Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus

Post-translational modifications of retrovirus gag gene-encoded polyproteins include proteolytic cleavage, phosphorylation, and glycosylation. To study the sequence of these events, we labeled JLS-V9 cells chronically infected with Rauscher murine leukemia virus in pulse-chase experiments with the radioactive precursors [3S]methionine, [14C]mannose, [3H]glucosamine, and [32P]phosphate. Newly synthesized gag polyproteins which incorporated label, and the modified products derived from them, were identified by immunoprecipitation of cell lysates with anti-p30 rabbit serum, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Pulse-chase experiments were carried out in the presence as well as in the absence of tunicamycin, an inhibitor of glycosylation. Among the three major polyproteins synthesized in the absence of tunicamycin, two were found to be glycosylated but not phosphorylated. These were designated gPr80gag and gP949"9. Both shared identical [3S]methionine peptides with Pr659'9 and p30. Of the two nonglycosylated precursors, Pr65gag and Pr759"9, only Pr659`9 was found to be detectably phosphorylated, and Pr759ag could be readily identified only when glycosylation was inhibited. On the basis of these results, a scheme for the post-translational modification of gag polyproteins is proposed. According to this scheme the gag gene-encoded polyproteins are processed from a common precursor, Pr759ag, by two divergent pathways: one leading through the intermediate Pr659'9 to internal virion components via cleavage and phosphorylation and the other via tunicamycin-sensitive mannosylation to the intermediate gPr809"9, which is further glycosylated to yield cell surface polyprotein gP94ga*.

Section: Discussionsupporting

confidence: 88%

Section: P94-mentioning

confidence: 88%

Post-translational modification of Rauscher leukemia virus precursor polyproteins encoded by the gag gene

Schultz¹,

Rabin²,

Oroszlan³

1979

“…Note also that all MCFs (with the possible exception of MCF Akv-J-C36) possess the MCF-specific T, oligonucleotide designated 101 that lies at the C-terminal end of Prpl5Ecoding sequences (20). By analogy with studies of Moloney murine leukemia virus, it seems likely that this T1 oligonucleotide encodes a region of Prpl5E that is cleaved off during the maturation of virus particles to yield mature pl5E and a small peptide (12,17,40).…”

Section: Resultsmentioning

confidence: 99%

Large RNase T1-resistant oligonucleotides encoding p15E and the U3 region of the long terminal repeat distinguish two biological classes of mink cell focus-forming type C viruses of inbred mice

Lung¹,

Hartley²,

Rowe³

et al. 1983

120

We used T1 oligonucleotide maps, in conjunction with available nucleotide sequences of appropriate C-type viruses, to identify regions of the viral genome that distinguish two biological classes of mink cell focus-forming (MCF) viruses described previously by Cloyd et al. (J. Exp. Med. 151:542-522, 1980). We found that leukemogenic MCF viruses from thymus differed from non-leukemogenic MCFs isolated from nonthymic neoplasms in nucleotide sequences encoding Prp15E and the U3 portion of the long terminal repeat (LTR). The thymic isolates possessed recombinant Prp15E genes, with the 5' to mid portion derived from their ecotropic parents and the extreme 3' portion invariably derived from their nonecotropic parents. These viruses probably derived the entire U3 portion of their LTRs from their nonecotropic parents. The nonthymic MCFs appeared to inherit their entire Prp15E coding region from their nonecotropic parents. We failed to detect consistent differences in gp70-coding sequences between the two groups of MCFs, but this may simply reflect limitations of the data. The studies presented here, in conjunction with studies from a number of labs indicating a role for MCF gp70 in leukemogenesis, indicate that three genetic elements, gp70, p15E, and the U3 portion of the LTR, may all play a role in determining the leukemogenic phenotype of type C viruses of high-leukemic inbred mice.

“…Removal of a leader peptide from Pr gp80 generates gp8O, which is further cleaved into two polypeptides, gp70 and Prpl5E. Prpl5E is further processed into pl2E (47,48).…”

Section: Figmentioning

confidence: 99%

Cas-Br-E murine leukemia virus: sequencing of the paralytogenic region of its genome and derivation of specific probes to study its origin and the structure of its recombinant genomes in leukemic tissues

Rassart¹,

Nelbach²,

Jolicoeur³

1986

The ecotropic Cas-Br-E murine leukemia virus (MuLV) and its molecularly cloned derivative pBR-NE-8 MuLV are capable of inducing hind-limb paralysis and leukemia after inoculation into susceptible mice. T, oligonucleotide fingerprinting, molecular hybridization, and restriction enzyme analysis previously showed that the env gene of Cas-Br-E MuLV diverged the most from that of other ecotropic MuLVs. To analyze proviruses in leukemic tissues, we derived DNA probes specific to Cas-Br-E sequences: two from the env region and one from the U3 long terminal repeat. With these probes, we found that this virus induced clonal (or oligoclonal) tumors and we documented the presence of typical mink cell focus-forming-type proviruses in leukemic tissues and the possible presence of other recombinant MuLV proviruses. Since the region harboring the determinant of paralysis was mapped within the pol-env region of the virus (L. DesGroseillers, M. Barrette, and P. Jolicoeur, J. Virol. 52:356-363, 1984), we performed the complete nucleotide sequence of this region covering the 3' end of the genome. We compared the deduced amino acid sequences of the pol carboxy-terminal domain and of the env gene products with those of other nonparalytogenic, ecotropic, and mink cell focus-forming MuLVs. This amino acid comparison revealed that this part of the pol gene product and the pl5E diverged very little from homologous proteins of other MuLVs. However, the Cas-Br-E gp7O sequence was found to be quite divergent from that of other MuLVs, and the amino acid changes were distributed all along the protein. Therefore, gp7O remains the best candidate for harboring the determinant of paralysis. The Cas-Br-E murine leukemia virus (MuLV) is an ecotropic retrovirus that was isolated from the brain of a paralyzed wild mouse (Mus musculus) trapped in Lake Casitas, Calif. (for a review, see references 16 and 17). It was shown to induce a progressive form of hind-limb paralysis and leukemia after inoculation into susceptible laboratory mice (16). Initial studies on the molecular structure of the Cas-Br-E MuLV genome revealed that its restriction map (5) and its T1 oligonucleotide fingerprinting pattern (29) were distinct from those of other ecotropic MuLVs isolated from inbred strains of mice. However, its genome appeared similar to the amphotropic MuLV genome except in the env gene, in which they diverged the most (1, 5, 29). Amphotropic MuLVs have also been isolated from wild mice, often from the same mice from which neurotropic MuLVs were isolated, and have been reported to be nonparalytogenic and weakly leukemogenic (16, 18, 36). In an effort to understand the molecular basis of the hind-limb paralysis, we first cloned the genome of Cas-Br-E MuLV and showed that this * Corresponding author. 9io that several determinants of leukemia seemed to be distributed along its genome (24). To monitor the fate of Cas-Br-E MuLV nucleic acids in infected tissues and to analyze its proviruses in infiltrated leukemic organs, we isolated probes which would not hybridize...