Functional characterization of cleavage-defective mutants of encephalomyocarditis virus.
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 degrees 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 cleavage-defective 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 wild-type capsid precursors with cell-free translation products of either wild-type or cleavage-defective mutant viral RNA led to similar products at 38 degrees C, indicating that the cleavage-defective mutant viral protease was not temperature sensitive. As a corollary to this, at 38 degrees C cleavage-defective mutant capsid precursors were not cleaved as completely as were wild-type capsid precursors by products of cell-free 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.
D002213:Capsid / Q000235:genetics* / Q000378:metabolism; D002474:Cell-Free System; D003513:Cycloheximide / Q000494:pharmacology; D004686:Encephalomyocarditis virus / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics; D013696:Temperature; D014764:Viral Proteins / Q000235:genetics*
D011498:Protein Precursors; D012367:RNA, Viral; D014764:Viral Proteins; D003513:Cycloheximide