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 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 (ambUL8) or in a partially dispensable gene, UL12, encoding alkaline nuclease (ambUL12). The growth of the mutants in Vero or Sup0 cells was either totally (ambUL8) or severely (ambUL12) 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 ambUL8 and ambUL12 failed to synthesize full-length UL8 and UL12 protein products, respectively. Western immunoblotting showed that the virus ambUL12 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 ambUL8 and ambUL12 are insufficient to allow their continuing propagation in the available Sup+ cells. Possible reasons are discussed.
Patel
A H
AH
MRC Virology Unit, Institute of Virology, Glasgow, UK. A.PATEL@VIR.GLA.AC.UK
Subak-Sharpe
J H
JH
Stow
N D
ND
Marsden
H S
HS
Maclean
J B
JB
Dargan
D J
DJ
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004265:DNA Helicases / Q000235:genetics; D019915:DNA Primase; D018259:Herpesvirus 1, Human / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D013937:Thymidine Kinase / Q000235:genetics; D014709:Vero Cells; D014764:Viral Proteins
D014764:Viral Proteins; D013937:Thymidine Kinase; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases