REVERSE-TRANSCRIPTASE AND PROTEASE ACTIVITIES OF AVIAN-LEUKOSIS VIRUSGAG-POL FUSION PROTEINS EXPRESSED IN INSECT CELLS

Protease (PR)-defective avian leukosis virus particles display 300-fol d-reduced levels of reverse transcriptase (RT) activity relative to wi ld-type particles. This observation suggests that during virion assemb ly RT is activated by proteolytic maturation of the Gag-Pol polyprotei n precursor. To study the relationship between proteolytic cleavage an d RT activation, we subjected PR-defective virion cores to digestion w ith purified viral PR and analyzed the structure of the major polypept ides produced as well as RT activity. Under conditions in which Gag pr ecursors were fully matured, the RT domain was only incompletely relea sed from the Gag-Pol precursor, remaining tethered to the upstream Gag domains PR or NC-PR. In the same reaction, RT activity was stimulated only three-fold, or 100-fold less than expected for a fully active RT . The poor activation suggested that the NC or PR domains could repres s RT activity. To test this idea, we constructed recombinant baculovir uses expressing 19 different fusion proteins with upstream Gag or down stream Pol sequences attached to RT. Each protein was partially purifi ed and assayed for its inherent RT activity. The results are consisten t with the idea that Gag sequences can inhibit RT activity but indicat e that the size of the Pol domain as well as the status of the PR doma in (wild-type or mutant) also can profoundly influence activity. Sever al of the constructed Gag-Pol fusion proteins contained a wild-type PR domain. Some of these underwent intracellular PR-mediated processing, while others did not. All proteins in which the PR domain was precede d by upstream Gag sequences showed specific proteolysis. By contrast, all proteins initiated with a methionine placed one residue upstream o f the natural N terminus of PR failed to show specific proteolysis. Am ino-terminal sequencing of one such protein yielded the correct amino acid sequence and showed that the initiating methionine was not remove d. One interpretation of these findings is that activation of PR requi res the generation of the precise N terminus of the mature PR.