Nuclear targeting of Semliki Forest virus nsP2.
The Semliki Forest virus-specific nonstructural protein nsP2 is transported into the nuclei of both infected and transfected BHK cells. The pentapeptide sequence P 648R 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 are rapidly inhibited in virus-infected cells, and nsP2 could be involved in these processes. It has been postulated that the inhibition of cellular replication could be due to viral NTPase activity. We have expressed 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 presence of single-stranded RNA, a typical feature of RNA helicases. The role of nsP2 in the nucleus was studied by creating a mutant virus SFV-RDR, which contained an altered NLS (PRDRV). The mutation affected neither the processing nor the stability of nsP2, but it did render nsP2 completely cytoplasmic. SFV-RDR was shown to be fully infectious, and no difference could be seen in the expression of viral proteins. In addition, the inhibition of host DNA synthesis was almost equally efficient in both wild-type and mutant-infected cells. The pathogenic properties of the mutant will be further studied.
Institute of Biotechnology, University of Helsinki, Finland.
M
M
Rikkonen
J
J
Peränen
L
L
Kääriäinen
D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D012672:Semliki forest virus / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014759:Viral Envelope Proteins