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SE21Q1b, a Rous sarcoma virus mutant which packages cellular rather than viral RNA, is competent for infection of quail cells and can transmit defective transforming retrovirus genes. Stably transformed recipient clones have been obtained by using this mutant.
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Infectious virus nucleic acids were extracted from the Hirt supernatant of chicken embryo fibroblasts infected for different times with the Schmidt-Ruppin strain of Rous sarcoma virus (RSV). Infectious DNA and DNA-RNA hybrid molecules could be recovered from 6 h after infection in experiments using 1.5 X 10(9) infected cells. Only small amounts of infectious virus DNA could be purified 6 h after infection whereas at 24 h approximately one infectious DNA molecule could be recovered for each input virus infectious unit. At 24 h, both infectious supercoiled and non-supercoiled molecules were found. The specific infectivity of the supercoiled fraction was less than that of the non-supercoiled fraction. Infectious supercoiled DNA could be recovered from 16 h after infection. Evidence is presented that both forms of unintegrated virus DNA may rest unintegrated for at least 8 days in the cell, though chronically infected cells were shown to contain less than one unintegrated molecule per 10(2) to 10(3) cells.
Preparations of Rous sarcoma virus (RSV) can form an infectious viral-antibody complex with antibodies raised against the major glycoprotein, gp85, isolated from avian myeloblastosis virus and Prague-RSV subgroup C. Binding of anti-gp85 antibodies to RSV can be demonstrated by the inhibition of focus-forming activity after addition of goat anti-rabbit immunoglobulin and by a shift in density of virions treated with anti-gp85 serum. Group- rather than subgroup- specific regions of viral gp85 appear to be the site of binding for infectious complex.
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To test the effect of long terminal repeat (LTR) regulatory sequences on the transforming capability of the v-myb oncogene from avian myeloblastosis virus (AMV), we have constructed replication-competent avian retroviral vectors with nearly identical structural genes that express v-myb from either AMV or Rous sarcoma virus (RSV) LTRs. After transfection into chicken embryo fibroblasts, virus-containing cell supernatants were used to infect chicken myelomonocytic target cells from preparations of 16-day-old embryonic spleen cells. Both wild-type AMV and the virus expressing v-myb from AMV LTRs (RCAMV-v-myb) were able to transform the splenocyte cultures into a population of immature myelomonocytic cells. The transformed cells expressed the p48v-Myb oncoprotein and formed compact foci when grown in soft agar. In contrast, the virus expressing v-myb from RSV LTRs (RCAS-v-myb) was repeatedly unable to transform the same splenocyte cells, despite being able to infect fibroblasts with high efficiency. This difference in the transforming activities of v-myb-expressing viruses with different LTRs most likely results from the presence of a factor (or factors) within the appropriate myelomonocytic target cell that promotes specific expression from the AMV but not from the RSV LTR.
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.
Cells transformed by Rous sarcoma virus take up 2-deoxyglucose at a faster rate than uninfected cells, under conditions where transformed and nontransformed cells grow at the same rate. In cells infected by a temperature-sensitive mutant, the stimulation of 2-deoxyglucose uptake is temperature dependent: the increase is observed at the permissive (36 degrees C), but not at the nonpermissive (41.5 degrees ) temperature. When infected cells are shifted from the nonpermissive temperature to the premissive temperature, the uptake of 2-deoxyglucose increases from a rate equal to that of uninfected cells to a rate equal to that of cells infected by the wild-type Schmidt-Ruppin Rous sarcoma virus. The reverse change occurs when the infected cells are shifted from the permissive to the nonpermissive temperature. By the use of cytosine arabinoside, an inhibitor of DNA synthesis, it was possible to show that DNA synthesis is neither required for the transformation, which occurs when the infected cells are shifted from the nonpermissive to the permissive temperature, nor for the phenotypic reversion, which occurs in the reverse shift.
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The mechanism of transfection by Rous sarcoma virus DNA was investigated by assaying DNA-mediated transformation under conditions which restricted secondary virus infection. Chicken embryo fibroblasts which were genetically resistant to virus infection as a result of the absence of receptors for virus penetration were also resistant to transformation by integrated or unintegrated Rous sarcoma virus DNA. In addition, DNA of replication-defective Bryan hightiter Rous sarcoma virus was noninfectious, and transformation by DNA of a temperature-sensitive DNA polymerase mutant was temperature sensitive. These results indicated that secondary virus infection was necessary for transformation by Rous sarcoma virus DNA. Since transformation was assayed by colony formation in soft agar, as well as by focus formation, the requirement for secondary virus infection was not an artifact of potential difficulty in detection of foci formed by division of single transformed cells. Therefore, it appeared that donor DNA did not stably transform recipient cells by direct integration. Instead, the results were consistent with the hypothesis that transfection of chicken embryo fibroblasts by Rous sarcoma virus DNA proceeded by transcription of donor DNA, formation of extracellular progeny virus, and secondary virus infection of sensitive cells.
The transforming protein of Rous sarcoma virus, pp60v-src, is known to be a tyrosine protein kinase, but the mechanism of cell transformation remains unclear. In further investigating pp60v-src structure and function, we have analyzed two temperature-sensitive (ts) Rous sarcoma virus src gene mutants, tsLA29 and tsLA32. The mutations in tsLA29 and tsLA32 map in the carboxy-terminal region and the amino-terminal half of pp60v-src, respectively, and encode mutant proteins with either temperature-labile (tsLA29) or -stable (tsLA32) kinase activities. Here we examined the intracellular processing and localization of these pp60v-src mutants and extended our characterization of transformation parameters expressed by cells infected by the Rous sarcoma virus variants. No obvious defects in functional integrity of the tsLA32 pp60v-src could yet be demonstrated, whereas the tsLA29 pp60v-src was perturbed not only in kinase activity, but also in aspects of protein processing and localization. Analysis of transformation parameters expressed by infected cells demonstrated the complete temperature lability of both mutants.
In cells infected with Vesicular Stomatitis virus (VSV) ts 1026 and superinfected with Rous Sarcoma virus (RSV) synthesis of vsrc mRNA and RSV env mRNA decreases. In these cells post-translational processing of RSV precursor proteins is impaired and small amounts of VSV antigens are detected.
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Rous sarcoma virus (RSV) proviruses integrated within the DNA of transformed rat cells frequently display duplications of variable segments of proviral DNA upstream of an intact provirus. The rearrangement in the A11 clone of transformed rat cells consists of a partial duplication of both viral and cellular DNA segments whose origin is a region of approximately 4 kb encompassing the 3' virus-cell junction. Transposition of this DNA appears to have occurred at or after virus integration by a mechanism involving at least two recombination events. In every case examined, including A11, the transcriptional organization of the original provirus has been conserved and viral RNA expression appears to occur normally. The frequency of such rearranged proviruses in the DNA of transformed rat cells suggests that upstream rearrangements may influence provirus expression.
The oncogene of Rous sarcoma virus (v-src) arose by transduction of a cellular gene (c-src). In an effort to explore the mechanism of transduction, we have identified the splice acceptor site used in the genesis of mRNA for v-src, shown that an equivalent site is used in the splicing of mRNA for c-src, and determined the nucleotide sequence from the boundaries of homology between v-src and c-src. Our data indicate that (i) only a portion of c-src is represented within v-src, (ii) the leftward recombination between the genome of the transducing virus and c-src occurred in an intron of the cellular gene, (iii) v-src is in part a spliced version of the corresponding portion of c-src, and (iv) nucleotide sequences represented once in the genome of the transducing virus become duplicated to flank v-src. These findings indicate that the first step in transduction is probably recombination between DNA forms of the transducing viral genome and c-src and otherwise support the prevailing model for transduction by retroviruses. The carboxyl termini of the proteins encoded by v-src and c-src differ appreciably. An unidentified domain of 127 or 128 nucleotides is located at different positions in the genomes of two strains of RSV and gives evidence of being a foreign element that entered the viral genomes by genetic transposition independent of the transduction of src.
Focus formation by Rous sarcoma virus (RSV) was significantly enhanced when virus was incubated with the saline fraction of chloroform extracted chicken egg yolk, prior to infecting chicken embryo cells. The enhancement was restricted to members of RSV subgroups B, C and D and was proportional to yolk dilution. Subgroup A virus was never affected. In all, 108 yolk samples from specific pathogen free chickens were investigated. Of these 78 to 97% stimulated focus formation. RSV(RAV-50) of subgroup D which was stimulated up to tenfold, was the most strongly affected strain. The enhancing principle was shown to be a specific yolk factor, and its effect remained constant even after several years' storage. Crude yolk specimens showed essentially the same enhancing property. The chemical nature of the yolk factor is still unknown. It must, however, be taken into account when assaying for avian leukosis virus antibodies.