primarily in a nonionic detergent-soluble compartment, whereas P110 partitioned almost exclusively with a nonionic detergent-insoluble or crude cytoskeletal fraction. The tyrosine-specific protein kinase activity previously observed in immunoprecipitates which presumably contained both P110 and P105 was found predominantly in the P110-containing immunoprecipitates made from the cytoskeletal fraction and was essentially absent from the P105-containing immunoprecipitates prepared from the soluble fraction. Individual analysis of 32P-labeled P110 and P105 prepared by this fractionation technique revealed that P110 contained more phosphotyrosine per mole of protein than did P105. Examination of the tryptic peptide maps of 32P-labeled P110 and P105 suggested that the additional phosphotyrosine in P110 resulted from phosphorylation at discrete sites within the protein. From these experiments, we conclude that PRC II-infected cells contain two discrete forms, P105 and P110, of the transforming protein and that each of these proteins exhibits distinct structural and functional characteristics.

Two structurally and functionally different forms of the transforming protein of PRC II avian sarcoma virus.

The primary translation product of the PRC II avian sarcoma virus genome is a protein of 105,000 daltons (P105), and we have previously shown that approximately 50% of the P105 molecules are converted to molecules of 110,000 daltons (P110) by posttranslational modification. Fractionation of PRC II-infected cells showed that P105 was contained primarily in a nonionic detergent-soluble compartment, whereas P110 partitioned almost exclusively with a nonionic detergent-insoluble or crude cytoskeletal fraction. The tyrosine-specific protein kinase activity previously observed in immunoprecipitates which presumably contained both P110 and P105 was found predominantly in the P110-containing immunoprecipitates made from the cytoskeletal fraction and was essentially absent from the P105-containing immunoprecipitates prepared from the soluble fraction. Individual analysis of 32P-labeled P110 and P105 prepared by this fractionation technique revealed that P110 contained more phosphotyrosine per mole of protein than did P105. Examination of the tryptic peptide maps of 32P-labeled P110 and P105 suggested that the additional phosphotyrosine in P110 resulted from phosphorylation at discrete sites within the protein. From these experiments, we conclude that PRC II-infected cells contain two discrete forms, P105 and P110, of the transforming protein and that each of these proteins exhibits distinct structural and functional characteristics.

Integration of avian sarcoma virus DNA in chicken cells

We have used restriction endonucleases and hybridization reagents specific for various regions of the avian sarcoma virus (ASV) genome to assess the structure and sites of integration of proviruses found in chicken cells, the natural host for thiss viris, after infection in culture. Colonies of ASV-transformed chicken fibroblasts were shown to contain new proviruses located at from one to several sites in the host genome; the sites of integration appeared to differ in each of the nine colonies studied. Fractionation of chromosomes from lymphoblastoid cells infected in mass culture with ASV also failed to reveal preferred sites for insertion of ASV proviruses. Thus, in chicken cells, as well as in heterologous cells[ Hughes S. H. et al. (1978) . Cell 15, 1397–1410; Sabran, J. L., et al. (1979) . J. Virol. 29, 170–185; Martin, S. et al. (1979) . Virology, 96, 530–546; Gilmer, T., and Parsons, J. T. (1979). J. Virol. 32, 762–770], many regions of cellular DNA can accomodate an ASV provirus, and these regions are distributed over several chromosomes. Although proviruses are found at many sites in cellular DNA, the structure of proviral DNA appears to be invariant in infected chicken cells. Viral DNA is linked to cellular DNA near or at the extremities of a ca. 330-nucleotide sequence repeated at the ends of unintegrated linear DNA. The repeated structure is composed of sequences derived from both the 3′ and the 5′ ends of viral RNa. The resulting structure for ASV DNA can be written “cell DNA-3′5′- gag-pol-env-src-3′5′-cell DNA,” as similarly deduced from studies of ASV proviruses in nonpermissive, heterologous host cells[ Hughes, S. H., et al. (1978) . Cell 15, 1397–1410]. The colonies examined in detail were obtained by suspending cells in agar 3 to 6 days after infection. Some but not all of these colonies appeared to be clonal, as judged by the physical mapping of integrated proviruses. Colonies obtained from cells placed in agar directly after infection did not appear clonal by this criterion; explanations for this anomalous behavior are considered.

Structure of viral DNA and RNA in mammalian cells infected with avian sarcoma virus

The expression of retrovirus genes varies from one species of host to another and is probably regulated by cellular mechanisms. Thus, avian fibroblasts infected with avian sarcoma virus (ASV) produce virus and acquire a neoplastic phenotype, whereas ASV-infected mammalian (cells produce little or no virus and only rarely become neoplastic. We have explored the origins of these variations by analysing the viral DNA and RNA in several lines of mammalian cells infected by ASV. Our work was facilitated by recently developed techniques that permit the fractionation, detection and characterization of extremely small quantities of viral nucleic acids. Each line of ASV-infected mammalian cells contained either one or two copies of ASV provirus. Nevertheless, the amounts of stable viral RNA produced in these cells varied over a range of at least 100-fold. In permissive chicken cells, the four genes of ASV ( gag, pol, env and src) appear to be expressed by means of three viral messenger RNAs ( gag/ pol, M r 3.3 × 10 6; env, M r 1.8 × 10 6; and src, M r 1.1 × 10 6). The same viral mRNAs were found in ASV-transformed mammalian cells, although the relative amounts of the three species were different; the gag/ pol mRNA predominated in permissive cells, the src, mRNA in mammalian cells. The previous description of these RNAs was revised by demonstrating that the src mRNA apparently contains a 5′ untranslated region composed of two distinct elements: a nucleotide sequence transposed from the 5′ end of the viral genome, and a nucleotide sequence contiguous to the 5′ boundary of src in the viral genome. We encountered several novel forms of viral mRNA: two distinctive src mRNAs in the same clone of cells; an env RNA appreciably larger than usual; and two mRNAs that appear to be encoded by the extreme right-hand end of the ASV provirus and adjacent cellular DNA. The first of these anomalies persisted when virus was rescued from the mammalian cells and then propagated in permissive cells, whereas the second and third anomalies were not apparent during the replication of rescued virus. Viral gene expression was attenuated in ASV-infected hamster cells that had reverted from a transformed to a normal phenotype: in particular, the amount of src mRNA was reduced by almost 100-fold. The revertant cells and their transformed siblings contained single identical proviruses located at apparently identical sites within the host genome. Thus, the change in viral gene expression that accompanies and perhaps causes reversion is not due to either translocation of the provirus or independent origins of the cell lines. We conclude that the enzymatic mechanisms for the genesis of ASV mRNAs are widely distributed among vertebrates: in particular, phylogenetically disparate host cells may utilize the same signals within the ASV genome for the splicing and polyadenylation of viral RNA: even anomalous signals can be interpreted identically across a broad phylogenetic range. However, the regulation of these mechanisms varies from one cell to another, and consistent differences may occur in the maturation of viral RNA within individual clones of transformed cells. In at least some instances, transcription may be initiated within the ASV provirus but continue uninterrupted into adjacent cellular DNA; thus, the mere insertion of viral DNA into the host genome might induce the expression of previously silent cellular genes. Attenuation of viral gene expression by the host cell can lead to reversion of the cell from a transformed to a normal phenotype. Our data indicate that the host modulates ASV gene expression principally by regulating the production of viral mRNAs. but the mechanism of this regulation remains to be elucidated.

Molecular mechanism for the capture and excision of the transforming gene of avian sarcoma virus as suggested by analysis of recombinant clones.

Structural analysis of two cDNA clones, derived from reverse transcripts of avian sarcoma virus 21S mRNA's, reveals unusual features in the organization and expression of the integrated avian sarcoma virus (ASV) proviral DNA and predicts a mechanism for recombination events that will lead to either the capture or the excision of the transforming gene of this virus. The latter is supported by our observation that there is an extensive homologous region on either side of the transforming gene that will allow site-specific deletion or integration to occur. Comparison of the clone derived from the src-specific 21S mRNA coding for the transforming gene product to that derived from the env-specific 21S mRNA coding for the envelope glycoprotein show that the common c region present at the 3' terminus of the ASV genome is 326 bases long. Within this c region are nucleotide sequences that may play key roles in the life cycle of this virus. These regulatory sequences include (i) probable promoter sites for the initiation of transcription, (ii) a polyadenylation signal, and (iii) a sequence that is complementary to the 3' termini of both the env and the src regions, which will allow the generation of transformation-defective deletions.

Recombinants between avian sarcoma virus genome and chicken helper factor gene of the host cell: cloning by transfection.

Chicken cells of chicken helper factor-positive (chf+) phenotype were infected with a cloned (envE-free) Rous sarcoma virus, subgroup D, and examined for the presence of parent and recombinant proviruses by transfection in chicken and turkey cells, respectively. It was found that most parent virus DNA is integrated into the host cell genome during the first 18 hr after infection, and no significant integration occurs between 18 and 72 hr after infection. On the other hand, no recombinant virus DNA was detected at 18 hr, although both unintegrated and integrated (provirus) forms of this DNA occurred 72 hr after infection. Recombination proviruses were also found in chronically virus-infected chf+ cells but not in chf- cells lacking virus-related RNA. Our results show that recombinants between the exogenous virus and endogenous chf gene can be cloned from the DNA of the host cell by transfection and suggest that a second replicative cycle of the virus is required to generate such recombinants.

Binding of Escherichia coli RNA polymerase to a specific site located near the 3′-end of the avian sarcoma virus genome

We have isolated a unique 35 base pair region of avian tumor virus DNA that binds specifically to Escherichia coli RNA polymerase holoenzyme. Studies with various size classes of viral DNA coupled with restriction enzyme mapping data indicate that the binding site is located in the large terminal repeat of the viral genome and is within the first 50 nucleotides of the heteropolymeric region corresponding to the 3′-end of the virion RNA.

Avian retroviruses that cause carcinoma and leukemia: identification of nucleotide sequences associated with pathogenicity.

Avian myelocytomatosis virus (MC29V) is a retrovirus that transforms both fibroblasts and macrophages in culture and induces myelocytomatosis, carcinomas, and sarcomas in birds. Previous work identified a sequence of about 1,500 nucleotides (here denoted onc(MCV)) that apparently derived from a normal cellular sequence and that may encode the oncogenic capacity of MC29V. In an effort to further implicate onc(MCV) in tumorigenesis, we used molecular hybridization to examine the distribution of nucleotide sequences related to onc(MCV) among the genomes of various avian retroviruses. In addition, we characterized further the genetic composition of the remainder of the MC29V genome. Our work exploited the availability of radioactive DNAs (cDNA's) complementary to onc(MCV) (cDNA(MCV)) or to specific portions of the genome of avian sarcoma virus (ASV). We showed that genomic RNAs of avian erythroblastosis virus (AEV) and avian myeloblastosis virus (AMV) could not hybridize appreciably with cDNA(MCV). By contrast, cDNA(MCV) hybridized extensively (about 75%) and with essentially complete fidelity to the genome of Mill Hill 2 virus (MH2V), whose pathogenicity is very similar to that of MC29V, but different from that of AEV or AMV. Hybridization with the ASV cDNA's demonstrated that the MC29V genome includes about half of the ASV envelope protein gene and that the remainder of the MC29V genome is closely related to nucleotide sequences that are shared among the genomes of many avian leukosis and sarcoma viruses. We conclude that onc(MCV) probably specifies the unique set of pathogenicities displayed by MC29V and MH2V, whereas the oncogenic potentials of AEV and AMV are presumably encoded by a distinct nucleotide sequence unrelated to onc(MCV). The genomes of ASV, MC29V, and other avian oncoviruses thus share a set of common sequences, but apparently owe their various oncogenic potentials to unrelated transforming genes.

The nucleotide sequence of an untranslated but conserved domain at the 3' end of the avian sarcoma virus genome.

The genomes of numerous avian retroviruses contain at their 3' termini a conserved domain denoted "c". The precise boundaries and function of "c" have been enigmas. In an effort to resolve these issues, we determined the sequence of over 900 nucleotides at the 3' end of the genome of the Schmidt-Ruppin subgroup A strain of avian sarcoma virus (ASV). We obtained the sequence from a suitable fragment of ASV DNA that had cloned into the single-stranded DNA phage M13mp2. Computer-assisted analysis of the sequence revealed the following structural features: i) the length of "c" - 473 nucleotides; ii) the 3' terminal domain of src, ending in an amber codon at the 5'boundary of "c"; iii) terminator codons that preclude continuous translation from "c"; iv) suitably located sequences that may serve as signals for the initiation of viral RNA synthesis and for the processing and/or polyadenylation of viral mRNA; v) a repeated sequence that flanks src and that could facilitate deletion of this gene; vi) repeated sequences within "c"; and vii) unexplained homologies between sequences in "c" and sequences in several other nucleic acids, including the 5' terminal domain of the ASV genome, tRNATrp and its inversion, the complement of tRNATrp and its inversion, and the 18S RNA of eukaryotic ribosomes. We conclude that "c" probably does not encode a protein, but its sequence may nevertheless serve several essential functions in viral replication.

Unusual features in the nucleotide sequence of a cDNA clone derived from the common region of avian sarcoma virus messenger RNA.

We have constructed a recombinant plasmid containing a 700-base pair (bp) cDNA copy of the common region present at the 3' end of Schmidt-Ruppin avian sarcoma virus (ASV) 21S mRNA. The cDNA was inserted into plasmid pBR322 at the Pst I site by the G-C tailing method. A restriction map of the cloned insert from a recombinant plasmid pSRI indicates that it corresponds to the 3' end of the ASV genome. R-loop analysis with ASV genomic RNA indicates that the insert is colinear with the ASV genome over most of its length. The sequence of 331 bp at the 3' end of the DNA insert was determined and shows that the insert contains extra sequences not found at the 3' end of ASV genomic RNA. Following the terminally redundant sequence of 20 bp that has been found at the extreme 3' end of genomic RNA is a sequence of 79 bp that is almost identical to that located immediately next to the 20-bp repeat at the 5' end of ASV genomic RNA. This is followed by 18 bp of unique sequence, possibly of host origin. The structure of the clone suggests that ASV mRNA may differ from genomic RNA at its 3' end and that 21S mRNA is transcribed from integrated ASV DNA and contains at its 3' end sequences derived both from the 5' end of the ASV genome and from host DNA adjacent to the site of integration. The presence of termination codons in all three reading frames suggests that the common region probably does not contain coding sequences. However, the presence of sequences that resemble probable promoter sites supports the possibility that this region may be involved in the regulation of transcription.

Avian erythroblastosis virus: transformation-specific sequences form a contiguous segment of 3.25 kb located in the middle of the 6-kb genome

Several foci of chicken embryo fibroblasts transformed by avian erythroblastosis virus (AEV) strain ES-4 were found to produce virus progeny containing the RNA of the replication-defective AEV in excess over the RNA of the helper virus. The size of AEV RNA was determined by methylmercury-agarose gel electrophoresis and electron microscopy to be 28 S or 6 kb. About 40 to 45% of this RNA is homologous by RNA-DNA hybridization to the RNA of other chicken leukosis and sarcoma viruses; the rest of the genome is AEV specific. These AEV specific sequences, which presumably contain the genetic information responsible for transformation form a contiguous stretch of 3.25 kb, located by heteroduplex mapping in the center of the 6 kb genome between two segments of 1.06 kb at the 3′ end and 1.64 kb at the 5′ end which are homologous to the genome of avian sarcoma virus. From the length of the region showing homology between AEV and avian sarcoma virus at the 5′ end of the genome and from the known sequence composition of the AEV-specific 75K protein (Hayman et al., 1979), it can be deduced that the initiation point for the N terminus of the gag protein p19 is located about 1.0 kb from the 5′ end of the genome in avian oncoviruses. Nonproducing AEV-transformed chicken embryo fibroblasts were also isolated. Infectious AEV could be rescued from these cells only with chicken leukosis viruses; unrelated avian retroviruses were ineffective, probably because AEV requires complementation in the gag and pol genes, in addition to env.

Elongation of DNA complementary to the 5' end of the avian sarcoma virus genome by the virion-associated RNA-dependent DNA polymerase.

RNA-dependent DNA synthesis in a virion-associated reaction has been described as being dependent upon the detergent concentration used for disruption of the virion. In this study, the Triton X-100 concentration was found to affect the elongation of the initially synthesized DNA complementary to the last approximately 100 nucleotides at the 5' end of the RNA (cDNA100). Whereas elongation of cDNA100 increased with time of incubation at the optimal detergent concentration, this process was retarded at higher detergent concentrations. At the optimal detergent concentration, elongated DNA was of low chemical complexity, indicating that extension of cDNA100 occurred at a unique site on the RNA. Higher than optimal detergent concentrations resulted in nonspecific elongation and in DNA of high chemical complexity. This was shown by oligopyrimidine tract analysis. Furthermore, actinomycin D was observed to inhibit the elongation of cDNA100 at the optimal detergent concentration. The nature of the elongation process was elucidated by analysis of DNA synthesized in a virion-associated reaction in the presence of bacteriophage Qbeta RNA. At the optimal detergent concentration DNA complementary only to avian sarcoma virus RNA was synthesized, whereas at higher concentrations DNA was copied from both avian sarcoma virus and Qbeta RNA. We conclude that the elongation mechanism of cDNA100 is affected by the detergent concentration and elongation is unspecific at higher than optimal detergent concentrations. The mechanism by which the nonionic detergent stimulates DNA synthesis has not yet been resolve. We assume that other factors in addition to DNA polymerase are involved in elongation of cDNA100.

Initiation of DNA synthesis by the avian retrovirus reverse transcriptase in vitro: nature and location of the oligodeoxycytidylic acid primer binding site.

We have investigated the use of oligodeoxycytidylic acid [oligo(dC)] as a primer for the initiation of DNA synthesis by the avian retrovirus reverse transcriptase in vitro, employing the viral RNA genome as template. The addition of oligo(dC)(12-18) to viral 35S RNA results in a stimulation of DNA synthesis by the viral RNA-directed DNA polymerase comparable to that observed when oligo(dT) is employed as a primer. Under similar conditions neither oligo(dA)(12-18) nor oligo(dG)(12-18) was active as primer for transcription of the avian retrovirus genome. Several different approaches have been employed to localize the oligo(dC)(12-18) binding site on the viral genome, including isolation of poly(A)-containing fragments, competition hybridization, and RNase H hydrolysis. These analyses indicate that oligo(dC)(12-18) binds to a site approximately 2,000 to 3,000 nucleotides from the 3' terminus of the genome of transforming strains of avian sarcoma viruses and approximately 700 to 1,000 nucleotides from the 3' terminus of nontransforming avian retroviruses. Therefore, the major site of initiation of DNA synthesis by oligo(dC)(12-18) appears to be in the vicinity of the 3' end of the env gene and the 5' end of the src gene, although the presence of minor initiation sites located elsewhere on the viral genome cannot be excluded by these data. Characterization of oligonucleotides after pancreatic RNase hydrolysis and poly(C)-Sepharose chromatography of viral RNA directly demonstrates the presence of oligoguanylic acid residues in the avian sarcoma virus genome. DNA sequences transcribed from the oligo(dC) primer appear to be conserved in all of the avian leukosis-sarcoma viruses tested. The use of oligo(dC) as a tool for the production of specific complementary DNA probes is discussed.

Detection of the Viral Sarcoma Gene Product in Cells Infected with Various Strains of Avian Sarcoma Virus and of a Related Protein in Uninfected Chicken Cells

Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.

Cell-free translation of avian sarcoma virus RNA: Suppression of the gag termination codon does not augment synthesis of the joint gag/pol Product

The RNA genome of avian sarcoma virus (ASV) and 38S polyadenylated RNA from cells producing ASV directed the cell-free synthesis of two viral gene products: Pr76 gag, the precursor to structural proteins of the virion core, and P180 gag/pol, a joint product of two viral genes that is the putative precursor of viral reverse transcriptase. To test the hypothesis that P180 gag/pol is synthesized by read-through of the termination codon for gag, we determined the effect of purified yeast suppressor tRNAs on cell-free translation from gag and pol. Although the synthesis of P180 gag/pol was not enhanced by the suppressor tRNAs, the use of amber suppressor tRNA reduced the synthesis of Pr76 gag and resulted in the synthesis of a new 80, 000 dalton polypeptide (P80) that contained the antigenic determinants of gag. Studies using chemical inhibitors of initiation of translation demonstrated that P80 was generated by the addition of amino acids to the carboxy terminus of Pr76 gag. We conclude that the gag gene of ASV is terminated by an amber codon (UAG), and that a second termination signal occurs ∼120 nucleotides downstream. Our data indicate that suppression of a single termination codon does not suffice for the production of P180 gag/pol. Thus it is doubtful that suppression by tRNA is responsible for the synthesis of this protein. The mechanism by which P180 gag/pol is synthesized in infected cells remains uncertain.

At least 104 nucleotides are transposed from the 5′ terminus of the avian sarcoma virus genome to the 5′ termini of smaller viral mrnas

Cells producing avian sarcoma virus (ASV) contain at least three virus-specific mRNAs, two of which are encoded within the 3′ half of the viral genome. Each of these viral RNAs can hybridize with single-stranded DNA (cDNA 5′) that is complementary to a sequence of 101 nucleotides found at the 5′ terminus of the ASV genome, but not within the 3′ half of the genome. We proposed previously ( Weiss, Varmus and Bishop, 1977) that this nucleotide sequence may be transposed to the 5′ termini of viral mRNAs during the genesis of these RNAs. We now substantiate this proposal by reporting the isolation and chemical characterization of the nucleotide sequences complementary to cDNA 5′, in the genome and mRNAs of the Prague B strain of ASV. We isolated the three identified classes of ASV mRNA (38, 28 and 21S) by molecular hybridization; each class of RNA contained a “capped” oligonucleotide identical to that found at the 5′ terminus of the ASV genome. When hybridized with cDNA 5′ each class of RNA gave rise to RNAase-resistant duplex hybrids that probably encompassed the full extent of cDNA 5′. The molar yields of duplex conformed approximately to the number of virus-specific RNA molecules in the initial samples; hence most if not all of the molecules of virus-specific RNA could give rise to the duplexes. The duplexes prepared from the various RNAs all contained the capped oligonucleotide found at the 5′ terminus of the viral genome and had identical “fingerprints” when analyzed by two-dimensional fractionation following hydrolysis with RNAase T1. In contrast, RNA representing the 3′ half of the ASV genome did not form hybrids with cDNA 5′. We conclude that a sequence of more than 100 nucleotides is transposed from the 5′ end of the ASV genome to the 5′ termini of smaller viral RNAs during the genesis of these RNAs. Transposition of nucleotide sequences during the production of mRNA has now been described for three families of animal viruses and may be a common feature of mRNA biogenesis in eucaryotic cells. The mechanism of transposition, however, and the function of the transposed sequences are not known.