Mutations in the nuclear localization signal of nsP2 influencing RNA synthesis, protein expression and cytotoxicity of Semliki Forest virus.
The cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues (648)RRR(650) with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations.
Tamm
Kristi
K
Estonian Biocentre, Riia 23, 51010, Tartu, Estonia.
Merits
Andres
A
Institute of Technology, University of Tartu, Nooruse 1, 50411, Tartu, Estonia.|Estonian Biocentre, Riia 23, 51010, Tartu, Estonia.
Sarand
Inga
I
Estonian Biocentre, Riia 23, 51010, Tartu, Estonia.
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003588:Cytopathogenic Effect, Viral; D015967:Gene Expression Regulation, Viral; D009154:Mutation; D019913:Nuclear Localization Signals / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis*; D012672:Semliki forest virus / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D019913:Nuclear Localization Signals; D012367:RNA, Viral; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase