Cleavage‐site preferences of Sindbis virus polyproteins containing the non‐structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo

• Published in: The EMBO journal Vol. 9; no. 8; pp. 2631 - 2638
• Main Authors: Groot, R. J., Hardy, W. R., Shirako, Y., Strauss, J. H.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 01.08.1990
• Subjects: Animals; Base Sequence; Biological and medical sciences; Capsid - genetics; Capsid - metabolism; Cells, Cultured; Chick Embryo; Codon - genetics; Endopeptidases - metabolism; Fibroblasts - enzymology; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Viral; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Plasmids; Polymerase Chain Reaction; Protein Biosynthesis; Protein Processing, Post-Translational; RNA, Viral - genetics; Sindbis Virus - enzymology; Sindbis Virus - genetics; Transcription, Genetic; Translation. Translation factors. Protein processing; Viral Core Proteins - genetics; Viral Core Proteins - metabolism; Viral Nonstructural Proteins; In vitro transcription; Molecular processing; In vitro translation; Enzyme; Cleavage site; Proteinase; Togaviridae; Precursor; Sindbis virus; Virus; Infection; Proteins; Immunoprecipitation reaction; Regulation(control); Specificity; Mutagenesis; Alphavirus; Mechanism of action
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1990.tb07445.x

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.