title: Amino acid mutations in the replicase protein nsP3 of Semliki Forest virus cumulatively affect neurovirulence.
abstract: It has been shown previously that an avirulent Semliki Forest virus (SFV) clone, rA774, engineered to carry the nsP3 gene of the virulent clone SFV4 becomes highly neurovirulent and is lethal for adult BALB/c mice. rA774, like several other alphaviruses, has an opal termination codon close to the 5' end of nsP3 (aa 469), while SFV4 has an arginine residue at this position. Mutation of the opal codon to an arginine residue increases the virulence of rA774 but does not reconstruct the severe neurovirulence of SFV4. Additionally, nsP3 amino acid sequences differ between these two strains by eight amino acids and by a deletion of seven amino acids in the C-terminal third of rA774 nsP3. This study shows that neurovirulence can be reconstituted gradually by exchanging individual amino acids and is fully retained when combinations of two nsP3 mutations, V(11)-->I and L(201)-->F, V(11)-->I and D(249)-->N, A(48)-->E and G(70)-->A or T(435)-->A and F(442)-->L, are introduced into an rA774 derivative carrying R(469). The critical role of the arginine codon for neurovirulence was confirmed further by the acquisition of a fully lethal phenotype following the introduction of R(469) into a moderately virulent rA774 recombinant carrying the SFV4 nsP1 and nsP2 genes. In conclusion, virulence determinants in SFV are distributed over a wide region of the nonstructural genes.
authors.affiliation: Department of Biochemistry and Pharmacy, Åbo Akademi University and Turku Immunology Centre, PO Box 66, 20521 Turku, Finland.
authors.forename: Minna
authors.initials: M
authors.lastname: Tuittila
authors.affiliation: Department of Biochemistry and Pharmacy, Åbo Akademi University and Turku Immunology Centre, PO Box 66, 20521 Turku, Finland.
authors.forename: Ari E
authors.initials: AE
authors.lastname: Hinkkanen
mesh_terms: D018354:Alphavirus Infections / Q000821:virology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D005260:Female; D005814:Genes, Viral; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D016601:RNA-Binding Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D012672:Semliki forest virus / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014774:Virulence / Q000235:genetics
chemical_list: C414175:Nsp3 protein, semliki forest virus; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase