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Department of Biochemistry and Pharmacy, Åbo Akademi University and Turku Immunology Centre, PO Box 66, 20521 Turku, Finland
Correspondence
Minna Tuittila
minna.tuittila{at}abo.fi
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, V11
I and L201F, V11I and D249N, A48E and G70A or T435A and F442L, are introduced into an rA774 derivative carrying R469. The critical role of the arginine codon for neurovirulence was confirmed further by the acquisition of a fully lethal phenotype following the introduction of R469 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.
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