Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo.
The non-structural proteins of Sindbis virus, nsP1, 2, 3 and 4, are produced upon cleavage of polyproteins P123 and P1234 by a proteinase residing in nsP2. We used cell free translation of SP6 transcripts to study the proteolytic activity of nsP2 and of nsP2-containing polyproteins. To generate polyprotein enzymes, a set of plasmids was made in which cleavage sites were eliminated and new initiation and termination codons introduced by in vitro mutagenesis. As a substrate, we used a polyprotein in which the nsP2 proteinase had been inactivated by a single amino acid substitution. All nsP2-containing polyproteins cleaved the nsP1/2 site in trans. However, proteinases containing nsP1 were unable to cleave the nsP2/3 site. Furthermore, only proteinases containing nsP3 could cleave the nsP3/4 site. These differences in cleavage site specificity result in a temporal regulation of processing in vivo. At 1.7 h post infection P123 and nsP4 accumulated and only small amounts of P34 were found. However, at 4 h post infection P123 was processed rapidly and P34 was produced rather than nsP4. Since nsP4 is thought to be the viral RNA polymerase, the temporal regulation of the nsP4/P34 ratio may be responsible for the temporal regulation of RNA synthesis.
de Groot
R J
RJ
Division of Biology, California Institute of Technology, Pasadena 91125.
Hardy
W R
WR
Shirako
Y
Y
Strauss
J H
JH
D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D003062:Codon / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000235:genetics; D012845:Sindbis Virus / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins
D003062:Codon; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D010450:Endopeptidases