Mutational analysis of the proposed FG loop of poliovirus proteinase 3C identifies amino acids that are necessary for 3CD cleavage and might be determinants of a function distinct from proteolytic activity.
Mutations were introduced into a cDNA clone of poliovirus resulting in single-amino-acid substitutions within the region of the proposed FG loop of proteinase 3C. RNAs were made by in vitro transcription with T7 RNA polymerase and used to transfect HeLa cells. Virus viability was assessed as indicated by cell lysis. In parallel, RNAs were translated in vitro by using a HeLa cell lysate, and the patterns of the processed poly-proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Replacement of Lys-78, Arg-79, and Glu-81 had apparently no effect on virus viability and on proteolytic processing. In contrast, virus viability was abolished by mutation of Phe-83, Arg-84, Asp-85, Ile-86, and Arg-87. With respect to substitution of Phe-83, Asp-85, and Arg-87, these effects correlated with impaired processing of the 3CD cleavage site, separating 3C and 3D, and, to a lesser extent, of the P1 precursor. Replacement of Arg-84 and Ile-86, on the other hand, did not alter the processing pattern. Thus, the lethal effects in these mutant genomes may not have been caused by impaired processing. A special case was the mutant of Lys-82-Gln. Virus recovered from cells transfected with RNA carrying this mutation always contained an A-to-G transition which resulted in the replacement of glutamine for arginine. Our data suggest that residues in the proposed FG loop of proteinase 3C influence 3CD cleavage and that they are determinants of a function unrelated to proteolytic processing.
Hämmerle
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T
Department of Microbiology, School of Medicine, State University of New York at Stony Brook 11794-8621.
Molla
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A
Wimmer
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D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017955:Poliovirus / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins
D000596:Amino Acids; D004279:DNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases