Suppression of amber nonsense mutations of herpes simplex virus type 1 in a tissue culture system

MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK We have investigated the ability of monkey kidney cell lines (SupD3 and SupD12) inducibly expressing an amber suppressor tRNA ser to suppress amber nonsense mutations in three genes of herpes simplex virus type 1 (HSV-1). HSV-1 mutant TK4, which contains a nonsense mutation in the non-essential viral thymidine kinase (TK) gene, synthesized a full-length TK polypeptide at about 30% of the wild-type (wt) level in induced SupD3 cells but not in the parental non-suppressor (Sup0) cells. Using complementing cells, we constructed HSV-1 mutants carrying nonsense mutations in an essential gene, UL8, encoding a protein essential for viral DNA replication ( amb UL8) or in a partially dispensable gene, UL12, encoding alkaline nuclease ( amb UL12). The growth of the mutants in Vero or Sup0 cells was either totally ( amb UL8) or severely ( amb UL12) impaired, whereas in cells expressing suppressor tRNA the mutants produced infectious virus. However, the yields were much lower than obtained with wt HSV-1. In Vero or Sup0 cells the mutants amb UL8 and amb UL12 failed to synthesize full-length UL8 and UL12 protein products, respectively. Western immuno-blotting showed that the virus amb UL12 produced full-length UL12 protein in SupD12 cells which yielded a level of 25.9% of the alkaline nuclease activity of the wt HSV-1 control. Our results show that the levels of suppression of the nonsense mutations in amb UL8 and amb UL12 are insufficient to allow their continuing propagation in the available Sup + cells. Possible reasons are discussed. * Author for correspondence. Fax +44 141 337 2236. e-mail A.PATEL@VIR.GLA.AC.UK Received 26 July 1995; accepted 23 October 1995.