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An intact 5.7-kb provirus of the avian erythroblastosis virus E26 has been molecularly cloned for comparisons with avian myeloblastosis virus (AMV) and other avian tumor viruses. E26 and AMV transform hemopoietic cells exclusively. Both cause myeloblastosis, but E26 also causes erythroblastosis. Sequence analysis of the proviral DNA showed that: The tripartite transforming gene of E26 forms a contiguous reading frame of 1046 codons, including 272 gag, 283 mybE, and 491 ets codons. No subgenomic ets-specific mRNA was detected in E26-infected cells. By contrast, the onc gene of AMV consists almost entirely of a mybA sequence expressed via subgenomic mRNA that extends over the 5' and 3' ends of mybE. mybE is only slightly diverged from the mybA homolog of AMV and even less from the cellular proto-myb sequence with no characteristic mutation that sets apart the two viruses from proto-myb. The U5 region of the long terminal repeat (LTR) of E26 and AMV are colinear and differ only in scattered point mutations. The U3 region of the E26 LTR is different from that of AMV but is colinear and closely related with that of avian carcinoma virus MH2 and also with that of Prague Rous sarcoma virus (RSV), except for an unexpected 16-nucleotide substitution of 22 RSV nucleotides. Upstream of the 3' LTR, the c region of E26 appears to be the same as that of RSV for 70 nucleotides and very similar to those of AMV and MH2 for about 20 to 30 nucleotides. Since the U3s of E26, MH2 and RSV are very closely related and neither MH2 nor RSV show a particular erythroblast tropism, it is possible that the U3 does not play a critical role in the erythroblast tropism of E26. Electrophoretic size analyses of chicken DNA digested with restriction enzymes indicate that DNA fragments totaling over 50 kb hybridize with viral ets DNA.

A nonconditional mutant of the Schmidt-Ruppin strain of Rous sarcoma virus (subgroup A) that fails to synthesize infectious progeny virus is described. Nonproducing cells transformed by this mutant LA 7365 have functional viral src and env genes. The gag gene of LA 7365 is defective. There is no complementation with a gag ts mutant, and pulse-chase experiments show that the proteolytic processing of the gag precursor pr76 is strongly inhibited in mutant-infected cells. Whether there is an additional lesion in the pol gene of LA 7365 could not be determined from the available data.

A variant of Moloney murine sarcoma virus (Mo-MSV) reported to behave like a nondefective sarcoma virus was subjected to biological and biochemical analyses to determine whether its alleged helper-independence could be confirmed. When plated at low multiplicity the virus was shown to readily generate (four out of six) transformed clones which failed to produce virus unless superinfected with helper leukemia virus. The RNA of the parental virus stock was compared electrophoretically to that from clones which produced virus after the initial infection (producer clones) or after superinfection with Mo-murine leukemia virus (MLV) (nonproducer clones). All clones contained a MSV-specific 30 S RNA species. In addition, virus from one producer clone also contained 38 S MLV RNA at a high relative concentration, indicating that the original Mo-MSV stock must have contained such an RNA species. However, the original virus stock as well as virus from another producer clone contained 38 S MLV RNA at a low, uncertain relative concentration. A hypothesis consistent with these and previous data suggests that the Mo-MSV variant investigated here is defective and contains helper leukemia virus at a low concentration. This explains (i) the ready generation of nonproducer clones by infection at low multiplicity, (ii) the difficulty in detecting helper leukemia virus 38 S RNA in the original virus stock, and (iii) the low complexity (approximately 1.9 × 10 6 daltons) of the MSV-specific 30 S RNA. These results are compatible with the properties reported for a defective MSV genome, but incompatible with those of a nondefective MSV genome. The MSV-specific RNA components of different clonal isolates of Mo-MSV differed from each other in size, ranging between 2.1 and 1.6 × 10 6 . The Harvey sarcoma virus-specific RNA was 1.9 × 10 6 , that of Kirsten sarcoma virus was 2.5 × 10 6 , and the spleen focus forming component of Friend virus was 2.0 × 10 6 . The sarcoma- or transformation-specific RNA components of all transforming viruses tested here were smaller than the 38 S RNA of helper leukemia viruses of 3.1 × 10 6 .

Replication-defective deletion mutants of Rous sarcoma virus (RSV) have been described which transform cells in culture and elaborate envelope (−) defective particles. The env deletions of two clonal variants of the Bryan strain of RSV, RSV(−)3, and RSV(−)16, and of a replication-defective variant of Schmidt-Ruppin RSV (SRN8) were analyzed by fingerprinting oligonucleotides hybridized by a molecularly cloned env DNA probe that spans from near the 3′ end of pol to the 3′ end of env . It was observed that all three replication-defective RSV strains are essentially complete env deletions but retain the 3′ end of pol . Based on a common pol-src junction oliogonucleotide that may reflect a homologous sequence repeated at both ends of env in nondefective RSV, the env deletions of RSV(−)3 and 16 appear to be isogenic. The original deletion may have involved recombination between these sequences. The absence of this oligonucleotide in SRN8 indicates that the env deletion of SRN8 has different borders and represents an independent env deletion of nondefective RSV. All three defective RSVs have the genetic structure gag-pol-src . This genetic structure is consistent with the need for a complete gag to make a particle and with the assumption that an independent src gene rather than a gag - or gag-pol-src hybrid gene functions in transformation. It is suggested that a complete pol is not necessary for, but may assist, virus particle formation.

Two glycoprotein components containing glucosamine, galactose, and fucose were isolated from different strains of Rous sarcoma virus (RSV) and from Rauscher mouse leukemia virus (MLV). The molecular weight of the major glycoprotein of RSV was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) to be between 90,000 and 105,000 daltons, depending on the strain of RSV. The minor glycoprotein had an approximate molecular weight of 37,000. Both glycoproteins have higher molecular weights than the proteins of the group-specific antigen of avian tumor viruses. The glycoproteins represent between 10% and 20% of the radioactive protein of purified virus. Disruption of RSV with SDS yields glycoprotein components with sedimentation coefficients between 4 S and 2 S. The glycoprotein derived from Tween 20 disrupted virus was obtained in a 4-2 S form and in an 8 S form. The 8 S component consists predominantly of a multimeric aggregate of the two viral glycoprotein components, and some group-specific antigen. Because both the 8 S and the 4-2 S glycoprotein derived from Tween 20 disrupted RSV specifically inhibited virus neutralizing antibody and because the glycoproteins of different strains of RSV had different electrophoretic mobilities, it was concluded that the glycoproteins of avian tumor viruses are part of or identical with the viral type- or subgroup-specific antigen.

The glycoproteins of several Rous sarcoma virus (RSV) strains were found to have a slightly lower electrophoretic mobility in polyacrylamide gels than those of corresponding leukosis viruses of the same subgroup and strain. The glycoproteins of T5, a temperature-sensitive mutant of Schmidt-Ruppin RSV affecting the transforming ability of the virus, had a lower electrophoretic mobility in virus propagated at the permissive temperature than in virus propagated at the nonpermissive temperature. Further the glycopeptides of viruses released from transformed and nontransformed cell were compared. Viral glycopeptides were prepared by exhaustive pronase digestion of viral glycoproteins. It was found that the glycopeptides of all viruses released from transformed cells were larger (MW ≈ 5100) than those of viruses (MW ≈ 3900) released from normal cells. These differences were not strain- or subgroup-specific, as all viruses belonging to different subgroups and strains, which were released from transformed cells, had large glycopeptides. The same was true for glycopeptides, which were all small, of viruses released from phenotypically normal cells. The roles of cellular and viral genes in determining the size of viral glycoproteins and glycopeptides are discussed.

The transforming (onc) genes of retroviruses contain specific sequences, derived from as yet poorly defined, normal cellular genes, termed proto-onc genes. Proto-onc genes must be defined to explain their docility compared to the oncogenicity of the viral derivatives. Here we set out to determine the borders of the chicken proto-fps gene from which the onc genes of avian Fujinami (FSV) and PRC sarcoma viruses (PRCSV) are derived. These onc genes are hybrids of an element from the gag gene of retroviruses (delta gag) linked to a 2.8-kb domain from proto-fps. To identify the 5' border of proto-fps we have sequenced 1.5 kb beyond the 5' border of overlap with viral fps utilizing a proto-fps clone derived previously. A possible promoter was identified that maps 736 nucleotides from this border. The 736 nucleotides contain two possible exons with 121 codons, and short regions of homology with the delta gag termini of FSV and PRCII. A translation stop codon and an adjacent polyadenylation signal were identified just prior to the 3' border of overlap with viral fps within a 1.15-kb sequence of a newly isolated proto-fps clone. Comparing four exons within this 1.15 kb proto-fps sequence with known fps equivalents of FSV and PRCSV, we have detected strain-specific, but no common point mutations in each viral genome. A 3.3-kb polyadenylated proto-fps mRNA was detected in chicken liver RNA by gel electrophoresis and hybridization with proto-fps DNA. We conclude that the coding capacity of proto-fps is just over 3 kb, consistent with the size of the putative proto-fps protein of 98 kDa and hence slightly larger than that of viral fps. Thus proto-fps and the viral delta gag-fps genes each contain distinct 5' regulatory and coding sequences and share the 3' terminal fps domains. It is suggested that this difference, rather than scattered point mutations, is responsible for the oncogenic function of the viral genes and the unknown cellular function of proto-fps.

Kirsten sarcoma virus-xenotropic leukemia virus complex (Ki-MSV(MLV)) produced by transformed NRK rat cells was shown to replicate in duck cells. Infected duck cells assumed a transformed morphology and produced virus at about the same rate as NRK rat cells. The RNAs of Ki-virus produced by duck and rat cells were indistinguishable by comparative electrophoresis and fingerprinting of RNase T1-resistant oligonucleotides. The viral proteins p10, p12, p15, and p30 of rat- and duck-derived virus were electrophoretically indistinguishable. Electrophoretic evidence suggested that the viral glycoprotein gp70 of rat-propagated virus is slightly larger than that of duck-propagated virus. Preliminary analysis indicated that the glycoprotein from rat-produced virus contains larger glycopeptides than the corresponding glycoprotein from duck-produced virus. It is suggested that the size difference between the viral glycoproteins from different cells is host-controlled.

The avian sarcoma viruses FSV, PRCII, PRCIIp, and PRCIV share a related class of hybrid onc genes (Δ gag-fps ) defined by a specific nucleotide sequence fps and by Δ gag-fps proteins of different sizes. Among these viruses, PRCII appears to have a lower tumorigenic potential than the others. Here we have compared fibroblast-transforming function and onc gene structure of these viruses. The fibroblast transforming ability of PRCII was lower than those of FSV, PRCIIp, and PRCIV. By gel electrophoresis the genomic RNA of PRCII measured 3.5 kb and those of FSV, PRCIIp, and PRCIV 4.5 kb; the Δ gag-fps protein of PRCII measured 105 kilodaltons (kd), that of FSV 140 kd, and those of PRCIIp and PRCIV about 150 kd. By fingerprinting viral RNAs hybridized with molecularly cloned viral DNA the Δ gag regions of PRCII and PRCIIp were defined to be 1.45 kb and that of FSV to be 1.3 kb. Fingerprint analysis of viral RNA-proto fps DNA hybrids showed the fps regions (approximately 2.8 kb) of FSV and PRCIIp to be isogenic. Compared to FSV and PRCIIp, the fps sequence of PRCII lacked a 1-kb region which maps between 0.3 and 1.3 kb from the 5′ end of fps in FSV and PRCIIp. Based on oligonucleotide analysis, the shared fps complements of PRCII and PRCIIp were indistinguishable while that of FSV differed from those of the PRC viruses in scattered point mutations amounting to 1–2% of the RNA. Since all other regions of PRCII are isogenic with those of the highly tumorigenic variants PRCIIp, PRCIV, and FSV, it is concluded that the low fibroblast-transforming and oncogenic potential of PRCII reflects the internal fps deletion. Since the fps deletion reduces but does not eliminate transforming function, we suggest that the complete onc genes of viruses in the FSV' subgroup include either several functional, or a regulatory and a functional fibroblast transforming domain. It has been reported that the 3′ domains of the onc genes of viruses in the Fujinami subgroup and the onc genes of certain feline sarcoma viruses are distantly related. Since full transforming potential of the avian viruses depends on the 5′ fps region not shared with the feline sarcoma viruses, we suggest that despite their structural homology, the avian and feline onc genes must have functionally different domains.

The RNAs and proteins of five laboratory strains of defective avian acute leukemia virus MC29, differing in passage history and in some cases in their helper viruses and oncogenic spectra, were investigated for genetic variation. Earlier work based on homology with nondefective viruses of the avian tumor virus group has distinguished in 5.7-kb (kilobase) MC29 RNA a 5′ group-specific section, an internal MC29-specific section, and a 3′ group-specific section, and has determined that 5′ and internal sections together are translated into a nonstructural, gag gene-related 110K (kilodaltons) protein. The RNAs of the five MC29 strains analyzed here shared 20 conserved T 1 -oligonucleotides and differed in specific sets selected from 10 variable oligonucleotides. Since some, but not all, variable and some conserved MC29-oligonucleotides were shared with helper viruses, and since the 5′ cap-oligonucleotides of MC29 RNA rescued from the same nonproducer cell varied with that of the helper virus RNA, it is concluded that genetic variation of MC29 includes recombination with helper virus. Oligonucleotide maps of the five MC29 RNAs located 8 variable oligonucleotides in their 3′ group-specific sections, only conserved oligonucleotides in their internal-specific section, and mostly conserved oligonucleotides in their 5′ group-specific section (except for variable 5′ cap-oligonucleotides and the lack of an internal oligonucleotide in one strain). The 110K proteins of the MC29 strains were electrophoretically indistinguishable, which is in accord with the conserved nature of their templates. The ratio of MC29 RNA to helper virus RNA in virions was found to reflect, by a constant, the ratio of 110K MC29 to helper viral proteins in transformed cells. MC29 RNA is proposed to consist of two genetic units: one made of the highly conserved 5′ and internal-specific RNA sections, which codes for the 110K protein and appears to serve a primary function in oncogenicity; the other made of the variable 3′ group-specific RNA sections which may affect oncogenicity indirectly.

The avian Fujinami sarcoma virus (FSV) contains a hybrid transforming gene (Δ gag-fps ) with a 5′ 1.3-kb portion derived from the gag gene of avian retroviruses and a 3′ 2.8-kb portion ( fps ) derived from a cellular prototype. A lambda recombinant DNA clone carrying fps sequences within a 16-kb insert of cellular DNA, termed lambda proto- fps clone 12, has been selected from a chicken DNA library for comparison with the viral onc gene. Mapping of endonuclease-resistant proto- fps DNA fragments and hybridization with cloned viral DNA located FSV-related sequences at the 3′ end of the insert within a region of about 4.25 kb. Alignment of endonuclease-resistant proto- fps and viral DNA fragments relative to common RNase T 1 -resistant oligonucleotide sequences of viral RNA, identified by fingerprinting DNA-RNA hybrids, indicated: (i) that proto- fps is colinear with viral fps but is interrupted by 1.75 kb of scattered sequences unrelated to viral fps ; (ii) that among the nine endonuclease sites compared, proto- fps and viral fps share one Pvu II, one Bam HI, and possibly a Kpn I site at homologous locations, and that they each have unique endonuclease sites and common sites at unique locations; (iii) that within 12 kb upstream from the 5′ boundary of overlap with viral fps , proto- fps lacks gag -related sequences; and (iv) that proto- fps clone 12, like several others isolated by us, lacks at the 3′ end an equivalent of the 3′ 10 to 20% of viral fps . The eight endonuclease site-map coordinates of proto- fps and viral DNA also divided 44 fps oligonucleotides of viral RNA into 9 map segments. We conclude that the onc gene of FSV differs from proto- fps in Δ gag and in multiple point mutations, compatible with a transforming function for the viral gene and a normal function for the cellular sequence homolog. Since proto- fps is unrelated to essential virion genes, the onc gene of FSV must have originated from cellular proto- fps by rare, illegitimate recombination.

Pseudotype preparations of the defective avian oncovirus MH2 contain a large excess of helper virus when grown in fibroblasts but relatively more MH2 when replicated in macrophages. Such macrophage grown virus was used for an accurate size determination of the genomic RNA of MH2. This 28 S RNA species contains between 5700 and 5900 nucleotides and has a molecular weight of 1.9 × 106.

The sequence of 500 nucleotides of a cellular proto- fps region that includes the 5′ boundary of overlap with the fps sequence of Fujinami sarcoma virus (FSV) was determined. It was found that the potential reading frame of the proto- fps region continues over the 5′ boundary of overlap with viral fps for 44 codons which do not include an AUG translation initiation codon. A potential upstream exon of proto- fps of over 91 codons and a potential splice acceptor at the site of fps /proto- fps overlap have been identified. It is concluded that the boundary of overlap between proto- fps and viral fps cannot represent the 5′ end of the coding region of proto- fps . The proto- fps gene starts either in the upstream exon(s) not shared with FSV or at least 51 codons downstream of the boundary of overlap with FSV. In addition, the 5′ coding region of proto- fps differs from the (Δ gas-fps ) hybrid transforming gene of FSV in the absence of the retroviral gag gene-derived sequence.

We have investigated the RNAs of two avian sarcoma viruses recovered (rASV) from tumors induced in chickens by a deletion mutant of Schmidt-Ruppin Rous sarcoma virus (SR-RSV) that had lost part, but not all, of its sarcoma gene (src). The RNAs of the rASVs had the same size as SR-RSV RNA and were larger than the predominant RNA species of the partial src deletion mutant, if measured by electrophoresis in polyacrylamide gels. Fingerprinting of RNase T,-resistant oligonucleotides indicated that the rASVs shared one src gene oligonucleotide with SR-D which was also present in the partial src deletion mutant of SR-RSV. The two rASVs shared one other, probable src oligonucleotide, that was not found in SR-RSV, and SR-RSV contained a src oligonucleotide not found in the rASVs. However, the distinctive src oligonucleotide of the rASVs was structurally closely related to that of SR-RSV. We conclude that the src genes of the rASVs and that of SR-RSV are closely related. Possible mechanisms by which a partial src deletion may recover a complete src gene are discussed in view of our results.

The 50-70 S RNA complex of a clone of Kirsten murine leukemia virus (Ki-MLV), propagated in NIH 3T3 cells, as well as that of a Moloney (Mo)-MLV, propagated in a mouse thymus-bone marrow cell line, contained an approximately 30 S RNA of unknown biological activity in addition to 38 S genomic RNA. The approximately 30 S RNA shares with 38 S virion RNA the ability to form 50-70 S RNA complexes and to be packaged by virus particles. The morphology of the cell lines propagating these viruses was indistinguishable from that of uninfected controls.

The proteins of purified avian tumor viruses, including the Bryan Rous sarcoma and Rous-associated virus (RSV (RAV)), avian myeloblastosis virus (AMV), the “defective” ∗ NP-particle (H. L. Robinson, 1967) which is also known as RSV(0) (Vogt, 1967) and the nondefective Schmidt-Ruppin RSV(SR-RSV) were isolated by the phenol method. Each amino acid-labeled virus was found to contain two major and one minor radioactive proteins. These three proteins correspond to about 60–80% of the total radioactivity of amino-acid labeled RSV(RAV), RSV(0), and SR-RSV. Polyacrylamide gel electrophoresis under two different conditions showed that each virus contained the same three proteins in similar relative amounts. The two major proteins had a high complement-fixation titer with antibody against the group-specific antigen of the avian leukosis viruses, suggesting that the group-specific antigen of the avian leukosis viruses consists of at least two proteins. The “defective” RSV(0) isolated from NP-cultures Hanafusa et al., 1963 , Hanafusa et al., 1964 ; H. L. Robinson, 1967) contained all the proteins of the group-specific antigen detectable by the described methods. From this it is concluded that the “defectiveness” of the Bryan strain of RSV is not based on a lack of the group-specific antigen in the virus particle.