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Published August 1990 | public
Journal Article

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.

Additional Information

© Oxford University Press. Received on February 20, 1990; revised on April 27, 1990. We thank Ellen G.Strauss for helpful comments and for assistance in the preparation of the manuscript. We thank C. M. Rice for supplying the plasmid pToto1000.S, Randy Levinson for generously providing the plasmid pToto.Cys481-Gly and Richard Kuhn for stimulating discussions and for supplying the plasmid pTotoS7. Furthermore, we thank Alexander van der Bliek for helpful suggestions concerning the PCR experiments and Michael Harrington for advice on densitometry. This work has been supported by Grants AI10793 and AI20612 from the National Institutes of Health and by Grant DMB8617372 from the National Science Foundation. R. J. de Groot was supported by a fellowship of the European Molecular Biology Organization (ALTF 280-1988).

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August 19, 2023
October 19, 2023