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

Murphy, Edwin C.; Kopchick, John J.; Watson, Kenneth F.; Arlinghaus, Ralph B.

doi:10.1016/0092-8674(78)90204-0

Cited by 62 publications

(57 citation statements)

References 27 publications

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“…The absorbed antisera inhibited the in vitro RT reaction and it mainly precipitated an 80,000 molecular weight polypeptide (p80Pol) from virions. The latter is similar in size to the purified polymerase protein (1,5,13,14).…”

Section: Resultsmentioning

confidence: 56%

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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Section: Resultsmentioning

confidence: 56%

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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“…The frequency of frameshifting and the ratio of gag to gag-pol protein are relatively constant (Murphy et al, 1978;Jacks & Varmus, 1987;Jacks et al, 1988) and the maintenance of that ratio may be important for the production of infectious virus particles. A major function of the gag protein is assembly of the virus core.…”

Section: Introductionmentioning

confidence: 99%

Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study

Hoshikawa¹,

Kojima²,

Yasuda³

et al. 1991

Journal of General Virology

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“…Translation of the 35S viral RNA in vitro in crude reticulocyte extracts also resulted in a ratio of about 1 : 20 between these two polyprotein products (Murphy et aL, 1978 ;Philipson et al, 1978). Addition of yeast amber suppressor tRNA and to a lesser extent ochre, dramatically changed this ratio to increase the amount of Pr200 gag po~ (Philipson et al, 1978).…”

Section: Introductionmentioning

confidence: 99%

Glutamine Starvation of Murine Leukaemia Virus-infected Cells Inhibits the Readthrough of the gag-pol Genes and Proteolytic Processing of the gag Polyprotein

Gloger

Panet

1986

Journal of General Virology

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

Cited by 62 publications

References 27 publications

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

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

Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study

Glutamine Starvation of Murine Leukaemia Virus-infected Cells Inhibits the Readthrough of the gag-pol Genes and Proteolytic Processing of the gag Polyprotein

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