Molecular analysis of a neurovirulent herpes simplex virus type 2 strain with reduced thymidine kinase activity

Thymidine kinase (TK) of herpes simplex virus (HSV) has been identified as one of the factors responsible for its virulence. We have previously isolated acyclovir (ACV)-resistant HSV type 2 (HSV-2), strain YS-4 C-1, by simple plaque cloning from a clinical isolate. Although YS-4 C-1 had extremely low TK activity, it retained high virulence in mice. To determine the mechanism of the reduction of TK activity, a molecular analysis of the YS-4 C-1 TK gene was performed. YS-4 C-1 produced TK mRNA, which was indistinguishable both in size and amount from that of wild-type strains. However, the YS-4 C-1 TK had a single amino acid change from serine to asparagine at amino acid residue 182 of the TK polypeptide, which was caused by a single nucleotide mutation. It was situated within a highly conserved region (162-194) and close to the putative nucleoside-binding site (169-177), one of the three active centers of TK. In order to confirm the effect of this missense mutation on both the TK activity and neurovirulence, the mutation was introduced into the TK genes of wild-type strains. Although all the recombinants were altered to ACV-resistant viruses with reduced TK activity, they retained high neurovirulence for mice. Our study thus suggested that this mutant TK, in spite of low activity, might play a role in the neurovirulence of HSV-2.