Avian Sarcoma Virus Genome Research Articles

Cycloleucine blocks 5'-terminal and internal methylations of avian sarcoma virus genome RNA

Cycloleucine, a competitive inhibitor of ATP: L-methionine S-adenosyltransferase in vitro, has been used to reduce intracellular concentrations of S-adenosylmethionine and by this means to inhibit virion RNA methylation in chicken embryo cells that are infected with B77 avian sarcoma virus. Under conditions of cycloleucine treatment, where virus production as measured by incorporation of radioactive precursors or by number of infectious particles is not significantly affected, the internal m6A methylations of the avian sarcoma virus genome RNA are inhibited greater than 90%. The predominant 5'-terminal structure in viral RNA produced by treated cells in m7G(5')pppG (cap zero) rather than m7G-(5')pppGm (cap 1). It appears from these results that internal m6A and penultimate ribose methylations are not required for avian sarcoma RNA synthesis and function. Furthermore, these methylations are apparently not required for transport of genome RNA to virus assembly sites. The insensitivity of the 5'-terminal m7G methylation to inhibition by cycloleucine suggests that the affinity of S-adenosylmethionine for 7-methylguanosine methyltransferase is significantly greater than for the 2'-0-methyltransferases or the N6-methyltransferases.

Evidence for splicing of avian sarcoma virus 5′-terminal genomic sequences onto viral-specific RNA in infected cells

The 5'-terminal nucleotide sequences of the avian sarcoma virus (ASV) genome are transcribed by the reverse transcriptase in vitro into a DNA transcript that represents the entire distance ( approximately 100 nucleotides) between the tRNA(Trp) primer molecule and the 5' terminus. We have used these DNA(100) transcripts in hybridization reactions with ASV-specific RNA from infected avian cells and find nucleotide sequences complementary to these transcripts on all of the various size classes of viral mRNA identified. Similar hybridization results were obtained with a specific DNA transcript complementary to viral genomic nucleotide sequences between the tRNA(Trp) primer molecule and up to, but not including, the terminal redundant sequences (DNA(70)), indicating that the observed hybridization of DNA(100) to all size classes of viral RNA in infected cells did not reflect hybridization of DNA(100) to the terminal redundant sequences at the 3' end of the viral genome. Escherichia coli RNase H hydrolysis of RNA.DNA hybrids consisting of genomic 35S RNA obtained from virus and DNA(100) transcripts indicated that viral genomic sequences complementary to these DNA transcripts were not present at sites distal to the ends of the RNA genome and therefore not adjacent to the corresponding gene sequences representing the various species of viral mRNA from infected cells. These studies suggest that the 5'-terminal genomic nucleotide sequences, or a portion thereof, are somehow added or "spliced" onto each ASV-specific mRNA species in infected cells either during or after transcription of proviral DNA for some as yet undetermined purpose.

RNase H hydrolysis of the 5' terminus of the avian sarcoma virus genome during reverse transcription.

NUCLEOTIDE sequence analyses of the ends of the avian retro-virus genome 1–6 have confirmed predictions of the terminally redundant nature of the viral RNA7,8. These studies have also provided the basis for several models of retrovirus proviral DNA synthesis, all of which have implicated the RNase H activity associated with viral reverse transcriptase in a functional role in the continued and uninterrupted DNA transcription of the viral RNA genome1–8. According to these models DNA synthesis initiates on the tRNAtrp primer molecule located close to the 5′ end of the viral genome9,10 and transcription proceeds to the terminus. Presumably, at this time a suitable substrate is available for the retrovirus RNase H, which is a processive exoribonuclease requiring an unblocked terminus of the RNA moiety of RNA:DNA hybrids for activity11–13. Release of the terminally repeated nucleotides from the hybrid region of the viral genome would create ‘sticky ends’ with the tRNAtrp-initiated DNA at the 5′ end capable of hybridising to the terminally redundant RNA genomic sequences at the 3′ end of the same or a second 35S RNA subunit. This latter reaction would facilitate uninterrupted transcription from the 5′ to the 3′ end of the viral genome resulting in genome-length DNA transcripts15–20. We have recently obtained evidence indicating that DNA transcripts much longer than the distance between the tRNAtrp primer molecule and the 5′ end of the viral genome and containing nucleotide sequences representing the 3′ region of the viral genome can be synthesised by the reverse transcriptase in vitro21. Thus, it seems that DNA synthesis initiated at the 5′ end of the viral genome continues at the 3′ end in enzymatic reactions in vitro. If the avian sarcoma virus (ASV) reverse transcriptase-associated RNase H activity is required for the continued transcription of the viral genome at the 3′ end as proposed in above models, then release of 5′ terminally-located ribonucleotides should be apparent during DNA synthesis in vitro. We report here that ribonucleotides are indeed released from the viral RNA genome during reverse transcription, and that hydrolysis occurs at a specific site near the 5′ terminus. These studies exemplify the first demonstration of RNase H hydrolysis occurring during reverse transcription of the retro-virus RNA genome in vitro and implicate functional role for this activity during replication of retroviruses.

Identification of a polypeptide encoded by the avian sarcoma virus src gene.

Two techniques were used to search for the polypeptide encoded by the avian sarcoma virus (ASV) src gene. First, antiserum from rabbits bearing ASV-induced fibrosarcomas was used to immunoprecipitate a transformation-specific antigen from ASV-transformed chick embryo fibroblasts. This antigen has an apparent molecular weight (Mr) of 60,000. Second, the 3' one-third of the ASV genome, selected by oligo(dT)-cellulose chromatography and sucrose gradient sedimentation, was translated in a mRNA-dependent reticulocyte cell-free lysate. This RNA species programmed the synthesis of a polypeptide that comigrated with the transformation-specific antigen of Mr 60,000 immunoprecipitated from transformed cells. The methionine-containing tryptic peptides from the polypeptides of Mr 60,000 obtained from translation in vitro and from immunoprecipitation were found to be identical upon two-dimensional fractionation.

Transmission of B77 Virus Double Mutant LA334 Markers by Transfection

Successful transfection of Brown Leghorn chicken fibroblasts was carried out with DNA isolated from duck cells transformed by the LA334 mutant of avian sarcoma virus B77. Transfection of duck cells was negative. The four viruses recovered after transfection were all temperature-sensitive for transformation. Two were fully temperature-sensitive for replication, as shown by analysis of virus replication, by characterization of virus particles produced at the nonpermissive temperature using density gradient centrifugation, and by electron microscopic examination. The other two viruses were only partially temperature-sensitive for replication. The results suggest that both the src and gag regions of the avian sarcoma virus genome are transferred simultaneously during transfection, probably by a single integral provirus copy.

The size and genetic composition of virus-specific RNAs in the cytoplasm of cells producing avian sarcoma-leukosis viruses

The genome of avian sarcoma virus (ASV) contains four known genes: gag, encoding structural proteins of the viral core; pol, encoding the viral RNA-directed DNA polymerase; env, encoding the glycoprotein(s) of the viral envelope; and src, which is responsible for neoplastic transformation of the host cell. We have located these genes on virus-specific RNAs in cells productively infected with both nondefective and defective strains of ASV by using molecular hybridization with DNAs complementary to specific portions of the ASV genome. The cytoplasm of cells producing nondefective ASV contains three species of polyadenylated virus-specific RNA, each of which has chemical polarity identical to that of the viral genome. The largest species has a molecular weight of 3.3 × 10 6 daltons and a sedimentation coefficient of 38S, encodes all four viral genes, and is probably identical to the viral genome. A second species has a molecular weight of 1.8 × 10 6 daltons and a sedimentation coefficient of 28S, and encodes the 3′ half of the viral genome, including env, src and a genetically silent region known as “c.” The smallest species has a molecular weight of 1.2 × 10 6 daltons and a sedimentation coefficient of 21S, and encodes only src and “c.” All three species of virus-specific RNA contain nucleotide sequences at least partially homologous to a sequence of 101 nucleotides found at the extreme 5′ end of the ASV genome. This sequence may not be present in the portions of the ASV genome which encode the 28S and 21S virus-specific RNAs, and hence may be joined to these RNAs during their maturation from precursor molecules. The size and genetic composition of virus-specific RNAs in cells producing defective deletion mutants reflect the nature of the deletion. Deletions of either src or env eliminate the 28S virus-specific RNA, leaving a 21S RNA (which contains either env and “c” in the case of src deletions or src and “c” in the case of env deletions) and a 35S RNA which is probably identical to the viral genome. Based on these and related results, we propose a model for viral gene expression which conforms to previous suggestions that eucaryotic cells initiate translations only at the 5′ termini of messenger RNAs.

Occurrence of partial deletion and substitution of the src gene in the RNA genome of avian sarcoma virus

The genome size of 20 transformation-defective (td) viruses derived from different strains of Rous sarcoma viruses [Prague (subgroups A and C), Schmidt-Ruppin (subgroups A and D) (SR-D), Bratislava 77, and Carr-Zilber subgroup D)] was examined by polyacrylamide gel electrophoresis. All of the td viruses except td SR-D have 35S RNA of the same size-i.e., class b RNA. Two of five td SR-D viruses examined have a slightly larger RNA, corresponding to a td deletion that is about 25% smaller than that of class b RNA. However, the RNase T(1)-oligonucleotide fingerprints of all the td SR-D viruses are identical, lacking two sarcoma-specific oligonucleotides. The fingerprints of these viruses also showed a minor oligonucleotide present at very low concentration. A study of heteroduplex molecules formed between genome-length cDNA made from wild-type SR-D and 35S RNA of td SR-D showed a deletion loop of 2.0 and 1.5 kilobases, respectively, at the map position of the src gene for these two classes of td SR-D viruses, confirming the results of polyacrylamide gel electrophoresis. In addition, some heteroduplex molecules with a substitution loop of 0.6-0.7 kilobase at the same site as the deletion loop were observed in all five of the td SR-D viruses. We conclude that some of the td SR-D viruses have a partially deleted src gene and that all of the td SR-D viruses have incorporated heterologous sequences of distinct length in some RNA molecules at the position of the src gene. The nature and origin of these heterologous sequences are discussed.

Translation of avian type C virus messenger RNA: Evidence for multiple initiation sites

The avian sarcoma virus genome has been shown to consist of four genes arranged in the following order: 5′- gag pol-env-src-3′. In the present study, the pol gene product was found to be expressed in avian tumor virus-infected cells at several-hundred-fold lower levels than the translational products of the gag and env genes. Moreover, a translational block or possible deletion involving the carboxy-terminal region of the gag gene in chicken embryo cells had no apparent influence on the level of expression of the env gene. These findings indicate the involvement of multiple initiation sites for translation of the avian type C viral genome.

Integration of avian sarcoma virus DNA sequences in transformed mammalian cells.

DNA from six avian sarcoma virus (ASV)-transformed mammalian cell lines was digested with the restriction endonucleases EcoRI, Xho I, or Sal I, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filter strips, and hybridized with specific ASV [32P]cDNA probes. DNA from all of the ASV-transformed cell lines yielded three common virus-specific DNA fragments (2.4, 1.8, and 1.3 X 10(6) daltons) upon cleavage with EcoRI. Xho I appeared to cleave at least once within the integrated provirus and yielded a common fragment of 3.3 X 10(6) daltons as well as a second virus-specific DNA fragment whose size varied from 4.0 to 5.0 X 10(6) daltons in the different transformed cell lines. Sal I did not cleave within the provirus and yielded a single major virus-specific fragment of about 11 X 10(6) daltons in all transformed lines examined. Using specific cDNA probes, we show that the 1.8 X 10(6)-dalton EcoRI fragment contains sequences homologous to the 3' end of the viral RNA as well as to the src region of the viral genome. These studies clearly demonstrate that the same region on the ASV genome is utilized for provirus integration in different ASV-transformed cell lines.

Characterization of dna complementary to nucleotide sequences adjacent to poly(A) at the 3′-terminus of the avian sarcoma virus genome

We have prepared DNA complementary to a sequence of 300–400 nucleotides adjacent to the poly(A) at the 3′-terminus of the genome of avian sarcoma virus (ASV) DNA was transcribed from denatured ASV RNA following initiation on the primer (dT) 12–18. We demonstrated that homopolymer synthesis was not a significant feature of our enzymatic reaction, and we proved that most if not all the initiations on (dT) 12–18 occurred at or near the 3'-terminus of the viral RNA. The presence of oligo(dT) on cDNA 3, accelerates hybridization between cDNA 3, and polyadenylated viral RNA; this artifact can be eliminated by including a competing homopolymer [poly(dT) or poly(A)l in the reaction mixtures. Nucleotide sequences complementary to cDNA 3, occur only once or twice in the genome of avian sarcoma viruses, are present in the genomes of virus strains from subgroups A, B, C, D, E, and F of avian leukosis-sarcoma viruses, and are conserved even when large deletions affect an adjacent viral gene ( src) By contrast, we find little or no complementarity between cDNA 3, and the genomes of golden pheasant virus (subgroup G of avian leukosis-sarcoma viruses), mouse mammary tumor virus, and the Moloney strains of murine sarcoma virus and murine leukemia virus. cDNA 3, can be used to identify the 3′-terminus of the avian sarcoma virus genome by molecular hybridization and will therefore be a useful reagent for the analysis of viral replication.

Characterization of dna complementary to nucleotide sequences at the 5′-terminus of the avian sarcoma virus genome

The 5′-end of the genome of avian sarcoma virus (ASV) contains a sequence of nucleotides which is the initial template for transcription by RNA-directed DNA polymerase. We have prepared DNA complementary to this sequence (cDNA 5,) and have characterized the sequence by chemical analysis and molecular hybridization. The length of cDNA 5, (00 nucleotides) provides an estimate of the distance from the primer for DNA synthesis (tRNAtrp) to the 5′-terminus of the genome. As reported previously by other investigators, transcription of DNA from the genome of ASV in vitro can be arrested at preferred sites short of the 5′-terminus by reduction in the concentration of either dCTP or dGTP. The nucleotide sequence complementary to cDNA 5, is similar but not identical in the genomes of two strains of ASV, and homologous sequences are present in the genomes of other avian leukosis-sarcoma viruses. By contrast, we find little or no complementarity between cDNA 5, and the genomes of golden pheasant virus (subgroup G of avian leukosis-sarcoma virus), Moloney murine leukemia virus, and Moloney murine sarcoma virus. This pattern of homologies mirrors that found at the 3′termini of RNA tumor virus genomes. A nucleotide sequence complementary to most or all of cDNA 5, occurs within the 3′-half, but not at the 3′-terminus, of the ASV genome; we suggest that this sequence may be a ribosome-binding site. cDNA 5, constitutes the bulk (usually greater than 75%) of DNA synthesized under conventional conditions with detergent-activated virions. Consequently, the use of this DNA to analyze homologies among retrovirus genomes is of limited value.

Nucleotide sequence at the 5' terminus of the avian sarcoma virus genome.

Transcription of DNA from the RNA genome of avian sarcoma virus by RNA-directed DNA polymerase in vitro initiates on a primer (tRNATrp) located near the 5'-terminus of the viral genome. One of the major products of transcription is a single-stranded DNA chain complementary to a sequence of 101 nucleotides immediately distal to the site of initiation of DNA synthesis. We have determined the complete nucleotide sequence of this transcribed chain for the Prague strain of avian sarcoma virus, a partial sequence of the transcribed chain for the Bratislava 77 strain of avian sarcoma virus, and the sequence of a DNA transcript that is shorter than the transcribed single-stranded chain. Our data define the location of tRNATrp on the genome of avian sarcoma virus and provide the sequence of 119 nucleotides at the 5'-terminus of the genome. Portions of this sequence may be involved in the binding of RNA-directed DNA polymerase, the initiation of translation from viral messenger RNA, the extension of RNA-directed DNA synthesis from the 5'- to the 3'-terminus of viral RNA, and the integration of viral DNA into the host genome.

Nucleotide sequence that binds primer for DNA synthesis to the avian sarcoma virus genome

Initiation of transcription from the genome of avian sarcoma virus by RNA-directed DNA polymerase in vitro requires tRNAtrp as a primer. The tRNA is bound to the viral genome by a sequence of 16 contiguous nucleotides (U-C-A-C-G-U-C-G-G-G-G-U-C-A-C-Cp), beginning with the penultimate base at the 3' terminus of the primer and extending through the acceptor stem into loop IV of the tRNA. Consequently, the native conformation of the tRNA must be disrupted by the binding of primer to the viral genome. The binding sequence does not include two adjacent residues of pseudouridine in loop IV, which distinguish the primer from many other tRNAs, and the 3' terminal adenosine of primer may also be excluded from base pairing with the viral genome.

Suppression of the Avian Sarcoma Virus Genome in 8-Azaguanine-Resistant, Transformed, Hamster Cells1

The avian sarcoma virus genome (Schmidt-Ruppin strain) in transformed hamster cells resistant to 8-azaquanine [Ha(SR)AG-50] was strongly suppressed. The suppression was genetically stable and could not be overcome by attempts at induction with 5-iodo-2'-deoxyuridine. Fusion of hamster cells, which had suppressed virus genome, with chicken Rous-associated virus (RAV-1)-preinfected cells easily rescued the sarcoma virus. The rescued virus had envelope properties of RAV-1, as determined by viral interference, virus neutralization, and plating on genetically resistant chicken cells. By repeatedly cloning the rescued virus, we determined that virus recombined in the rescue experiment and that the recombinant virus had the envelope properties of helper virus used for its rescue. Cells with suppressed avian sarcoma virus genome were suitable for preparation of different recombinant viruses.

The defective maturation of viral progeny with a temperature-sensitive mutant of avian sarcoma virus

LA334 (previously ts 75) has been identified as a mutant exhibiting two genetic lesions affecting late functions of the avian sarcoma virus genome. One causes the transformed phenotype to be temperature sensitive, and the other induces a rapidly reversible inhibition of progeny virus production at the nonpermissive temperature. The nature of the latter defect has been investigated in this study with the following findings: (1) no new protein synthesis was needed to initiate synthesis of infectious virus after a shift from the nonpermissive to the permissive temperature; (2) significant amounts of physical particles (20 to 70% of that produced at the permissive temperature) were produced at the nonpermissive temperature; and (3) intracellular accumulation of structures resembling budding virus, approximately 80% of which were aberrant, were observed in mutant-infected cells at the nonpermissive temperature. The possibility that a defect in the synthesis of the viral envelope glycoprotein, gp85, would account for these findings prompted an investigation of the expression of gp85 in LA334-infected cells. Intracellular synthesis of gp85 in normal amounts was shown by immunoprecipitation; however, only inefficient interference against superinfecting virus could be observed with mutant-infected cells at the nonpermissive temperature. Immunoferritin electron microscopic observations also indicated the presence of envelope glycoprotein in association with budding virions of normal morphology, but in contrast, little or no glycoprotein was associated with the budding structures of aberrant morphology. It is concluded that the primary virus defect is not related to expression of gp85; rather, on the basis of the abnormal core assembly, it is proposed that a defect exists in one of the core structural proteins. This hypothesis is discussed in the light of known properties of the mutant and of certain recent observations on the composition of the noninfectious virus produced by infected cells at the nonpermissive temperature.

Evidence of methylation of B77 avian sarcoma virus genome RNA subunits

B77 avian sarcoma virus RNA was labeled with (methyl-3H) methionine under conditions that prevent non-methyl incorporation of 3H radioactivity into purine rings. From the determined values for the extent of methylation of 4S RNA isolated from infected chicken embryo cells, it was estimated that 30 to 40S RNA subunits that results from heat denaturation of the 60 to 70S RNA contain approximately 21 methyl groups, of which 14 to 16 are present at internal positions as N6 -methyladenosine residues. In addition, each of the virion RNA subunits appears to contain about two methyl groups in the "capped" 5' -terminal structure m7G(5')ppp(5') gm. These properties are consistent with the hypothesis that the 30 to 40S genome RNA os oncornaviruses also serves an mRNA function in infected cells.

Reassociation of 4 S and 5 S RNA's with the genome of avian sarcoma virus

A tryptophan tRNA derived from the host cell serves as primer for the initiation of DNA synthesis by the reverse transcript ase of avian sarcoma virus (ASV). We have characterized the binding of primer to the viral genome by studying the reassociation of low molecular weight RNA's with high molecular weight subunits of ASV-RNA. Primer can be annealed to a limited number (two to four) of sites on the viral genome to reconstitute a template fully active with reverse transcriptase. The annealing is specific and accurate; none of the other 4 S RNA's found in ASV will reassociate with viral genome under any of the conditions tested, and the reconstructed complex denatures with the same T m as the native complex between primer and viral genome. These results further substantiate our previous conclusion that the primer in ASV is a single species of tRNA, although the normal host cell may contain other 4 S RNA's which could also serve as primer if they were included in the viral genome during assembly. Chicken ribosomal 5 S RNA also reanneals to the ASV genome, presumably by binding to the sites originally occupied by 5 S RNA in viral 70 S RNA.

Avian sarcoma virus transformed hamster cells made resistant to ethidium bromide.

ABSTRACT Hamster cells transformed with the Schmidt-Ruppin strain of avian sarcoma virus were selected for resistance to ethidium bromide (EB). The resistant cell lines proliferated in the presence of up to 30 μg/ml EB. From avian sarcoma virus-transformed hamster cells already resistant to bromodeoxyuridinu (BrdU), ethidium bromide-resistant cells which were able to grow in 10μg/ml EB were also prepared. These cells remain deficient in thymidine kinase activity and are suitable for selective preparation of hybrid cells. The EB resistance was genetically stable. The EB-resistant cell lines, and doubly resistant cells (BrdU, EB) showed no differences in mitochondrial ultrastructure compared with the original cell lines. Thymidine incorporation into mitochondrial DNA was not influenced by EB resistance. All resistant cell lines, including the doubly resistant cell line, contained the avian sarcoma virus genome. The number of cells needed for positive rescue experiments for avian sarcoma virus genome by cell fusion with permissive chicken embryo cells was the same as with the original cell lines. The single EB-resistant cell lines contained R-type virus-like particles, while in BrdU-rcsistant and doubly resistant cells the R-type particles were absent. The possible nature of EB resistance is discussed.

Avian sarcoma virus transformed hamster cells resistant to bromodeoxyuridine and deficient in thymidine kinase activity.

Hamster cells transformed with the Schmidt-Ruppin strain of avian sarcoma virus were selected for resistance to 5-bromodeoxyuridine (BUDR). The resistant cell lines Ha(SR)BU-25 and Ha(SR)BU-100, proliferated in the presence of 25 and 100 μg/ml BUDR, respectively. The resistant cells were deficient in thymidine kinase activity. They did not grow in HATG medium and did not incorporate labelled thymidine into DNA. Several single-cell clones were isolated in medium containing 100 μg/ml BUDR from Ha(SR)BU-100 cells. These isolated cell clones differed in morphology and modal number of chromosomes from each other. None of the clones incorporated thymidine into DNA. The BUDR resistant cells were tested for their tumorigenicity in young hamsters. The number of cells needed for induction of tumor growth was higher with the BUDR resistant cells than with original clone of Ha(SR) cells. All clones of thymidine kinase deficient cells were tested for the presence of the virus genome by fusion with chicken embryo cells. All clones of Ha(SR)BU-100 cells contained the virus genome, and infectious virus could be rescued from these cells. Therefore, the genome of avian sarcoma virus can persist in virogenic hamster cells growing in the presence of 5-bromodeoxyuridine. These virogenic cells deficient in thymidine kinase activity are useful for preparation of cell hybrids.