Open AccessBiosynthesis of reverse transcriptase from Rauscher murine leukemia virus by synthesis and cleavage of a gag-pol read-through viral precursor polyprotein
Author(s) -
John J. Kopchick,
Ghazi A. Jamjoom,
Kenneth F. Watson,
Ralph B. Arlinghaus
Publication year - 1978
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.75.4.2016
Subject(s) - antiserum , reverse transcriptase , microbiology and biotechnology , biology , rna directed dna polymerase , cycloheximide , murine leukemia virus , virology , biochemistry , rna , chemistry , virus , antigen , protein biosynthesis , gene , genetics
Reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase) from Rauscher leukemia virus is synthesized in infected cells by way of a read-through poly- rotein of 200,000 molecular weight. This polyprotein (Pr200gag-pol ) was precipitated by antiserum to RT; in a previous study all the monospecific antisera togag proteins recognized Pr200gag-pol . Pr200gag-pol contains both p30 and RT peptide sequences. Intermediate RT-related precursors of 145,000 (Pr145pol ), 135,000 (Pr135pol ), and 125,000 (Pr125pol ) molecular weights were specifically recognized by precipitation from infected cell extracts by antiserum to RT. These proteins shared methionine-containing tryptic peptide sequences with a virion polypeptide of 80,000 molecular weight (p80pol ) precipitate by antiserum to RT. Purification of active RT enzyme from virions labeled with [3 H]methionine showed that p80pol was the major component, based on analysis by gel electrophoresis and tryptic peptide mapping experiments. A polypeptide (Pr80pol ), similar in size to mature viral p80pol , was also precipitated from infected cells by antiserum to RT. Its peptide map was nearly identical to that of virion p80pol . Pulse-chase studies showed that Pr80pol , Pr125pol , and Pr135pol were stable polypeptides, whereas Pr200gag-pol and Pr145pol were unstable precursors. Pulse-chase studies with the protein synthesis inhibitor, cycloheximide, showed that the processing of Pr200gag-pol occurred for a short time in the absence of protein synthesis.