SYNTHESIS AND PROCESSING OF THE INTERNAL STRUCTURAL PROTEINS OF RETROVIRUSES: SITE OF SYNTHESIS, EVIDENCE FOR MULTIPLY-CHARGED SPECIES, AND ANALYSIS OF A MUTANT DEFECTIVE IN PROCESSING

Abstract

In this repot we review the evidence that synthesis and processing of Pr76 gag , the precursor to the core proteins (products of the gag gene) of avian retroviruses, is both necessary and sufficient to produce a virus particle. In addition, we describe the results of two-dimensional polyacrylamide gel electrophoretic analysis of Pr76 gag and its cleavage products. When the two-dimensional gel system is used in conjunction with protein blotting and antibody detection on nitrocellulose sheets, we observe multiply-charged species of both precursor and product proteins. Comparison with 32 PO 4 and 35 S-methionine labelled proteins indicates that much of the heterogeneity is probably due to phosphorylation. We also report results of an investigation into the site of synthesis of Pr76 gag . Immunoprecipitation across sucrose gradients containing polyribosomes derived from cytoplasmic and membrane fractions of infected cells indicates that Pr76 gag is found exclusively on cytoplasmic polyribosomes. Both Pr76 gag and, what we presume to be related incomplete nascent polypeptide chains, are localized in the 450S region of the gradient and are released by treatment with EDTA. Finally, we describe studies on a deletion mutant which, while failing to produce either reverse transcriptase or envelope glycoprotein, synthesizes pp60 src and a 63,000 dalton protein (P63) related to retrovirus core proteins. P63 contains an approximately 13,000 dalton internal deletion (in p27) as determined by tryptic mapping and analysis of products of in vitro cleavage by the gag-specific virion protease. The deletion does not appear to extend into the region of the p15 cleavage protease. P63 is resistant to normal processing and no product proteins are detectable even in cells superinfected with non-defective viruses. We conclude that the internal deletion in P63 has rendered this protein defective for processing either by inducing a conformational change, removal of a cleavage site, or both.