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J. Biol. Chem., Vol. 278, Issue 43, 41636-41645, October 24, 2003
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Regulation of the Sequential Processing of Semliki Forest Virus Replicase Polyprotein*
¶||
From the
Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, P. O. Box 56, 00014 Helsinki, Finland and Estonian Biocentre and Institute of Molecular and Cellular Biology, Riia Street 23, 51010 Tartu, Estonia
The replication of most positive-strand RNA viruses and retroviruses is regulated by proteolytic processing. Alphavirus replicase proteins are synthesized as a polyprotein, called P1234, which is cleaved into nsP1, nsP2, nsP3, and nsP4 by the carboxyl-terminal protease domain of nsP2. The cleavage intermediate P123+nsP4 synthesizes minus-strand copies of the viral RNA genome, whereas the completely processed complex is required for plus-strand synthesis. To understand the mechanisms responsible for this sequential proteolysis, we analyzed in vitro translated Semliki Forest virus polyproteins containing noncleavable processing sites or various deletions. Processing of each of the three sites in vitro required a different type of activity. Site 3/4 was cleaved in trans by nsP2, its carboxyl-terminal fragment Pro39, and by all polyprotein proteases. Site 1/2 was cleaved in cis with a half-life of about 20–30 min. Site 2/3 was cleaved rapidly in trans but only after release of nsP1 from the polyprotein exposing an "activator" sequence present in the amino terminus of nsP2. Deletion of amino-terminal amino acids of nsP2 or addition of extra amino acid residues to its amino terminus specifically inhibited the protease activity that processes the 2/3 site. This sequence of delayed processing of P1234 would explain the accumulation of P123 plus nsP4, the early short-lived minus-strand replicase. The polyprotein stage would allow correct assembly and membrane association of the RNA-polymerase complex. Late in infection free nsP2 would cleave at site 2/3 yielding P12 and P34, the products of which, nsP1–4, are distributed to the plasma membrane, nucleus, cytoplasmic aggregates, and proteasomes, respectively.
Received for publication, July 11, 2003 , and in revised form, August 11, 2003.
* This work was supported by the Academy of Finland (Grants 8397 and 201687), Biocentrum Helsinki, and Helsinki University Research Funds, as well as the Estonian Science Foundation (Grant 5055). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ An International Senior Research Fellow of the Wellcome Trust.
|| To whom correspondence should be addressed. Tel.: 358-9-19159400; Fax: 358-9-19159560; E-mail: leevi.kaariainen{at}helsinki.fi.
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