NUCLEAR TARGETING OF SEMLIKI FOREST VIRUS NSP2

The Semliki Forest virus-specific nonstructural protein nsP2 is transp orted into the nuclei of both infected and transfected BHK cells. The pentapeptide sequence P (648)R RRV is an essential part of the nuclear localization signal (NLS) of nsP2, the middle arginine being the most critical residue for nuclear targeting. Host DNA and RNA syntheses ar e rapidly inhibited in virus-infected cells, and nsP2 could be involve d in these processes. It has been postulated that the inhibition of ce llular replication could be due to viral NTPase activity. We have expr essed and purified nsP2 in E. coli using the highly efficient T7 based expression system. Purified nsP2 was shown to have ATPase and GTPase activities, and these specific activities were increased in the presen ce of single-stranded RNA, a typical feature of RNA helicases. The rol e of nsP2 in the nucleus was studied by creating a mutant virus SFV-RD R, which contained an altered NLS (PRDRV). The mutation affected neith er the processing nor the stability of nsP2, but it did render nsP2 co mpletely cytoplasmic. SFV-RDR was shown to be fully infectious, and no difference could be seen in the expression of viral proteins. In addi tion, the inhibition of host DNA synthesis was almost equally efficien t in both wild-type and mutant-infected cells. The pathogenic properti es of the mutant will be further studied.