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Suppression of amber nonsense mutations of herpes simplex virus type 1 in a tissue culture system
A H Patel1, J H Subak-Sharpe, N D Stow
1MRC Virology Unit, Institute of Virology, Glasgow, UK. A.PATEL@VIR.GLA.AC.UK
Abstract:
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.
Insights
Monkey kidney cells expressing suppressor tRNA can partially suppress amber nonsense mutations in herpes simplex virus type 1 (HSV-1) genes. However, the suppression levels are insufficient for efficient viral propagation, impacting viral DNA replication and alkaline nuclease activity.
Area of Science:
- Molecular Virology
- Genetics
- Cell Biology
Background:
- Nonsense mutations terminate protein synthesis prematurely, posing challenges for studying essential viral genes.
- Amber suppressor tRNAs can potentially rescue protein expression from nonsense mutations.
- Herpes Simplex Virus type 1 (HSV-1) possesses essential and non-essential genes crucial for its lifecycle.
Purpose of the Study:
- To investigate the efficacy of amber suppressor tRNA in monkey kidney cell lines for suppressing HSV-1 nonsense mutations.
- To assess the impact of suppressed mutations on viral gene expression and replication.
- To evaluate the potential for using suppressor cell lines for HSV-1 mutant propagation.
Main Methods:
- Construction of HSV-1 mutants with amber nonsense mutations in thymidine kinase (TK), UL8, and UL12 genes.
- Utilized inducible monkey kidney cell lines (SupD3, SupD12) expressing amber suppressor tRNA(Ser).
- Assessed viral gene product synthesis and viral infectivity using Western immunoblotting and plaque assays.
Main Results:
- Partial restoration of full-length thymidine kinase (TK) polypeptide in SupD3 cells.
- Infectious virus production from UL8 and UL12 nonsense mutants in suppressor cells, albeit at reduced yields.
- Demonstrated synthesis of full-length UL12 protein in SupD12 cells, with 25.9% alkaline nuclease activity compared to wild-type.
Conclusions:
- Amber suppressor tRNA can partially suppress nonsense mutations in HSV-1 genes, enabling limited viral propagation.
- The observed suppression levels are insufficient for efficient propagation of essential (UL8) and partially dispensable (UL12) HSV-1 mutants.
- Further research is needed to understand the limitations and potential applications of suppressor cell lines in virology.