SUPPRESSION OF AMBER NONSENSE MUTATIONS OF HERPES-SIMPLEX VIRUS TYPE-1 IN A TISSUE-CULTURE SYSTEM

We have investigated the ability of monkey kidney cell lines (SupD3 an d SupD12) inducibly expressing an amber suppressor tRNA(ser) to suppre ss amber nonsense mutations in three genes of herpes simplex virus typ e 1 (HSV-1). HSV-I mutant TK4, which contains a nonsense mutation in t he non-essential viral thymidine kinase (TK) gene, synthesized a full- length TK polypeptide at about 30% of the wild-type (wt) level in indu ced SupD3 cells but not in the parental nonsuppressor (Sup0) cells. Us ing complementing cells, we constructed HSV-1 mutants carrying nonsens e mutations in an essential gene, UL8, encoding a protein essential fo r viral DNA replication (ambUL8) or in a partially dispensable gene, U L12, encoding alkaline nuclease (ambUL12). The growth of the mutants i n Vero or Sup0 cells was either totally (ambUL8) or severely (ambUL12) impaired, whereas in cells expressing suppressor tRNA the mutants pro duced infectious virus. However, the yields were much lower than obtai ned with wt HSV-1. In Vero or Sup0 cells the mutants ambUL8 and ambUL1 2 failed to synthesize full-length UL8 and UL12 protein products, resp ectively. Western immuno-blotting showed that the virus ambUL12 produc ed full-length UL12 protein in SupD12 cells which yielded a level of 2 5.9 % of the alkaline nuclease activity of the wt HSV-1 control. Our r esults show that the levels of suppression of the nonsense mutations i n ambUL8 and ambUL12 are insufficient to allow their continuing propag ation in the available Sup(+) cells. Possible reasons are discussed.