Reverse transcriptase and protease activities of avian leukosis virus Gag-Pol fusion proteins expressed in insect cells.
Protease (PR)-defective avian leukosis virus particles display 300-fold-reduced levels of reverse transcriptase (RT) activity relative to wild-type particles. This observation suggests that during virion assembly RT is activated by proteolytic maturation of the Gag-Pol polyprotein precursor. To study the relationship between proteolytic cleavage and RT activation, we subjected PR-defective virion cores to digestion with purified viral PR and analyzed the structure of the major polypeptides produced as well as RT activity. Under conditions in which Gag precursors were fully matured, the RT domain was only incompletely released 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 repress RT activity. To test this idea, we constructed recombinant baculoviruses expressing 19 different fusion proteins with upstream Gag or downstream Pol sequences attached to RT. Each protein was partially purified and assayed for its inherent RT activity. The results are consistent with the idea that Gag sequences can inhibit RT activity but indicate that the size of the Pol domain as well as the status of the PR domain (wild-type or mutant) also can profoundly influence activity. Several 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 preceded by upstream Gag sequences showed specific proteolysis. By contrast, all proteins initiated with a methionine placed one residue upstream of the natural N terminus of PR failed to show specific proteolysis. Amino-terminal sequencing of one such protein yielded the correct amino acid sequence and showed that the initiating methionine was not removed. One interpretation of these findings is that activation of PR requires the generation of the precise N terminus of the mature PR.
Stewart
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Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Vogt
V M
VM
D000818:Animals; D001354:Avian Leukosis Virus / Q000201:enzymology* / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D003673:Defective Viruses / Q000378:metabolism; D010450:Endopeptidases / Q000032:analysis*; D004789:Enzyme Activation; D015685:Fusion Proteins, gag-pol / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D015750:Genes, gag; D015751:Genes, pol; D009036:Moths; D009154:Mutation; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D011994:Recombinant Proteins / Q000378:metabolism; D012191:Retroviridae Proteins / Q000378:metabolism*; D014771:Virion / Q000378:metabolism
D015685:Fusion Proteins, gag-pol; D011994:Recombinant Proteins; D012191:Retroviridae Proteins; C040520:avian retrovirus proteins; D012194:RNA-Directed DNA Polymerase; D010450:Endopeptidases