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

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Abstract
Reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase) from Rauscher leukemia virus is synthesized in infected [mouse] cells by way of a read-through polyprotein of 200,000 MW. This polyprotein (Pr200gag-pol) was precipitated by antiserum to RT; in a previous study all the monospecific antisera to gag 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) MW 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 MW (p80pol) precipitable by antiserum to RT. Purification of active RT enzyme from virions labeled with [3H]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, but 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.