Ralph B. Arlinghaus

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.

Cell-free synthesis of a precursor polyprotein containing both gag and pol gene products by Rauscher murine leukemia virus 35S RNA

Abstract Translation of Rauscher murine leukemia virus (RLV) 35S RNA in a mRNA-dependent protein-synthesizing system results in the synthesis of polypeptides with apparent molecular weights of 200,000, 75,000 and 65,000 daltons. Each of these polypeptides was immunoprecipitable with anti-p30 serum, while only the 200,000 dalton size class was specifically recognized by antiserum prepared against purified reverse transcriptase. intracellular Pr200 gag-pol has been shown to contain p30 tryptic peptide sequences(Arcement et al., 1976) and to share antigenic determinants with the gag proteins p30, p15, p12 and p10 and the reverse transcriptase (Jamjoom, Naso and Arlinghaus, 1977). These results and others (Jamjoom et al., 1977) led us to the conclusion that Pr200 gag-pol is the initial translation product that leads to the formation of mature reverse transcriptase. In the present study, nine of eleven methionine-containing tryptic peptides found in a anti-reverse transcriptase-precipitable 80,000 dalton molecular weight virion polypeptide (p80 pol ) were contained in Pr200 gag-pol . In addition, virion p80 pol co-migrates in SDS-polyacrylamide gels with the major polypeptide found in partially purified preparations of active reverse transcriptase. The in vitro synthesized 200,000 dalton polypeptide was identical to intracellular Pr200 gag-pol as determined by comparing ion-exchange profiles of methionine-labeled tryptic peptides. The frequency of synthesis of the in vitro synthesized Pr200 gag-pol was 125 to 120 that of the combined synthesis of the 65,000 and 75,000 dalton gag precursors. A similar ratio of Pr200 gag-pol to Pr65 gag and Pr80 gag has been observed in viral infected cells. Thus 35S viral genomic RNA is an mRNA for both gag and pol gene products. Experiments with the arginine analogue canavanine suggest that the usual termination signal occurs after the translation of Pr80 gag . We propose that the Pr200 gag-pol results from an occasional read-through (5% frequency) of a single termination codon at the end of the gag gene.

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.

Partial characterization of a Moloney murine sarcoma virus 85,000-dalton polypeptide whose expression correlates with the transformed phenotype in cells infected with a temperature-sensitive mutant virus

Abstract The viral proteins specified by a Moloney murine sarcoma virus (Mo-MuSV) with a temperature-sensitive mutation in its transforming gene were examined. Normal rat kidney cells infected with this replication-defective virus have transformed cell characteristics at 33° but revert to a normal phenotype at 40°. At the temperature permissive for transformation, the cells contained an 85,000-dalton protein (P85) which had antigenic determinants of p15, pp12, and p30, and also tryptic peptides characteristic of p15 and p30 as well as additional unidentified tryptic peptides. P85 was only detectable at the permissive temperature. A 58,000-dalton protein (P58) was also detected. It had both antigenic determinants and tryptic peptides of p15, pp12, and p30. P58 was seen at both temperatures. Phosphorylation experiments indicated that P58 is a phosphoprotein whereas 32 P-labeled P85 was not observed. Temperature shift experiments showed that newly synthesized P85 was first detected between 2 and 3 hr following transfer of cultures to 33°. Morphological and biochemical changes indicative of transformation occurred 8 or more hr after temperature shift. These results are consistent with the interpretation that P85 contains peptide sequences derived from both the gag gene and the MuSV-acquired or src gene sequences.

A selective temperature-sensitive defect in viral RNA expression in cells infected with a ts transformation mutant of murine sarcoma virus

Abstract We investigated the nature of the defect in the temperature-sensitive mutant of Moloney murine sarcoma virus (Mo-MuSV), termed ts 110. This mutant has a temperature-sensitive defect in a function required for maintenance of the transformed state. A nonproducer cell clone, 6m2, infected with ts110 expresses P85 and P58 at 33°C, the transformed temperature, but only P58 is detected at the restrictive temperature of 39°C. Shift-up (33°C → 39°C) and in vitro experiments have established that P85 is not thermolabile for immunoprecipitation. Previous temperature-shift experiments (39°C → 33°C) have shown that P85 synthesis resumes after a 2–3 hr lag period. Temperature shifts (39°C → 33°C) performed in the presence of actinomycin D prevented the synthesis of P85, whereas P58 synthesis did not decline for 5 hr, suggesting that P58 and P85 are translated from different mRNAs. The shift-up experiments also indicated that, once made, the RNA coding for P85 can function at the restrictive temperature for several hours. MuSV- ts 110-infected cells superinfected with Mo-MuLV produced a ts110 MuSV-MuLV mixture. Sucrose gradient analysis of virus subunit RNAs revealed a ∼28S and a ∼35S peak. Electrophoresis of the ∼28S poly(A)-containing RNA from ts110 virus in methyl mercuric hydroxide gels resolved two RNAs with estimated sizes of 1.9 × 10 6 and 1.6 × 10 6 daltons, both smaller than the wild type MuSV-349 genomic RNA (2.2 × 10 6 daltons). RNA in the ∼28S size class from virus preparations harvested at 33°C was found to translate from P85 and P58, whereas, the ∼35S RNA yielded helper virus Pr63 gag . In contrast, virus harvested at 39°C was deficient in P85 coding RNA only. Peptide mapping experiments indicate that P85 contains P23 sequences, a candidate Moloney mouse sarcoma virus src gene product. Taken together, these results suggest that two virus-specific RNAs are present in ts 110-infected 6m2 cells and rescued ts 110 pseudotype virions at 33°C, one coding for P85, whose expression can be interfered with by shifting the culture to 39°C; the other coding for P58, whose expression is unaffected by temperature shifts. P85 is a candidate gag-src fusion protein, while P58 contains gag sequences only.

Cell-free synthesis of rauscher murine leukemia virus "gag" and "gag-pol" precursor polyproteins from virion 35 s rna in a mrna-dependent translation system derived from mouse tissue culture cells.

Abstract Using a mRNA-dependent cell-free protein synthesis sytem derived from mouse tissue culture cell extracts by treatment with micrococcal nuclease, we have examined the capacity of 35S genomic RNA from Rauscher leukemia virus (RLV) to code for the synthesis of viral proteins. Analysis of the polypeptide product by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that RLV 358 RNA directed the synthesis of RLV-specific polypeptides of 55,000, 65,000, 75,000, and 200,000 apparent molecular weights, identical in size to the known intracellular precursors of the RLV mature polypeptides. None of the individual mature virus proteins appeared to be synthesized. If canavanine, an arginine analogue, was substituted for arginine under conditions for cell-free protein synthesis, a RLV-specific polypeptide with a molecular weight of approximately 80,000 was synthesized at the expense of the 65,000 and 75,000 MW polypeptides. Monospecific antisera directed against p30, p15, p12, and p10 recognized the 65,000, 75,000, 80,000 and 200,000 MW polypeptides, indicating that each shared antigenic determinants with all of these “gag” proteins. Using the appropriate authentic RLV precursor polypeptides as standards, comparative tryptic maps were performed with the [ 3 H]tyrosine or [ 35 Snine-labeled in vitro -synthesized 65,000 and 200,000 MW polypeptides. The 65,000 MW polypeptide was found to be identical to Pr658 gag , 2 a 65,000 MW RLV gag protein precursor obtained from infected cells by immunoprecipitation and gel electrophoresis. The 200,000 MW in vitro -synthesized polypeptide was found to contain methionine-labeled tryptic peptides characteristic of the RLV reverse transcriptase (“pol”) and p30.