Rous Sarcoma Virus Subgroup Research Articles

Research Note: Rous sarcoma growth differs among congenic lines containing major histocompatibility (B) complex recombinants

New arrangements of chicken major histocompatibility complex (MHC) class I BF and class IV BG genes are created through recombination. Characterizing the immune responses of such recombinants reveals genes or gene regions that contribute to immunity. Inbred Line UCD 003 (B17B17) served as the genetic background for congenic lines, each containing a unique MHC recombinant. After an initial cross to introduce a specific recombinant, 10 backcrosses to the inbred line produced lines with 99.9% genetic uniformity. The current study compared Rous sarcoma virus (RSV) tumor growth in 5 congenic lines homozygous for MHC recombinants (003.R1 = BF24-BG23, 003.R2 = BF2-BG23, 003.R4 = BF2-BG23, 003.R5 = BF21-BG19, and 003.R13 = BF17-BG23). Two experiments used a total of 70 birds from the 5 congenic lines inoculated with 20 pock forming units of RSV subgroup C at 6 wk of age. Tumor size was scored 6 times over 10 wk postinoculation followed by assignment of a tumor profile index (TPI) based on the tumor size scores. Tumor growth over time and rank transformed TPI values were analyzed by least squares ANOVA. Tumor size increased over the experimental period in all genotypes through 4 wk postinoculation. After this time, tumor size increased in Lines 003.R1, plateaued in Lines 003.R2, 003.R4, and 003.R13, and declined in 003.R5. Tumor growth over time was significantly lower in Line 003.R5 compared with all other genotypes. In addition, Line 003.R5 chickens had significantly lower TPI values compared with Lines 003.R2, 003.R4, and 003.R13. The TPI of Line 003.R1 did not differ significantly from any of the other genotypes. The BF21 in Line 003.R5 produced a greater response against subgroup C RSV tumors than did BF24, found in 003.R1; BF2 found in 003.R2 and R4 as well as BF17 found in 003.R13.

Genetic analysis of Rous sarcoma virus (subgroup A) induced tumourigenesis and performance of synthetic broiler stocks in relation to group-specific antigen shedding

An investigation was made using two high-performance meat lines, synthetic male line (SML) and synthetic dam line (SDL), to assess the susceptibility pattern in response to subgroup A Rous sarcoma virus (RSV) and the effect of group-specific (gs) antigen shedding in eggs, as evidence of avian leukosis virus infection effects on production and fitness traits. The ar gene frequency was observed to be at 0.68 +/- 0.04 and 0.66 +/- 0.06 for SDL and SML lines, respectively. Synthetic broiler lines were fivefold less sensitive to pock expression in chorioallantoic membranes (CAMs) as compared with a White Leghorn (WL-2A) reference line, in response to Bryan standard RSV (subgroup A). It was also observed that gs antigen shedding had neither influence on pock response in CAMs nor on induced liver tumour death. The prevalence of gs antigen shedding, however, fluctuated from generation to generation, ranging from 16.66 to 69.23% in SDL and 20.68 to 51.85% in SML, indicating an exogenous infection. Analysis of the relationship of gs antigen shedding in eggs with some fitness traits and overall flock production traits provided strong evidence that fitness traits such as fertility, hatchability, and production traits, such as 6-week body weight and egg weight, were significantly influenced by gs shedding.

Genetic susceptibility of indigenous chicks to subgroup A Rous sarcoma virus inoculated via the chorioallantoic membrane

An investigation was made using chicks of two Indian indigenous breeds of fowl, Kadaknath and Aseel, to ascertain genetic resistance to infection by Rous sarcoma virus of subgroup A. A standard inoculation dose of 0.2 ml virus containing 1000 pock forming units ml −1 was injected via the chorioallantoic membrane (CAM) into the 11-day-old embryos that were subsequently hatched. The sensitivity of the two indigenous breeds was compared with the highly susceptible exotic White Leghorn (WL) strain maintained in the laboratory. The Kadaknath breed was about three-fold and Aseel, about six-fold less sensitive than the WL strain, indicating superiority of the indigenous breeds over the exotic breed of fowl. Most of the CAM-susceptible chicks died of liver tumour (LT) and most of the CAM-resistant chicks survived. However, conversely associated tumour phenotype subclass chicks, i.e. CAM-susceptible LT-negative chicks that survived and CAM-resistant LT-positive chicks that died, occurred consistently in the three breeds of fowl. Nevertheless, the overall survival potential of Kadaknath chicks measured up to 8 weeks post-hatching was greater than that of Aseel chicks. Neither transformation of embryonic tissue prior to hatching nor the visceral metastasis including liver conformed with the degree of CAM-infection as measured by number of pocks on CAMs.

Genetic analysis of the Rous sarcoma virus subgroup D env gene: mammal tropism correlates with temperature sensitivity of gp85

Subgroup D avian sarcoma and leukosis viruses can penetrate a variety of mammalian cells in addition to cells from their natural host, chickens. Sequences derived from the gp85-coding domain within the env gene of a mammal-tropic subgroup D virus (Schmidt-Ruppin D strain of Rous sarcoma virus [SR-D RSV]) and a non-mammal-tropic subgroup B virus (Rous-associated virus type 2) were recombined to map genetic determinants that allow penetration of mammalian cells. The following conclusions were based on host range analysis of the recombinant viruses. (i) The determinants of gp85 that result in the mammal tropism phenotype of SR-D RSV are encoded within the 160 codons that lie 3' of codon 121 from the corresponding amino terminus of the gp85 protein. (ii) Small linear domains of the SR-D RSV gp85-coding domain placed in the subgroup B background did not yield viruses with titers equal to that of the subgroup D virus in a human cell line. (iii) Recombinant viruses that contained subgroup D sequences within the hr1 variable domain of gp85 showed modest-to-significant increases in infectivity on human cells relative to chicken cells. A recombinant virus that contained three fortuitous amino acid substitutions in the gp85-coding domain was found to penetrate the human cell line and give a titer similar to that of the subgroup D virus. In addition, we found that the subgroup D virus, the mutant virus, and recombinant viruses with an increased mammal tropism phenotype were unstable at 42 degrees C. These results suggest that the mammal tropism of the SR-D strain is not related to altered receptor specificity but rather to an unstable and fusogenic viral glycoprotein. A temperature sensitivity phenotype for infectivity of mammalian cells was also observed for another mammal-tropic avian retrovirus, the Bratislava 77 strain of RSV, a subgroup C virus, but was not seen for any other avian retrovirus tested, strengthening the correlation between mammal tropism and temperature sensitivity.

Genetic analysis of the Rous sarcoma virus subgroup D env gene: mammal tropism correlates with temperature sensitivity of gp85.

Genetic analysis of the Rous sarcoma virus subgroup D env gene: mammal tropism correlates with temperature sensitivity of gp85.

Ducks: a new experimental host system for studying persistent infection with avian leukaemia retroviruses

Long-term persistence of the avian leukosis virus (ALV), the transformation-defective mutant of Prague strain Rous sarcoma virus subgroup C (td PR-C) was established in heterologous duck hosts after infection in mid-embryogenesis. Transient viraemia was observed for about 4 weeks after hatching and was lost in most of the infected ducks by about 6 months. Loss of viraemia was accompanied by the increasing synthesis of virus-neutralizing antibodies. In spite of strong virus-neutralizing antibodies, virus was detected by the cocultivation assay in duck tissues throughout the observation period up to 5 years. In the viraemic phase of infection, we found integrated proviruses in various tissues, preferentially in stomach muscle tissue and in the thymus. The long-term persistence of virus was frequently accompanied by liver necrosis and neoplastic diseases. Injection of td PR-C virus into early embryos resulted in more pronounced infection accompanied by an increased copy number of viral DNA per cell, high mortality and remarkable atrophy of thymus tissue in infected ducklings.

A Variant Schmidt‐Ruppin Strain of Rous Sarcoma Virus with Increased Affinity for Mammalian Cells

SR‐RSV‐D(H), a variant virus with extremely high tropism for mammalian cells, was isolated by passage of the Schmidt‐Ruppin strain of Rous sarcoma virus of subgroup D (SR‐RSV‐D) through hamster cells. This variant virus has acquired an altered envelope glycoprotein, encoded by the env gene, that has high affinity for receptors on the surface of mammalian cells. The variant virus transforms rat cells at about 100 times the efficiency of the parental virus, SR‐RSV‐D(S), as assayed by focus formation. Addition of amphotericin B (Fungizone) to the medium at a concentration of 0.2 μg/ml completely inhibited rat cell transformation by SR‐RSV‐D(H), possibly by blocking virus penetration into the cells, whereas the drug showed no inhibitory effect on transformation of chick embryo flbroblast (CEF) cells by the variant virus or on transformation of rat cells by the parental virus. The efficiency of transformation of rat cells by the variant virus was much less than its efficiency of transformation of CEF cells. Analysis of infection of rat cells suggested that the virus can infect rat cells as efficiently as CEF cells but that rat cells were not transformed by the virus as fully as CEF cells because of inefficiency of some post‐penetrational step involved in viral gene expression. The finding that El AY cells, rat cells expressing adenovirus El A gene, were transformed by SR‐RSV‐D(H) as efficiently as CEF cells supports this conclusion and suggests that expression of the E1A gene in rat cells may overcome the defect in the transforming step(s) in rat cells.

Phosphorylation of the transforming protein of Rous sarcoma virus: direct demonstration of phosphorylation of serine 17 and identification of an additional site of tyrosine phosphorylation in p60v-src of Prague Rous sarcoma virus

We provide direct evidence that serine 17 is the major site of serine phosphorylation in p60v-src, the transforming protein of Rous sarcoma virus, and in its cellular homolog, p60c-src. The amino acid composition of the tryptic peptide containing the major site of serine phosphorylation in p60v-src was deduced by peptide map analysis of the protein labeled biosynthetically with a variety of radioactive amino acids. Manual Edman degradation revealed that the phosphorylated serine in this peptide was the amino terminal residue. These data are consistent only with the phosphorylation of serine 17. The major site of serine phosphorylation in chicken p60c-src, the cellular homolog of p60v-src, is contained in a tryptic peptide identical to that containing serine 17 in p60v-src of Schmidt Ruppin Rous sarcoma virus of subgroup A. Serine 17 is therefore also phosphorylated in p60c-src. The p60v-src protein encoded by Prague Rous sarcoma virus was found to contain two sites of tyrosine phosphorylation. The previously unrecognized site of tyrosine phosphorylation may be tyrosine 205 or possibly tyrosine 208. Treatment of Prague Rous sarcoma virus-infected cells with vanadyl ions stimulated the protein kinase activity of p60v-src and increased the phosphorylation of tyrosine 416 but not the phosphorylation of the additional site of tyrosine phosphorylation.

Truncated gag-related proteins are produced by large deletion mutants of Rous sarcoma virus and form virus particles.

Large deletion (LD) mutants of Prague strain Rous sarcoma virus subgroup B (PrB), derived by serial undiluted passage through chicken (C/E) cells, contain two deletions relative to wild-type virus. One of these joins gag sequences in the p12 coding region to env sequences in region encoding gp37; the other deletion spans the src region. Analysis of the viral proteins of QT6 cell clones containing only LD proviruses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major truncated gag-related phosphoprotein of 60,000 to 66,000 daltons (P63LD). P63LD was stable, but could be cleaved in vitro to the predicted products by p15gag. A second gag-related LD protein of about 68,000 to 74,000 molecular weight (P70LD) was also found which often reacted with an anti-gp37 serum. P70LD was unstable and may represent a short-lived gag-gp37 fusion protein. Finally, immunoprecipitation indicated that particles containing P63LD were shed from QT6-LD clones. Thin section preparations of these clones viewed in an electron microscope showed enveloped budding particles of "immature" morphology. Thus, the synthesis and release of particles from infected cells does not require cleavage of the gag precursor, nor does it require the presence of p15 or (most of) p12.

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.

Genetic Aspects of Rous Sarcoma-Induced Tumor Expression in Chickens

In part A of this study, six genotypes involving all combinations of the B blood group haplotypes, B1 , B12, and B15, in White Leghorn inbred line HN were compared for resistance to Rous sarcoma virus (RSV)-induced tumors. Results confirm the findings of others that tumor regression is controlled by genes (R-Rs, r-Rs) linked to the B complex. A comparison of tumor expression of the homozygotes, B1 B1, B12 B12, and B15 B15 demonstrates that R-Rs-12 linked to B12 and r-Rs-1 linked to B1 behave essentially as a fully dominant/recessive allelic pair, but for the allelic pair linked to B12 and B15 (i.e., R-Rs-12 and r-Rs-15) the former is incompletely dominant. In addition, the alleles, r-Rs-1 and r-Rs-15, linked to B1 and B15, respectively are each recessive to the B12-linked allele, R-Rs-12, but differ distinctly one from the other. These results demonstrate that these B complex-linked genes, controlling tumor expression, belong to a multiple allelic series.In part B, the question considered was whether or not a specific B complex-linked locus is required to regress a tumor induced by a specific RSV subgroup. Eight highly inbred Leghorn lines segregating for different B complex haplotypes plus two moderately inbred (40%) heavy-breed lines were studied. Each genetic group was challenged with each of three RSV subgroups, A, B, and C. Although the data were not sufficient to decisively choose either the single- or a multiple-locus hypothesis, the former gave a statistically satisfactory fit to the data. For the 10 different B complex haplotypes, 3 demonstrated significant differences in tumor regression between virus subgroups. This is considered evidence for multiple loci. However, when the results are pooled over the 10 haplotypes, the differences were not statistically significant. This would support the alternative hypothesis of a single B complex-linked locus controlling tumor regression.

Antiserum specific for the carboxy terminus of the transforming protein of Rous sarcoma virus

An antiserum specific for the carboxy terminus of p60src, the transforming protein of Rous sarcoma virus, was produced by immunization of rabbits with a conjugate of bovine serum albumin and the synthetic peptide NH2-Tyr-Val-Leu-Glu-Val-Ala-Glu-COOH. The carboxy-terminal six amino acids of this peptide correspond in sequence to that deduced for the carboxy terminus of the p60src of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup A. The p60src proteins of the several strains of Rous sarcoma virus and the cellular homolog of the viral transforming protein, p60c-src, comprise a polymorphic family of polypeptides. The anticarboxy-terminal serum reacted readily with the p60src proteins of three different strains of Rous sarcoma virus. In contrast, no precipitation of cellular p60c-src could be detected with this serum. This suggests that the viral p60src proteins have identical carboxy termini and that the carboxy terminus of cellular p60c-src may be different from that of viral p60src. The anticarboxy-terminal serum reacted poorly with the subpopulation of viral p60src which is present in a complex with two cellular phosphoproteins. Apparently, the presence of the two cellular proteins interferes with the recognition of p60src by the anticarboxy-terminal serum. It seems likely, therefore, that these two cellular proteins bind to the carboxy-terminal domain of p60src.

Age related resistance to avian leukosis virus. III. Infectious virus, neutralising antibody and tumours in chickens inoculated at various ages

Viraemia and neutralising antibodies were determined in chickens of six age-groups following inoculation with leukosis virus of subgroups A and B at the age of 1 day, and 2, 4, 6, 8 and 10 weeks respectively. The birds were kept in a filtered air positive pressure (FAPP) house. A seventh age-group, accommodated in a separate FAPP-house, was used as an untreated control. Serum samples, received at biweekly intervals between 1-17 weeks post-inoculation, from birds of the groups inoculated at 4, 6, 8 and 10 weeks of age, showed at 1 week post-inoculation a transient viraemia followed by neutralising antibodies at the later sampling times. Neutralising antibody to subgroup A virus was detected in nearly all birds tested; this was not so for antibody to subgroup B. In all four groups the average titre of the former antibody was higher than that of the latter. Midway through the laying period birds of each group inoculated with leukosis virus, and some of the uninoculated controls, were challenged by infection with either subgroup A or B virus. At termination of the experiment survivors from each group were tested for the presence of leukosis virus. The virus recovery was performed with plasma samples, white blood cell preparations and explant cultures of various organs. The plasma samples were all negative; the great majority of blood cell specimens received from birds inoculated early with leukosis virus were positive, whereas the majority of the preparations from the birds inoculated later remained negative. The organ explants from the two youngest age groups were mostly leukosis virus-positive, from the birds inoculated at 4 weeks of age the spleen and kidney explants contained leukosis virus whereas in the groups inoculated at 6, 8 and 10 weeks of age only the spleen explants of birds challenged with subgroup A virus In a subsidiary experiment, started 4 months after the challenge infection, four birds from each group (two challenged with leukosis virus of subgroup A and two with subgroup B) were accommodated in isolators. The birds were challenged again, this time with Rous sarcoma virus (RSV) of the homologous subgroup used for the previous challenge. The tests for virus just prior to the challenge showed leukosis virus only in the white blood cell preparations from the birds in the three youngest age groups; the birds from the older groups were virus-negative. The serological tests after challenge showed neutralising antibodies to both subgroups in birds of nearly all groups. Tumour formation at the site of injection was mainly observed in the chickens challenged with RSV of subgroup B. The virological and serological results as well as the tumour response show that the immune system of birds between 0-4 weeks of age is insufficiently developed to cope with a controlled exposure with leukosis virus, whereas in birds of 4-10 weeks of age an adequate immunological response has developed. The significance of the presence of leukosis virus in sera, plasma, white blood cell preparations and organ explant cultures is mentioned. In programmes for the control of lymphoid leukosis in reproductive stock the use of information on virus and neutralising antibodies is recommended.

Viral and cellular src genes contribute to the structure of recovered avian sarcoma virus transforming protein

Recovered avian sarcoma viruses (rASVs) were obtained from tumors induced by certain transformation-defective (td) mutants of Schmidt-Ruppin strain Rous sarcoma virus of subgroup A (SR-A). The genomes of these td SR-A mutants lack most but not all of the src gene. rASV genomes, however, possess intact src genes, which are largely derived from cellular genetic information, presumably an endogenous cellular gene called c- src, which shares considerable homology with the viral src. To further define the genetic origin of rASV src, we examined by tryptic peptide analysis the product of this gene, pp60 src, from rASV and SR-A, as well as the normal cellular homolog pp60 c-src. We found peptides unique to each putative “parental” protein present together in maps of rASV p60 src, demonstrating that the endogenous cellular c- src gene itself contributes to the structure of rASV pp60 src. Certain isolates of rASV encode pp60 srcs of altered apparent molecular weight. In these cases, the variation in structure was located in the amino-terminal portion of the protein. That such polymorphism can be tolerated suggests that this region of the protein is less critical to the ability of these agents to transform cells.

Mutant and recombinant avian retroviruses with extended host range.

Avian retroviruses of subgroups B and D efficiently infect chicken (C/E) but not turkey (T/BD) cells. We describe here three variants of subgroup B and D viruses that infect both cell types equally well. One of these viruses, NTRE-4, was a recombinant between transformation-defective Prague (Pr) strain Rous sarcoma virus (RSV) subgroup B and the endogenous virus RAV-0; the second, SR-DE-1, was a recombinant between Schmidt-Ruppin RSV subgroup D and defective endogenous virus information. T1 oligonucleotide fingerprint analysis of the genomes of these two viruses showed only a small alteration in the portion of the env gene responsible for subgroup specificity, as indicated by the presence of a single subgroup E oligonucleotide in an otherwise purely subgroup B or D gene. The third virus, hrBO1Pr-B, was a variant of Pr-RSV-B that did not appear to be a recombinant and whose altered host range we attribute to mutation. Analysis of the host range of all three viruses by infection of selectively resistant cells and by interference testing indicates that all use the subgroup B receptor on chicken cells and the subgroup E receptor on turkey cells. These viruses may be analogous to the polytropic recombinant viruses recently found to be associated with leukemia in some strains of mice.

The Influence of Host Adaptation of Rous Sarcoma Virus on the Transfecting Activity of its DNA Provirus

Mammalian cells transformed with either Prague strain Rous sarcoma virus of subgroup C (XC cells) or Schmidt-Ruppin strain Rous sarcoma virus of subgroup D (RSCH cells) yielded virus upon fusion with chick cells. Virus was also rescued by transfection of DNA from these cells on to chick cells. However, virus rescue did not occur upon transfection of duck cells, and fusion with duck cells led to virus rescue only from RSCH and not from XC cells. To investigate this restriction on the duck cells the non-defective Prague strain of Rous sarcoma virus of subgroup C (PR-RSV-C) was adapted for efficient replication in duck embryo cells (daPR-RSV-C) by long-term passage in vitro. However, a second PR-RSV-C isolate, rescued from the rat XC sarcoma line (XC DNA 940 virus), failed to adapt to growth in duck cells. When transformed with daPR-RSV-C, which replicates in duck cells as well as in brown leghorn embryo (BLEF) cells, duck cells yielded DNA which transfects fresh duck cells, in contrast to DNA isolated from chicken or duck cells transformed with parental PR-RSV-C.

Restitution of fibroblast-transforming ability in srcdeletion mutants of avian sarcoma virus during animal passage

The Schmidt-Ruppin strain of Rous sarcoma virus subgroup D (SR-D) gives rise to transformation defective ( td) mutants which have lost either all or almost all of the src gene (standard td or std viruses) or have only a partial deletion of src. These partial deletion mutants, designated ptd viruses, contain genomic RNA slightly larger than std isolates, and heteroduplex analyses suggest that ptd viruses retain approximately 25% of src from the 5′ end of that gene [ Lai et al. (1977) Proc. Natl. Acad. Sci. USA 74, 4781–4785 ]. Several ptd isolates of SR-D were injected into newly hatched chickens and after prolonged latent periods caused sarcomas in about 30% of the birds. The tumors occurred in internal organs away from the site of injection. Infectious sarcoma viruses isolated from these growths show the envelope markers of subgroup D are nondefective for replication and induce a transformation in vitro which is morphologically distinct from that of SR-D. Electrophoresis of 35 S genomic RNA from these recovered sarcoma viruses shows it to be of the size characteristic for nondefective sarcoma viruses. Fingerprint analysis of 32P-labeled RNA from one of the new sarcoma viruses detected all oligonucleotides present in ptd viruses, the src-specific oligonucleotides of SR-D, and one new oligonucleotide not present in SR-D. This new RNase T 1-resistant oligonucleotide and the src-specific oligonucleotides identical to those of SR-D map close to the 3′ end in the genome of the recovered sarcoma virus, which is the position expected for the src gene. These studies suggest that recovered avian sarcoma viruses have acquired cellular sequences which are closely related in structure and function to the viral src gene.

Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo

We have compared the polypeptide products of the src gene of several strains of Rous sarcoma virus produced by in vitro translation of heat-denatured 70S virion RNA in the nuclease-treated reticulocyte lysate with those present in chick cells transformed by these viruses. We have done this by immunoprecipitation, using sera from rabbits injected at birth with Schmidt-Ruppin Rous sarcoma virus. In vitro translation results in the synthesis of at least nine polypeptides which appear to be encoded by the src gene. These range in size from 17,000 to 60,000 daltons. The sera from tumor-bearing rabbits precipitated these polypeptides arising from the in vitro translation of RNA from Schmidt-Ruppin Rous sarcoma virus of both subgroup A and subgroup D and from one stock of Prague Rous sarcoma virus of subgroup C. In each case, all of this family of related polypeptides could be precipitated except the smallest, the 17,000-dalton polypeptide. No precipitation of analogous polypeptides resulting from the translation of RNA from other strains of Rous sarcoma virus was observed. Cells transformed by these three strains of Rous sarcoma virus contain easily detectable amounts of a polypeptide, p60src, essentially identical to the 60,000-dalton in vitro product. With one exception, they do not contain significant amounts of polypeptides analogous to the smaller in vitro products which can be precipitated by these sera. Cells transformed by one stock of Schmidt-Ruppin Rous sarcoma virus of subgroup A did contain a 39,000-dalton polypeptide, which was related, by peptide mapping, to the 60,000-dalton polypeptide and was similar in size to a precipitable in vitro product. The 60,000-dalton polypeptide present in transformed cells appeared to be phosphorylated 10 to 25 min after its synthesis, metabolically very stable, and not derived from a precursor polypeptide. All immunoprecipitates from transformed cells which contained p60src also contained an 80,000-dalton phosphoprotein. This polypeptide is unrelated to p60src, as determined by peptide mapping, and may well be a host cell polypeptide which is specifically associated with p60src.

Molecular events during the interaction of envelopes of myxo- and RNA-tumor viruses with cell membranes. A 270 MHz 1H nuclear magnetic resonance study

The nuclear magnetic resonance (NMR) spectra of chick embryo cells have been analyzed after exposure to Newcastle disease virus (NDV). Virions that contained the envelope glycoproteins in the cleaved form and, thus, had full biological activity have been compared to virions that had reduced infectivity due to the presence of uncleaved glycoprotein F. After exposure to infectious virus, drastic changes occurred in the signals assigned to choline and the hydrocarbon chains of fatty acids. These observations are interpreted to demonstrate alteration of the fluid lipid bilayer structure of the cell membranes. This is compatible with the concept of membrane fusion as a penetration mechanism for NDV. Virus containing uncleaved F glycoprotein did not alter the NMR spectra. This indicates that infection is blocked at the stage of penetration. Similar, though less pronounced, differences have been observed when the effects of highly infectious influenza virus containing the hemagglutinin in the cleaved form were compared to the effects of virus which had a lower infectivity due to the presence of uncleaved hemagglutinin. Thus, it appears that the hemagglutinin of influenza virus is involved in penetration and that cleavage is necessary for this function. Alterations of the NMR spectra of the membrane lipids have also been observed when susceptible chick embryo cells (C/E) were infected with Rous sarcoma virus of subgroup B. Such alterations did not occur when nonsusceptible cells (C/B) were used. Thus, infection appears to be blocked again at the stage of penetration.

Intracellular precursors to the major glycoprotein of avian oncoviruses in chicken embryo fibroblasts

A 96,000-dalton glycoprotein, p(96), was present in cell extracts obtained from gs-chf- chicken embryo fibroblasts infected with the avian RNA tumor viruses Rous-associated virus-2 subgroup B (RAV-2) and the Schmidt-Ruppin strain of Rous sarcoma virus subgroup A (SR-RSV-A), as well as from uninfected gsLchf+ (HE) cell extracts. It was not found in cell extracts from uninfected gs-chf- or gs+chf+ (HH) cells, nor from gs-chf- cells infected with envelope-deficient Bryan high-titer Rous sarcoma virus. Immunoprecipitation, kinetic, and biochemical data indicate the this polyprotein contains information that gives rise to the major virion glycoprotein gp85. A second polyprotein of 80,000 daltons, p/80), is also present in the RAV-2- and SR-RSV-A-infected gs-chf- cells. This second polyprotein contains less carbohydrate than p(96), and kinetic and biochemical data indicate that p(80) may be an immature form of p(96).