Site-specific protease activity of the carboxyl-terminal domain of semlikiforest virus replicase protein nsP2

The virus-specific components (nsP1-nsP4) of Semliki Forest virus RNA polym erase are synthesized as a large polyprotein (P1234), which is cleaved by a virus-encoded protease. Based on mutagenesis studies, nsP2 has been implic ated 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) specifi cally process P1234 and its cleavage intermediates. Analysis of cleavage pr oducts of in vitro synthesized P12, P23, and P34 revealed cleavages at site s 1/2,2/3, and 3/4. The cleavage regions of P1/2, P2/3, and P3/4 were expre ssed as thioredoxin fusion proteins (Trx12, Trx23, and Trx34), containing s imilar to 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, a nd 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 al most resistant to cleavage. The activity of Pro39 was inhibited by N-ethylm aleimide, Zn2+, and Cu2+, but not by EDTA, phenylmethylsulfonyl fluoride, o r pepstatin, in accordance with the thiol proteinase nature of nsP2.