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J. Biol. Chem., Vol. 276, Issue 33, 30786-30793, August 17, 2001
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From the Program in Cellular Biotechnology, Institute of
Biotechnology, Biocenter Viikki, P. O. Box 56, University of Helsinki,
FIN-00014 Helsinki, Finland
The virus-specific components (nsP1-nsP4) of
Semliki Forest virus RNA polymerase are synthesized as a large
polyprotein (P1234), which is cleaved by a virus-encoded protease.
Based on mutagenesis studies, nsP2 has been implicated as the protease
moiety of P1234. Here, we show that purified nsP2 (799 amino acids) and
its C-terminal domain Pro39 (amino acids 459-799) specifically process
P1234 and its cleavage intermediates. Analysis of cleavage products of
in vitro synthesized P12, P23, and P34 revealed cleavages
at sites 1/2, 2/3, and 3/4. The cleavage regions of P1/2, P2/3, and P3/4 were expressed as thioredoxin fusion proteins (Trx12, Trx23, and
Trx34), containing ~20 amino acids on each side of the cleavage sites. After exposure of these purified fusion proteins to nsP2 or
Pro39, the reaction products were analyzed by SDS-polyacrylamide gel
electrophoresis, mass spectrometry, and amino-terminal sequencing. The
expected amino termini of nsP2, nsP3, and nsP4 were detected. The
cleavage at 3/4 site was most efficient, whereas cleavage at 1/2 site
required 5000-fold more of Pro39, and 2/3 site was almost resistant to
cleavage. The activity of Pro39 was inhibited by
N-ethylmaleimide, Zn2+, and Cu2+,
but not by EDTA, phenylmethylsulfonyl fluoride, or pepstatin, in
accordance with the thiol proteinase nature of nsP2.
* Biocentrum Helsinki fellow. To whom correspondence
should be addressed. Tel.: 358-9-191-59400; Fax: 358-9-191-59560;
E-mail: leevi.kaariainen@helsinki.fi.
Site-specific Protease Activity of the Carboxyl-terminal Domain
of Semliki Forest Virus Replicase Protein nsP2*
*
This work was supported by Academy of Finland Grant 8397 and
by grants from the Technology Development Center and Center for International Mobility.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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