Functional characterization of cleavage-defective mutants of encephalomyocarditis virus

Abstract

We characterized seven temperature-sensitive capsid cleavage (cleavage-defective) mutants of encephalomyocarditis virus. Our experimental approach was to monitor in vitro proteolysis reactions of either wild-type or cleavage-defective mutant capsid precursors mixed with cell-free translation products (containing the viral protease) of either wild-type or mutant viral RNA. The cell-free translation reactions and in vitro proteolysis reactions were done at 38°C, because at this temperature cleavage of the capsid precursors was restricted in reactions containing cleavage-defective mutant viral RNA as the message, relative to those reactions containing wild-type viral RNA as the message. Wild-type or cleavagedefective mutant capsid precursors were prepared by adding cycloheximide to cell-free translation reactions primed with wild-type or mutant viral RNA, respectively, 12 min after the initiation of translation. In vitro proteolysis of wildtype capsid precursors with cell-free translation products of either wild-type or cleavage-defective mutant viral RNA led to similar products at 38°C, indicating that the cleavage-defective mutant viral protease was not temperature sensitive. As a corollary to this, at 38°C cleavage-defective mutant capsid precursors were not cleaved as completely as were wild-type capsid precursors by products of cellfree translation of wild-type viral RNA. The results from these in vitro proteolysis experiments indicate that all seven of the cleavage-defective mutants have capsid precursors with a temperature-sensitive configuration. High-molecular-weight precursor polypeptides are cleaved to functional proteins by a series of sequential proteolytic cleavages during the replication of picornaviruses (2-6). With encephalomyocarditis (EMC) virus, the capsid proteins 8, P, y, and a are generated by cleavage of the high-molecular-weight precursor polypeptide A, with polypeptides Dl, e, and possibly B