Mapping of the feline calicivirus proteinase responsible for autocatalytic processing of the nonstructural polyprotein and identification of a stable proteinase-polymerase precursor protein.
Expression of the region of the feline calicivirus (FCV) ORF1 encoded by nucleotides 3233 to 4054 in an in vitro rabbit reticulocyte system resulted in synthesis of an active proteinase that specifically processes the viral nonstructural polyprotein. Site-directed mutagenesis of the cysteine (Cys1193) residue in the putative active site of the proteinase abolished autocatalytic cleavage as well as cleavage of the viral capsid precursor, suggesting that this "3C-like" proteinase plays an important role in proteolytic processing during viral replication. Expression of the region encoding the C-terminal portion of the FCV ORF1 (amino acids 942 to 1761) in bacteria allowed direct N-terminal sequence analysis of the virus-specific polypeptides produced in this system. The results of these analyses indicate that the proteinase cleaves at amino acid residues E960-A961, E1071-S1072, E1345-T1346, and E1419-G1420; however, the cleavage efficiency is varied. The E1071-S1072 cleavage site defined the N terminus of a 692-amino-acid protein that contains sequences with similarity to the picornavirus 3C proteinase and 3D polymerase domains. Immunoprecipitation of radiolabeled proteins from FCV-infected feline kidney cells with serum raised against the FCV ORF1 C-terminal region showed that this "3CD-like" proteinase-polymerase precursor protein is apparently stable and accumulates in cells during infection.
Sosnovtseva
S A
SA
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Sosnovtsev
S V
SV
Green
K Y
KY
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017927:Calicivirus, Feline / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D002415:Cats; D002460:Cell Line; D002874:Chromosome Mapping; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D011498:Protein Precursors; D011506:Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases