Robert B. Naso

Further characterization of intracellular precursor polyproteins of Rauscher leukemia virus.

Abstract The identity and pathway of post-translational processing of viral primary gene products in cells infected with Rauscher leukemia virus were investigated in pulse-chase studies by precipitation with monospecific antisera and by the use of inhibitors of proteolytic cleavage. High molecular weight precursor polypeptides, designated Pr1a+b, of molecular weight of about 200,000, are precipitated both with antisera made against either one of the viral group-specific antigens (gag) p30, p15, p12, or p10 and with antisera made against the viral reverse transcriptase (RT). This indicates that these polypeptides contain determinants of both the “gag” and the polymerase (pol) gene products. Two pulse-labeled polypeptides, Pr3 (≅80,000–85,000 daltons) and its cleavage product Pr4 (≅70,000 daltons), are precipitated with anti-p30, -p15, -pl2, or -p10 sera, but not with anti-RT serum. Pr3 and Pr4 thus represent gag gene precursor polyproteins. Three other polypeptides, Pr RTl (≅145,000 daltons), Pr RT2 (≅135,000 daltons), and Pr RT3 (≅80,000–85,000 daltons), are precipitated with anti-RT serum only. Anti-gp69/71 serum precipitates the glycoprotein precursors Pr2a+b (≅90,000 daltons), but none of the above-mentioned polypeptides. Quantitative measurements of pulse-chase profiles and results obtained with the inhibitor TPCK and the arginine analog canavanine indicate that the proteolytic cleavage of Pr1a+b is the primary pathway for the formation of intermediate RT precursors Pr RT1, 2, 3. The same measurements did not clarify whether or not the cleavage of Pr1a+b is involved in the formation of gag gene products. However, they indicated that most of the detected amount of the gag gene precursors Pr3 and Pr4 is made by a mechanism that does not involve the cleavage of preformed Pr1a+b. A possible explanation of these results is suggested in a model in which translation proceeds from one initiation site to give rise either to a gag gene product or, less frequently, to a common gag-pol precursor that is made by overriding a termination site at the end of the gag gene.

Biosynthesis of Rauscher leukemia viral proteins: presence of p30 and envelope p15 sequences in precursor polypeptides.

Abstract Viral structural polypeptides p30 and a 17,000-dalton polypeptide, termed envelope p15, are formed in Rauscher leukemia virus (RLV)-infected N.I.H. Swiss mouse embryo fibroblasts by cleavage of high molecular weight precursor polypeptides. The evidence for this conclusion is based on the analysis of polypeptides precipitated from RLV-infected cells by antiserum directed against RLV structural proteins. High resolution sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE) of such immune precipitates from infected cells pulse-labeled with [ 35 S]methionine or pulse-labeled and then chased in unlabeled medium provides evidence that three size classes of unstable polypeptides are precursors to virion p30. They are: two polypeptides with an approximate molecular weight of 200,000 (termed Pr1 a and b ), an 80,000-dalton polypeptide (Pr3) and a 65,000-dalton polypeptide (Pr4). Ion-exchange chromatography of tryptic digests showed that methionine-containing tryptic peptides of p30 are present in these precursor polypeptides. Methionine-labeled tryptic peptide sequences of envelope p15 were present in a 90,000-dalton peptide fraction containing two components (Pr2 a and b ). The latter polypeptides comigrated with viral specific fucose-free glycoproteins not present in virions or uninfected cells.

Biosynthesis of Rauscher leukemia viral proteins

Antisera to disrupted Rauscher leukemia virus (RLV) or to the purified Rauscher viral 30,000 dalton polypeptide were used to specifically precipitate newly snythetized intracellular viral polypeptides from extracts of infected NIH Swiss mouse cells (JLS-V16). Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PHAGE) of extracts from cells pulse-labeled for 10-20 min with 35 S-methionine showed that immune precipitates contained none of the nonglycosylated internal structural polypeptides of mature viruses. The major viral-specific polypeptides labeled in 10 min included polypeptides of 180,000, 140,000, 110,000, 80,000, and 60,000 daltons with minor polypeptides of 65,000, 50,000, and 40,000 daltons. Labeling the intracellular virus-specific polypeptides with 14C-glucosamine indicated that the 180,000, 110,000, 80,000, and 60,000 dalton polypeptides were gylcosylated, and all but the 110,000 dalton polypeptides are contained in the mature virions. Based on pulse-chase experiments, it appears that at least 3 of the large polypeptides (140,000, 65,000, and 50,000 daltons) are precursors to the three major internal structural polypeptides of the mature virions.

“gag” polyprotein precursors of rauscher murine leukemia virus

Abstract The major polyprotein precursors of the four internal structural proteins of Rauscher murine leukemia virus, designated p30, p15, p12, and p10 (collectively termed “gag” proteins), have been characterized. Tryptic peptide sequences and antigenic determinants of the gag proteins are contained in precursor polyproteins Pr3 (≅80,000) and Pr4 (≅65,000 daltons). Two minor precursor polyproteins have also been identified and partially characterized. They are designated Pr5 (≅55,000) and Pr6 (≅45,000 daltons). Pr5 contains peptide sequences of p30, p15, and p12; Pr6 contains sequences of p30 and p12 but lacks p15 sequences. The presence or absence of p10 sequences in Pr5 and Pr6 remains to be established. The major pathway for formation of the gag proteins appears to be via synthesis and processing of Pr3. Antigenic determinants of the gag proteins (and p30 peptide sequences) are also contained in a polyprotein precursor termed Prl a +b (molecular weight, ≅200,000). The latter has also been shown to share antigenic determinants with the reverse transcriptase [ Jamjoom, G. A., Naso, R. B., and Arlinghaus, R. B. (1977) . Virology 78, 11-341.

Proteins of rauscher murine leukemia virus. Resolution of a 70,000-dalton, nonglycosylated polypeptide containing p30 peptide sequences.

Abstract A viral-specific polypeptide of molecular weight about 70,000 which does not incorporate radioactive glucosamine or fucose and which does not stain positively for carbohydrates is present in preparations of purified Rauscher murine leukemia virus. This polypeptide can be resolved from the major glycoprotein(s) previously designated gp69 71 by high resolution sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The 70,000-dalton polypeptide is present in Rauscher virus-infected cells but not in nonproducer, uninfected N.I.H. Swiss mouse embryo cells. The [ 35 S]methionine-labeled tryptic peptide patterns of the virion and the intracellular 70,000-dalton polypeptide are identical, and each contains [ 35 S]methionine-labeled tryptic peptides characteristic of virion p30, indicating a possible precursor relationship to p30.

The cell-free translation of Rauscher leukemia virus RNA into high molecular weight polypeptides

Rauscher leukemia virus (RLV) 65-S RNA, 35-S mengovirus RNA and reticulocyte A-rich RNA each stimulated cell-free protein synthesis in a JLS-V5 cell derived S-30 system. rRNA, however, was not stimulatory in this system. Of the stimulated protein products only those synthesized in response to added RLV RNA were immune-precipitable with anti-RLV rabbit serum. Furthermore, cell-free incubations with pactamycin at a concentration which specifically inhibits initiation and not elongation prevented the stimulation of amino acid incorporation in response to added RLV RNA. Analysis of the polypeptides synthesized by the cell-free system in response to reticulocyte A-rich RNA, showed them to be globin-like and, therefore, also mRNA specific. The RLV RNA-directed product included at least two classes of polypeptides (mol. wts of 140 000-185 000 and 50 000-75 000) both of which were larger than the group specific polypeptides of mature virions. None of the internal structural polypeptides of mature virions were synthesized in response to RLV RNA. The large molecular weight, viral-specific polypeptides are candidate precursor polyproteins which may represent the translational products of a polycistronic mRNA with a single initiation site.

BIOSYNTHESIS AND PROCESSING OF RAUSCHER LEUKEMIA VIRAL PRECURSOR POLYPROTEINS

ABSTRACT. Rauscher murine leukemia viral structural proteins were shown to be synthesized in infected cells by way of high molecular weight precursor polyproteins identified as Prl a+b (∼ 200,000 daltons), Pr2 a+b (∼ 90,000 daltons), Pr3 (∼ 80,000 daltons), Pr4 (∼ 65,000 daltons), Pr5 (∼ 55,000 daltons) and Pr6 (∼ 45,000 daltons). Tryptic peptide mapping experiments have shown that p30 is contained in precursors Pr1 a+b and Pr3 through Pr6. Viral protein p15 peptide sequences are present in Pr3 through Pr5 whereas p10 is contained in Pr3 and Pr4. Both p10 and p15, like p30, then must originate from Pr1 a+b Tryptic peptides of viral gp69/71 and p12 are found in Pr2 a+b and not in Pr1 a+b. Preliminary experiments indicate that the viral reverse transcriptase polypeptide (∼ 72,000–75,000 daltons) is contained in Pr1 a+b . Treatment of infected cells with inhibitors of proteolytic enzymes, such as TPCK, caused a build-up of Pr1 a+b and prevented the formation of mature structural proteins. A model for the biosynthesis of Rauscher viral proteins is presented.

Synthesis and cleavage of rauscher leukemia virus precursor proteins in synchronized cell

Abstract Viral precursor protein synthesis in cells chronically infected with Rauscher murine leukemia virus (R-MLV) was shown to occur in three distinct waves corresponding to the G 1 , middle S, and la2 phases of the cell cycle. During these times, the synthesis of viral-specific proteins amounted to between 4.5 and 5.5% of total cell protein synthesis in the pulse-labeled cells. The G, wave of viral precursor synthesis did not require new viral RNA synthesis, while the later two waves were apparently dependent upon the presence of newly made viral RNA. High resolution SDS-polyacrylamide gel electrophoresis of immunoprecipitates from pulse-labeled and pulsed, chase-incubated cells identified the viral-specific proteins made in each wave of synthesis as those precursors and viral cleavage products characteristic of nonsynchronized cells infected with R-MLV.