Purification of DNA complementary to nucleotide sequences required for neoplastic transformation of fibroblasts by avian sarcoma viruses.

We have prepared radioactive DNA (cDNA_sarc) complementary to nucleotide sequences which represent at least a portion of the viral gene(s) required for neoplastic transformation of fibroblasts by an avian sarcoma virus. The genetic complexity of cDNA_sarc (~1600 nucleotides) is sufficient to represent an entire cistron. The genomes of three independent isolates of avian sarcoma viruses share nucleotide sequences closely related to cDNA_sarc, whereas the sequences are absent from transformation-defective mutants of avian sarcoma viruses, several avian leukosis viruses, a non-pathogenic endogenous virus of chickens (Rous-associated virus-O), sarcoma-leukosis viruses of mice and cats, and mouse mammary tumor virus. We conclude that the transforming gene(s) of all avian sarcoma viruses have closely related or common genetic lineages distinct from the transforming genes in sarcoma viruses of other species. Our results conform to previous reports that transformation-defective variants of avian sarcoma viruses are mutants with identical regions deleted from each subunit of a polyploid genome.     

Comparison between the viral transforming gene (src) of recovered avian sarcoma virus and its cellular homolog.

Recovered avian sarcoma viruses are recombinants between transformation-defective mutants of Rous sarcoma virus and the chicken cellular gene homologous to the src gene of Rous sarcoma virus. We have constructed and analyzed molecular clones of viral deoxyribonucleic acid from recovered avian sarcoma virus and its transformation-competent progenitor, the Schmidt-Ruppin A strain of Rous sarcoma virus. A 2.0-megadalton EcoRI fragment containing the entire src gene from each of these clones was subcloned and characterized. These fragments were also used as probes to isolate recombinant phage clones containing the cellular counterpart of the viral src gene, termed cellular src, from a lambda library of chicken deoxyribonucleic acid. The structure of cellular src was analyzed by restriction endonuclease mapping and electron microscopy. Restriction endonuclease mapping revealed extensive similarity between the src regions of Rous sarcoma virus and recovered avian sarcoma virus, but striking differences between the viral src's and cellular src. Electron microscopic analysis of heteroduplexes between recovered virus src and cellular src revealed a 1.8-kilobase region of homology. In the cellular gene, the homologous region was interrupted by seven nonhomologous regions which we interpret to be intervening sequences. We estimate the minimum length of cellular src to be about 7.2 kilobases. These findings have implications concerning the mechanism of formation of recovered virus src and possibly other cell-derived retrovirus transforming genes.     

DNA sequence of the viral and cellular src gene of chickens. II. Comparison of the src genes of two strains of avian sarcoma virus and of the cellular homolog

The nucleotide sequence of the src gene and flanking regions of the Schmidt-Ruppin strain of Rous sarcoma virus (SR-A) was determined. The src region of SR-A was very homologous to that of recovered avian sarcoma virus (rASV1441), with only 17 differences among 1,578 nucleotides. The size of the predicted protein was 526 amino acids in both viruses, of which 6 amino acids were different. The differences in nucleotides and amino acids between the two viruses localized within the 5' two-thirds of the src coding region. There were also viruses localized within the 5' two-thirds of the src coding region. There were also some differences in the region flanking the 5' end of src. Since rASVs are considered to be recombinatns between deletion mutants of SR-A and cellular-src (c-src) sequences, several segments of c-src DNA were also sequenced to understand the molecular basis for the recombination. At 14 of 17 bases where SR-A and rASV1441 differed, rASV1441 had the same sequence as c-src. Three of these sequences corresponded to sequences of oligonucleotides which were previously identified in RNAs of nearly all isolates of rASV but which were absent in SR-A RNA. In the 5'-flanking sequences of the src gene, c-src was more similar to rASV1441 than to SR-A. These results confirm the cellular origin of the src sequences of rASVs and provide information about the possible sites of the recombination.     

Analysis of the src gene of sarcoma viruses generated by recombination between transformation-defective mutants and quail cellular sequences.

Tumors were produced in quails about 2 months after injection with a transformation-defective mutant of the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (SR-A), that retains a small portion of the src gene. Sarcoma viruses were isolated from each of five such tumors. A transformation-defective mutant which has a nearly complete deletion of the src gene was unable to induce tumors. The avian sarcoma viruses recovered from quail tumors (rASV-Q) had biological properties similar to those of the avian sarcoma viruses previously acquired from chicken tumors (rASV-C); these chicken tumors had been induced by the same transformation-defective mutants. Both rASV-Q and rASV-C transformed cells in culture with similar focus morphology and produced tumors within 7 to 14 days after injection into chickens or quails. The size of rASV-Q genomic RNA was indistinguishable from that of SR-A by polyacrylamide gel electrophoresis. The sequences of rASV-Q RNA genomes were analyzed and compared with those of the parental transformation-defective virus, SR-A and of rASV-C by RNase T1 fingerprinting and oligonucleotide mapping. We found that the src sequences of all five isolates of rASV-Q were identical to each other but different from those of SR-A and rASV-C. Of 13 oligonucleotides of rASV-Q identified as src specific, two were not found in either SR-A or rASV-C RNA. Furthermore, some oligonucleotides present in SR-A or rASV-C or both were absent in rASV-Q. No differences were found for the sequences outside the src region in any of the viruses examined. In addition, rASV-Q-infected cells possessed a 60,000-dalton protein specifically precipitable by rabbit serum raised against SR-D-induced tumors. The facts that the src sequences are essentially the same for rASV's recovered from one animal species and different for rASV's obtained from different species provide conclusive evidence that cellular sequences of normal birds were inserted into the viral genome and supplied to the resulting recombinant viruses genetic information for cell transformation.     

Identification of nucleotide sequences which may encode the oncogenic capacity of avian retrovirus MC29.

The retrovirus strain MC29 induces a variety of tumors in chickens, including myelocytomatosis and carcinomas of the kidney and liver. In addition, the virus can transform cultures of embryonic avian macrophages and fibroblasts. We have characterized the genome of MC29 virus and have identified nucleotide sequences that may encode the oncogenic potential ofthe virus. MC29 virus can replicate only with the assistance of a related helper virus. The defect in replication is apparently a consequence of a deletion in one or more viral genes: the haploid genome of the MC29 virus has a molecular weight of ca. 1.7 X 10(6), whereas the genome of the helper virus MCAV has a molecular weight of ca. 3.1 X 10(6). Although MC29 virus transforms fibroblasts in culture, its genome has no detectable homology with the gene src that is responsible for transformation of fibroblasts by avian sarcoma viruses. We prepared radioactive single-stranded DNA complementary to nucleotide sequences present in the genome of MC29 virus but not in the genome of MCAV (cDNA(MC29)). If they are contiguous, these sequences (ca. 1,500 nucleotides) are sufficiently complex to encode at least one protein. Homologous sequences were not detectable in several strains of avian sarcoma viruses or in an endogenous virus of chickens. Our findings confirm and extend recent reports from other laboratories and lead to the conclusion that MC29 virus may contain a previously unidentified gene(s) that is capable of transforming several distinct target cells. The evolutionary origins of this putative gene and its location on the viral genome can be explored with cDNA(MC29).     

Nucleotide Sequences Derived from Pheasant DNA in the Genome of Recombinant Avian Leukosis Viruses with Subgroup F Specificity

Recombination between viral and cellular genes can give rise to new strains of retroviruses. For example, Rous-associated virus 61 (RAV-61) is a recombinant between the Bryan high-titer strain of Rous sarcoma virus (RSV) and normal pheasant DNA. Nucleic acid hybridization techniques were used to study the genome of RAV-61 and another RAV with subgroup F specificity (RAV-F) obtained by passage of RSV-RAV-0 in cells from a ring-necked pheasant embryo. The nucleotide sequences acquired by these two independent isolates of RAV-F that were not shared with the parental virus comprised 20 to 25% of the RAV-F genomes and were indistinguishable by nucleic acid hybridization. (In addition, RAV-F genomes had another set of nucleotide sequences that were homologous to some pheasant nucleotide sequences and also were present in the parental viruses.) A specific complementary DNA, containing only nucleotide sequences complementary to those acquired by RAV-61 through recombination, was prepared. These nucleotide sequences were pheasant derived and were not present in the genomes of reticuloendotheliosis viruses, pheasant viruses, and avian leukosis-sarcoma viruses of subgroups A, B, C, D, and E. They were partially endogenous, however, to avian DNA other than pheasant. The fraction of these nucleotide sequences present in other avian DNAs generally paralleled the genetic relatedness of these avian species to pheasants. However, there was a high degree of homology between these pheasant nucleotide sequences and related nucleotide sequences in the DNA of normal chickens as indicated by the identical melting profiles of the respective hybrids.     

Purification of DNA complementary to the env gene of avian sarcoma virus and analysis of relationships among the env genes of avian leukosis-sarcoma viruses.

The env gene of avian leukosis-sarcoma viruses encodes a glycoprotein that determines the host range and surface antigenicitiy of virions. We have purified radioactive DNA (cDNAgp) complementary to at least a portion of the env gene for viral subgroups A and C; complementary DNA was synthesized with purified virions of wild-type avian sarcoma virus, and RNA from a mutant with a deletion in env was used to select DNA specific to env by molecular hybridization. The genetic complexity of cDNAgp for subgroup A (ca. 2,000 nucleotides) was sufficient to represent the entire deletion and most or all of the env cistron. The deletions in env in two independently isolated strains of virus (Bryan and rdNY8SR) overlap, and cDNAgp represents nucleotide sequences common to both deletions. By contrast, we could detect no overlap between deletions in env and deletions in the adjacent viral gene src. Laboratory stocks of viral subgroups A, B, C, D and E do not contain detectable amounts of env deletions when tested by molecular hybridization; hence, segregation of deletions in env is a less frequent event that the segregation of deletions in the viral transforming gene src (Vogt, 1971). We found extensive homology among the nucleotide sequences encoding the env genes of virus strains indigenous to chickens (subgroups A, B, C, D, and E) although subgorups B, D and E appear to differ slightly from subgroups A and C at the env locus. By contrast, viruses obtained from pheasant cells (subgroups F and G) have env genes with little or no relationship to env genes of chikcen viruses. According to available data, viruses of subgroup F arose by recombination between an avarian sarcoma virus and viral genes in the genome of ring-necked pheasants, whereas subgroup G viruses may be entirely endogenous to golden pheasants.     

Evidence for the common origin of viral and cellular sequences involved in sarcomagenic transformation.

The src genes of six different strains of avian sarcoma virus (ASV) were compared with those of a series of newly isolated sarcoma viruses, termed "recovery avian sarcoma viruses" (rASV's). The rASV's were isolated recently from chicken and quail tumors induced by transformation-defective (td) deletion mutants of Schmidt-Ruppin Rous sarcoma virus. The RNase T1-resistant oligonucleotide maps were constructed for the RNA genomes of different strains of ASV and td mutants. The src-specific sequences, characterized by RNase T1-resistant oligonucleotides ranging from 9 to 19 nucleotides long, were defined as those mapping between approximately 600 and 2,800 nucleotides from the 3' polyadenylate end of individual sarcoma viral RNAs, and missing in the corresponding td viral RNAs. Our results revealed that 12 src-specific oligonucleotides were highly conserved among several strains of ASV, including the rASV's, whereas certain strains of ASV were found to contain one to three characteristic src-specific oligonucleotides. We previously presented evidence supporting the idea that most of the src-specific sequences present in rASV RNAs are derived from cellular genetic information. Our present data indicate that the src genes of rASV's are closely related to other known ASVs. We conclude that the src genes of different strains of ASV and the cellular sarc sequences are of common origin, although some divergence has occurred among different viral src genes and related cellular sequences.     

The avian retrovirus env gene family: molecular analysis of host range and antigenic variants.

The nucleotide sequence of the env gp85-coding domain from two avian sarcoma and leukosis retrovirus isolates was determined to identify host range and antigenic determinants. The predicted amino acid sequence of gp85 from a subgroup D virus isolate of the Schmidt-Ruppin strain of Rous sarcoma virus was compared with the previously reported sequences of subgroup A, B, C, and E avian sarcoma and leukosis retroviruses. Subgroup D viruses are closely related to the subgroup B viruses but have an extended host range that includes the ability to penetrate certain mammalian cells. There are 27 amino acid differences shared between the subgroup D sequence and three subgroup B sequences. At 16 of these sites, the subgroup D sequence is identical to the sequence of one or more of the other subgroup viruses (A, C, and E). The remaining 11 sites are specific to subgroup D and show some clustering in the two large variable regions that are thought to be major determinants of host range. Biological analysis of recombinant viruses containing a dominant selectable marker confirmed the role of the gp85-coding domain in determining the host range of the subgroup D virus in the infection of mammalian cells. We also compared the sequence of the gp85-coding domain from two subgroup A viruses, Rous-associated virus type 1 and a subgroup A virus of the Schmidt-Ruppin strain of Rous sarcoma virus. The comparison revealed 24 nonconservative amino acid changes, of which 6 result in changes in potential glycosylation sites. The positions of 10 amino acid differences are coincident with the positions of 10 differences found between two subgroup B virus env gene sequences. These 10 sites identify seven domains in the sequence which may constitute determinants of type-specific antigenicity. Using a molecular recombinant, we demonstrated that type-specific neutralization of two subgroup A viruses was associated with the gp85-coding domain of the virus.     

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.     

3''-Terminal region of avian carcinoma virus MH2 shares sequence elements with avian sarcoma viruses Y73 and SR-A.

We determined the nucleotide sequence of the acute transforming avian retrovirus MH2 from an HgiAI site within the coding region of its oncogene, v-myc, to the KpnI site within the long terminal repeat. Comparison with published sequences from other retroviruses allowed us to identify all sequence elements in this region. We conclude that MH2 contains a unique assembly of 3'-terminal sequences, which includes part of the helper virus-derived SPC region of avian sarcoma virus Y73 and the complete F3 and F1 segments of Rous sarcoma virus strain SR-A.     

Induction of tumors and generation of recovered sarcoma viruses by, and mapping of deletions in, two molecularly cloned src deletion mutants.

td108 , a transformation-defective (td) deletion mutant of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup A (SR-A), was molecularly cloned. Two isolates of td viruses, td108 -3b and td108 -4a, obtained by transfection of the molecularly cloned td108 DNAs into chicken embryo fibroblasts, were tested for their ability to induce tumors and generate recovered avian sarcoma viruses ( rASVs ) in chickens. Both td viruses were able to induce tumors with a latency and frequency similar to those observed previously with biologically purified td mutants of SR-A. rASVs were isolated from most of the tumors examined. The genomic RNAs of those newly obtained rASVs were analyzed by RNase T1 oligonucleotide fingerprinting. The results showed that they had regained the deleted src sequences and contained the same set of marker src oligonucleotides as those of rASVs analyzed previously. The src oligonucleotides of rASVs are distinguishable from those present in SR-A. We conclude that those rASVs must have been generated by recombination between the molecularly cloned td mutants and the c-src sequence. The deletions in the td mutants were mapped by restriction enzyme analysis and nucleotide sequencing. td108 -3b was found to contain an internal src deletion of 1,416 nucleotides and to retain 57 and 105 nucleotides of the 5' and 3' src coding sequences, respectively. td108 -4a contained a src deletion of 1,174 nucleotides and retained 180 and 225 nucleotides of the 5' and 3' src sequences, respectively. Comparison of sequences in the 5' src and its upstream region of td108 -3b with those of SR-A, rASV1441 (a td108 -derived rASV analyzed previously), and c-src suggested that the 5' recombination between td108 and c-src occurred from 7 to 20 nucleotides upstream from the beginning of the src coding sequence.     

Nucleotide sequences of feline retroviral oncogenes (v-fes) provide evidence for a family of tyrosine-specific protein kinase genes

The nucleotide sequences encoding the transforming polyproteins of the Snyder-Theilen and Gardner-Arnstein strains of feline sarcoma virus (FeSV) have been determined. These sequences include a viral transforming gene (v-fes), derived from cellular proto-oncogene sequences (c-fes) of domestic cats by recombination with feline leukemia virus (FeLV). The v-fes sequences are predicted to encode a polypeptide domain strikingly similar to that specified by the transforming gene (v-fps) of the avian Fujinami sarcoma virus. In addition, the 3′ 0.8 kilobase pairs of v-fes encode amino acid sequences homologous to the carboxy-terminal portion of pp60^src, the transforming protein encoded by the avian Rous sarcoma virus src gene. Thus different feline and avian retroviral transforming genes, all of which encode functionally related proteins with associated tyrosine-specific kinase activities, must be derived from divergent members of the same protooncogene family.     

env Gene of Chicken RNA Tumor Viruses: Extent of Conservation in Cellular and Viral Genomes

The env gene of avian sarcoma-leukosis viruses codes for envelope glycoproteins that determine viral host range, antigenic specificity, and interference patterns. We used molecular hybridization to analyze the natural distribution and possible origins of the nucleotide sequences that encode env; our work exploited the availability of radioactive DNA (cDNA(gp)) complementary to most or all of env. env sequences were detectable in the DNAs of chickens which synthesized an env gene product (chick helper factor positive) encoded by an endogenous viral gene and also in the DNAs of chickens which synthesized little or no env gene product (chick helper factor negative). env sequences were not detectable in DNAs from Japanese quail, ring-necked pheasant, golden pheasant, duck, squab, salmon sperm, or calf thymus. The detection of sequences closely related to viral env only in chicken DNA contrasts sharply with the demonstration that the transforming gene (src) of avian sarcoma viruses has readily detectable homologues in the DNAs of all avian species tested [D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature (London) 260: 170-173, 1976] and in the DNAs of other vertebrates (D. Spector, personal communication). Thermal denaturation studies on duplexes formed between cDNA(gp) and chicken DNA and also between cDNA(gp) and RNAs of subgroup A to E viruses derived from chickens indicated that these duplexes were well matched. In contrast, cDNA(gp) did not form stable hybrids with RNAs of viruses which were isolated from ring-necked and golden pheasants. We conclude that substantial portions of nucleotide sequences within the env genes of viruses of subgroups A to E are closely related and that these genes probably have a common, perhaps cellular, evolutionary origin.     

Rate of divergence of cellular sequences homologous to segments of Moloney sarcoma virus.

The RNA genome of the Moloney isolate of murine sarcoma virus (M-MSV) consists of two parts--a sarcoma-specific region with no homology to known leukemia viral RNAs, and a shared region present also in Moloney murine leukemia virus RNA. Complementary DNA was isolated which was specific for each part of the M-MSV genome. The DNA of a number of mammalian species was examined for the presence of nucleotide sequences homologous with the two M-MSV regions. Both sets of viral sequences had homologous nucleotide sequences present in normal mouse cellular DNA. MSV-specific sequences found in mouse cellular DNA closely matched those nucleotide sequences found in M-MSV as seen by comparisons of thermal denaturation profiles. In all normal mouse cells tested, the cellular set of M-MSV-specific nucleotide sequences was present in DNA as one to a few copies per cell. The rate of base substitution of M-MSV nucleotide sequences was compared with the rate of evolution of both unique sequences and the hemoglobin gene of various species. Conservation of MSV-specific nucleotide sequences among species was similar to that of mouse globin gene(s) and greater than that of average unique cellular sequences. In contrast, cellular nucleotide sequences that are homologous to the M-MSV-murine leukemia virus "common" nucleotide region were present in multiple copies in mouse cells and were less well matched, as seen by reduced melting profiles of the hybrids. The cellular common nucleotide sequences diverged very rapidly during evolution, with a base substitution rate similar to that reported for some primate and avian endogenous virogenes. The observation that two sets of covalently linked viral sequences evolved at very different rates suggests that the origin of M-MSV may be different from endogenous helper viruses and that cellular sequences homologous to MSV-specific nucleotide sequences may be important to survival.     

Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics.

We determined the origin and evolutionary pathways of the PB1 genes of influenza A viruses responsible for the 1957 and 1968 human pandemics and obtained information on the variable or conserved region of the PB1 protein. The evolutionary tree constructed from nucleotide sequences suggested the following: (i) the PB1 gene of the 1957 human pandemic strain, A/Singapore/1/57 (H2N2), was probably introduced from avian species and was maintained in humans until 1968; (ii) in the 1968 pandemic strain, A/NT/60/68 (H3N2), the PB1 gene was not derived from the previously circulating virus in humans but probably from another avian virus; and (iii) a current human H3N2 virus inherited the PB1 gene from an A/NT/60/68-like virus. Nucleotide sequence analysis also showed that the avian PB1 gene was introduced into pigs. Hence, transmission of the PB1 gene from avian to mammalian species is a relatively frequent event. Comparative analysis of deduced amino acid sequences disclosed highly conserved regions in PB1 proteins, which may be key structures required for PB1 activities.     

Primary structure of p19 species of avian sarcoma and leukemia viruses.

The internal structural proteins of avian sarcoma and leukemia viruses are derived from a precursor polypeptide that is the product of the viral gag gene. The N-terminal domain of the precursor gives rise to p19, a protein that interacts with the lipid envelope of the virus and that may also interact with viral RNA. The C terminus of p19 from the Prague C strain of Rous sarcoma virus was previously assigned to a tyrosine residue 175 amino acids from the N terminus. We have used metabolic labeling and carboxypeptidase digestion to show that the C terminus of p19 is actually tyrosine 155. This implies the existence of a sixth gag protein 22 amino acids in length and located between p19 and p10 on the gag precursor. The p19 species of some recombinant avian sarcoma viruses and of the defective endogenous virus derived from the ev-1 locus migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as if they were about 4,000 daltons smaller than p19. We have elucidated the structure of these forms, called p19 beta, by analysis of the proteins and determination of the DNA sequence of the p19 region of the gag gene from ev-1 and ev-2. Esterification of carboxyl groups completely suppressed the differences in migration of p19 and p19 beta. Peptide mapping showed the altered mobility to be determined by sequences in the C-terminal cyanogen bromide fragment of the proteins. We conclude from the DNA sequence that a single glutamate-lysine alteration is responsible for the altered electrophoretic mobility.     

Construction and isolation of a transforming murine retrovirus containing the src gene of Rous sarcoma virus.

Recombinant murine retroviruses containing the src gene of the avian retrovirus Rous sarcoma virus were isolated. Such viruses were isolated from cells after transfection with DNAs in which the src gene was inserted into the genome of the amphotropic murine retrovirus 4070A. The isolated viruses had functional gag and pol genes, but they were all env defective since the src gene was inserted in the middle of the env gene coding region. Infectious transforming virus could be isolated only from cells transfected with DNA constructions in which the src gene was in the same polarity as that of a long terminal repeat of the amphotropic viral genome. These recombinant viruses encoded a pp60src protein with a molecular weight similar to that of the Schmidt-Ruppin strain of Rous sarcoma virus. In addition, the src protein(s) of these recombinant viruses was as active as protein kinases in the immune complex protein kinase assay. Intravenous injection of helper-independent Moloney and Friend murine leukemia virus pseudotypes of the src recombinant viruses into 6-week-old NIH Swiss mice resulted in the appearance of splenic foci within 2 weeks, splenomegaly and, later after infection (8 to 10 weeks), anemia. Infectious transforming virus could be recovered from the spleens of diseased animals. Such viruses encoded pp60src but not p21ras or mink cell focus-forming virus-related glycoproteins.