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

Arcement, L.J.; Karshin, W. L.; Naso, Robert; Jamjoom, Ghazi A.; Arlinghaus, Ralph B.

doi:10.1016/0042-6822(76)90504-3

Cited by 132 publications

(58 citation statements)

References 19 publications

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“…Therefore, we conclude that Prl5E, and quite probably its cleavage products, does not contain mannose or glucosamine and most likely is not glycosylated. The absence of labeling pl5E is in agreement with published reports that the p15E of R-MuLV is not glycosylated (14,21,23).…”

supporting

confidence: 81%

“…The cell extracts were allowed to react with the three antisera, and immunoprecipitates were subjected to electrophoresis on NaDodSOJpolyacrylamide gels (data not shown). As expected from the work ofothers (12,21,22), Pr8Oenv and gp70 are labeled with both sugars but neither Pr15E nor piSE is labeled with either. Therefore, we conclude that Prl5E, and quite probably its cleavage products, does not contain mannose or glucosamine and most likely is not glycosylated.…”

supporting

confidence: 55%

See 1 more Smart Citation

Sequence-specific antibodies show that maturation of Moloney leukemia virus envelope polyprotein involves removal of a COOH-terminal peptide.

Green¹,

Shinnick²,

Witte³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

141

113

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We followed maturation of the glycosylated envelope polyprotein Pr8Ovofa murine retrovirus by using antisera specific to subregions of the protein, including an antiserum directed against a synthetic peptide corresponding to the COOHterminus of Pr80. Shortly after synthesis and glycosylation, Pr8Ov is cleaved into two species, gp7O and Pr15E, that are found associated, perhaps through disulfide bonds, in infected cells. PrISE is further cleaved at the time of virus maturation to form virus protein pl5E. NH2-Terminal protein sequence analysis showed that PriSE had, an NH2 terminus in common with pl5E. Pr15E, but not plSE, is precipitated by antibody against the COOH-terminal peptide; hence, pl5E is missing a peptide at the COOH-terminus. Our data indicate that Pr15E is the predominant species in cells and pl5E is the major species in virus.The envelope (env) gene ofmurine retroviruses encodes a polymorphic family of proteins (1). The allele at this locus determines virus host range, and differences between highly leukemogenic viruses and their more benign relatives cluster within this gene (2-7). The primary protein product of the Moloney murine leukemia virus (Mo-MuLV) env gene is a glycosylated molecule that has an apparent Mr 80,000 (Pr80e"v) (8). The Pr80env polyprotein contains the information (NH2 to COOH terminal) for gp7O, the major envelope glycoprotein; pl5E, a second viral membrane protein; and R, a COOH-terminal species (9, 10). This report describes the intermediates and end products of Pr80env processing and their times of appearance. We present evidence that the COOH-terminal peptide is removed during the formation ofmature virions and may function in virus assembly..From the data presented here-and the work of others (9, 11-15), a scheme for env precursor processing emerges. After glycosylation of the primary env translation product, the resulting Pr80env molecule is cleaved into two products. One is derived from the NH2-terminal region and contains gp70; the other contains the COOH-terminal 196 amino acids. All of the carbohydrate chains are attached to the gp7O portion. (R-MuLV) were labeled with [3S]methionine similarly. Immune precipitation and one-dimensional gel electrophoresis were performed as described (13). Antisera used for immune precipitation were as follows: (i) anti-gp70, raised against gp7O purified from RMuLV (17); (ii) anti-plSE raised against pl5E purified from RMuLV (a gift from I. Fleissner); (iii) anti-plSE ascitic fluid from hybridoma 9E8, (a gift from R. Nowinski; ref. 18; and (iv) To 10 dul of the supernatant was added 1 A1 of bovine serum albumin in sample buffer (5 ,ug/ml) as an internal standard, 22 ,1 of sample dilution buffer (9.5 M urea/2% ampholines, pH 3.5-10/5% 2-mercaptoethanol/8% Nonidet P-40), and 8.25 mg of solid urea to bring the final concentration of urea to 9.5 M.Abbreviations: Mo-MuLV and R-MuLV, Moloney and Rauscher murine leukemia virus, respectively.

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supporting

confidence: 81%

supporting

confidence: 55%

Sequence-specific antibodies show that maturation of Moloney leukemia virus envelope polyprotein involves removal of a COOH-terminal peptide.

Green¹,

Shinnick²,

Witte³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

141

113

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“…The a and b polypeptides of Pr200gag-pol have identical methionine-labeled tryptic digest profiles under reducing conditions (L. J. Arcement and R. B. Arlinghaus, unpublished results). However, some minor differences in these polypeptides can be detected in the profiles of performic acid-oxidized tryptic peptides labeled with [35S]methionine (7). Pr135P"1 was isolated from cells pulse-labeled with [3H]methionine for 20 min followed by a 2.5-hr chase.…”

Section: Resultsmentioning

confidence: 99%

Biosynthesis of reverse transcriptase from Rauscher murine leukemia virus by synthesis and cleavage of a gag-pol read-through viral precursor polyprotein

Kopchick

Jamjoom

Watson

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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Reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase) from Rauscher leukemia virus is synthesized in infected cells by way of a read-through polyrotein of 200,000 molecular weight. This polyprotein (Pr200 gag-Pol) was precipitated by antiserum to RT; in a previous study all the monospecific antisera to gag proteins recognized Pr200 gag-Pol. Pr2JO gag-pol contains both p30 and RT peptide sequences. Intermediate RT-related precursors of 145,000 (Prl45 P0l), 135,000 (Prl35 P01), and 125,000 (PrI25 P0l) molecular weights were specifically recognized by precipitation from infected cell extracts by antiserum to RT. These proteins shared methionine-containing trtic peptide sequences with a virion polypeptide of 80,000 molecular weight (p80 P01) precipitable by antiserum to RT. Purification of active RT enzyme from virions labeled with [3H]methionine showed that p80 P01 was the major component, based on analysis by gel electrophoresis and tryptic peptide mapping experiments. A polypeptide (Pr80 P01), similar in size to mature viral p80 P01, was also precipitated from infected cells by antiserum to RT. Its peptide map was nearly identical to that of virion p80 P0I. Pulsechase studies showed that Pr80P01, Pr125 P01, and Pr135 P01 were stable polypeptides, whereas Pr200 gag-P0l and Pr145 P01 were unstable precursors. Pulse-chase studies wifh the protein synthesis inhibitor, cycloheximide, showed that the processing of Pr200 gag-pol occurred for a short time in the absence of protein synthesis.Type-C retrovirus genomes contain genes for their internal structural proteins, reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase), and envelope proteins.t These coding regions have been termed gag (group antigens), pol, and env, respectively (2). RT is present in much lower amounts in virus particles and in infected cells (3-5) than either the gag or env proteins. In this report we show that RT from Rauscher leukemia virus (RLV) is made in infected cells by synthesis and processing of a 200,000dalton read-through protein containing both gag and pol determinants. MATERIALS AND METHODSCells and Virus. RLV-infected NIH Swiss mouse embryo fibroblasts (JLS-V16) and BALB/C spleen-thymus (JLS-V5) cell lines were used in these studies. Virus was produced in infected JLS-V5 cells, whereas precursors were isolated from JLS-V16 cells.Labeling of Cells and Virus. These procedures have been described (3,6).Immune Precipitation and Peptide Mapping. Antisera to leukemia virus RT were supplied by the Office of Program Resources and Logistics of the National Cancer Institute. The sera were absorbed before use with uninfected cell proteins and disrupted virus (3). Immune precipitation was performed by indirect immune precipitation (3); tryptic digestion of precursors and viral proteins (7) was as described. Viral RT. RT was assayed as described (8). The active enzyme was purified as described (9). RESULTSImmunoprecipitation of Virion Proteins with Antiserum to RT. Absorbed antiserum to partially purified RT was...

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“…Specifically, cleavage of Pr659ag gives rise to p30, the major capsid protein, as well as pl 5, a hydrophobic, membrane-associated protein and two nucleic acid-associated proteins, p 12 and pl0 (Van Zaane et al, 1976;Arcement et al, 1976;Yoshinaka & Luftig, 1977a;Eisenman & Vogt, 1978). Although the molecular details of this process are not yet understood, it appears that both a virus-associated protease (Yoshinaka & Luftig, 1977b) and a kinase that specifically phosphorytates pl 2 residues on Pr659~ (Naso et al, 1979) are involved.…”

Section: Introductionmentioning

confidence: 99%

Murine Leukaemia Virus p30 Heterogeneity as Revealed by Two-dimensional Gel Electrophoresis Is Not an Artefact of the Technique

Katoh

Yoshinaka

Luftig

1984

Journal of General Virology

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SUMMARYWe have utilized two-dimensional (2D) gel electrophoresis [the first dimension being a linear pH gradient (5 to 8) and the second an 8 to 15% acrylamide gradient] to characterize the virion protein, p30, from several strains of purified murine leukaemia virus (MuLV). In all cases, we found that there was a predominant (70 to 90%) Coomassie Brilliant Blue-staining p30 spot, as well as several other species which differed in pI. The major p30 spot differed in pI among different MuLV strains and the minor spots varied depending on the host cell used to grow the virus. Specifically, (i) Moloney (M)-MuLV/NIH-3T3 showed two spots, a major one at pI 6.3 and a more acidic one, (ii) AKR/NIH-3T3, AKR/mouse embryo, and Gross/NIH-3T3 showed four spots, with the two basic, minor spots of AKR/NIH-3T3 appearing relatively decreased in intensity, and (iii) Rauscher (R)-MuLV/JLS-V9 (BALB/c) showed two spots, a major one with greater than 90% of the estimated Coomassie Brilliant Blue stain at a pI of 6.5 and a minor, acidic one. The major spots of AKR and M-MuLV viruses also differed in pI. The major spot of the AKR and Gross N-tropic viruses had a pI of 6.7 while that of NB-tropic virus M-MuLV had a pI of 6.3. The possibility that the heterogeneity observed in p30 was an artefact of the 2D gel technique had to be considered since urea was used to denature proteins in the first dimension of the gel. This possibility was made unlikely by our finding that another technique, chromatofocusing, gave the same results. Specifically, M-MuLV/JLS-V9 p30, when separated on chromatofocusing columns under non-denaturing conditions yielded three peaks, each of which directly corresponded to the three spots (pI : 6.1, 6-3, 6.6) observed on 2D gels. Furthermore, tryptic peptide maps of the major (pI 6-3) and one of the minor (pI 6-6) M-MuLV spots, although very similar in peptide composition, showed about five clearly defined differences. These results indicate (i) that the p30s of several N-and NB-tropic viruses are heterogeneous in pI, and (ii) for one particular MuLV, the p30 heterogeneity can be explained by a difference in amino acid composition. These findings of p30 charge heterogeneity may reflect either the presence of several different p30s in each virus particle and/or a heterogeneity in the virus population.

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

Cited by 132 publications

References 19 publications

Sequence-specific antibodies show that maturation of Moloney leukemia virus envelope polyprotein involves removal of a COOH-terminal peptide.

Sequence-specific antibodies show that maturation of Moloney leukemia virus envelope polyprotein involves removal of a COOH-terminal peptide.

Biosynthesis of reverse transcriptase from Rauscher murine leukemia virus by synthesis and cleavage of a gag-pol read-through viral precursor polyprotein

Murine Leukaemia Virus p30 Heterogeneity as Revealed by Two-dimensional Gel Electrophoresis Is Not an Artefact of the Technique

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