Cleavage between nsP1 and nsP2 initiates the processing pathway of Sindbis virus nonstructural polyprotein P123.
The cleavage between nsP1 and nsP2 and that between nsP2 and nsP3 in the Sindbis virus nonstructural polyproteins was studied with respect to order of processing and enzyme-substrate relationships, using site-specific mutants in which the cleavage sites had been altered. The penultimate Gly in nsP1 or nsP2 or both was substituted by Ala, Val, or Glu, and processing was studied in vitro. Substitution with Ala resulted in partial cleavage whereas substitution with Val or Glu totally abolished cleavage at the mutagenized site. Abolishment of cleavage at the nsP2/nsP3 site did not affect processing at the nsP1/nsP2 site in the precursor polyprotein P123, and nsP1 and P23 were produced. When cleavage at the nsP1/nsP2 site was abolished, however, processing at the nsP2/nsP3 site was also prevented and P123 accumulated. To investigate why cleavage at the nsP1/nsP2 site should be required for cleavage at the nsP2/nsP3 site, the mutagenized polypeptides were used as enzymes in trans-cleavage experiments. We found that P123 can cleave the nsP1/nsP2 site but not the nsP2/nsP3 site, whereas P23 can cleave the nsP2/nsP3 site very efficiently. Thus, cleavage at the nsP1/nsP2 site by P123 is required to produce an enzyme capable of cleaving the nsP2/nsP3 site. Release of nsP4 from P1234 appears to be independent of the other cleavages and occurs primarily immediately after translation. These mutations were also transferred into a full-length cDNA clone of Sindbis virus and virus was recovered. Mutants defective in the cleavage of the nsP2/nsP3 site were temperature sensitive, growing at a slightly reduced rate compared to wild-type virus at 30 degrees but growing poorly at 40 degrees. Mutants defective in the cleavage of both the nsP1/nsP2 site and the nsP2/nsP3 site were viable but grew poorly compared with wild-type at any temperature.
Shirako
Y
Y
Division of Biology, California Institute of Technology, Pasadena 91125.
Strauss
J H
JH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012689:Sequence Homology, Nucleic Acid; D012845:Sindbis Virus / Q000235:genetics*; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000235:genetics*; D017361:Viral Nonstructural Proteins
D015345:Oligonucleotide Probes; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins