Chikungunya virus (CHIKV)， genus Alphavirus， family Togaviridae， has a positive-stand RNA genome approximately 12 kb in length. In infected cells， the genome is translated into non-structural polyprotein P1234， an inactive precursor of the viral replicase， which is activated by cleavages carried out by the non-structural protease， nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First， we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second， the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third， substitution of Ser482 residue， recently re... More
Chikungunya virus (CHIKV)， genus Alphavirus， family Togaviridae， has a positive-stand RNA genome approximately 12 kb in length. In infected cells， the genome is translated into non-structural polyprotein P1234， an inactive precursor of the viral replicase， which is activated by cleavages carried out by the non-structural protease， nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First， we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second， the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third， substitution of Ser482 residue， recently reported to contribute to the protease activity of nsP2， with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth， Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast， trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth， Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus， CHIKV nsP2 is a cysteine protease.
