In vitro suppression of UAG and UGA mutants in the thymidine kinase gene of herpes simplex virus.

Abstract

We have studied the in vitro translation, in nuclease-treated reticulocyte lysates, of mRNA from cells infected with several thymidine kinase-deficient mutants of herpes simplex virus type 1. The addition of suppressor tRNAs from yeast resulted in suppression of the mutant property in the case of two mutants. Synthesis of enzymatically active viral thymidine kinase was restored by serine-inserting amber suppressor tRNA in the case of HSV TK4-and synthesis of the intact, but inactive, thymidine kinase protein was restored by serine-and leucine-inserting UGA suppressor tRNAs in the case of HSV TK43-. Read-through of the normal termination at the end of the thymidine kinase gene was promoted by UGA suppressor tRNAs. We conclude that HSV-TK4-is an amber (UAG) mutant and that HSV-TK43-is an opal (UGA) mutant.The isolation and characterization of nonsense mutants and nonsense suppressors in mammalian cells and their viruses will provide a powerful tool with which to analyze numerous viral biochemical and cellular functions. As yet, however, no nonsense suppressors have been reported in mammalian cells. In this report we describe unique nonsense mutants in a viral gene with a selectable phenotype which should make isolation of nonsense suppressors feasible.A nonsense mutation creates an in-phase polypeptide chain termination codon, such as UAG (amber), UAA (ochre), or UGA (opal), within the coding region of a structural gene. These codons result in the synthesis of shorter polypeptide products and usually loss of enzymatic or other activity. Suppressor mutations have been identified which result in the insertion of an amino acid at the position of the nonsense termination codon, which, in turn, leads to the synthesis of the complete polypeptide chain and restoration of functional activity (1).Several recent reports show that nonsense termination codons in eukaryotic cells or viruses can be suppressed by yeast suppressor tRNAs. In the first case, Gesteland et al. (2) showed that host range mutants of an adenovirus-simian virus 40 hybrid (AD2+ ND1) contain amber and ochre nonsense termination codons that can be suppressed by yeast suppressors in an in vitro cell-free suppression system. A second report described an ochre mutant within the hypoxanthine phosphoribosyltransferase gene (protein) of mouse cells (3). Another report described suppression of the natural amber termination codon between the gag and pol genes of murine leukemia virus RNA (4). Pelham (5) has presented evidence that a natural UAG termination codon in tobacco mosaic virus can be suppressed in vitro by amber suppressor tRNA from yeast.In this report we show that certain thymidine kinase (TK)-deficient mutants of herpes simplex virus (HSV) are nonsense mutants because the mutant property can be suppressed in vitro in a mammalian cell-free translation system using amber and opal suppressor tRNAs from yeast as well as having other properties expected of nonsense mutations. In one case the suppressed protein has enzymatic and immunological properti...

Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1.

“…The TK gene can code for a protein containing 376 amino acids with a molecular weight of 40,900. This also compares favorably with the estimated molecular weight of 40,000-44,000 obtained from NaDodSOjpolyacrylamide gel electrophoresis, (2)(3)(4)30 Table 2 indicates the number of times each codon is used and the overall amino acid composition. While all but four codons are used at least once, the distribution of codon usage is somewhat unusual.…”

Molecular Analysis of the Pyrimidine Deoxyribonucleoside Kinase Gene of Wild-type and Acyclovir-resistant Strains of Varicella-Zoster Virus

“…This loss of kinase activity for several substrates is consistent with an alteration in protein structure, such as that caused by premature termination. Similar nonsense mutations have been identified in TKdeficient HSV strains (Summers et al, 1975;Cremer et al, 1979). It was surprising that these VZV mutants independently derived in separate laboratories contained the same base substitution.…”

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

“…TK4 has a single base change within codon 44 of the thymidine kinase (TK) gene changing a glutamine codon (CAG) to amber (UAG) (Haarr et al, 1985). The TK4 mutant was selected because it has been well characterized and has previously been shown to be suppressed by a serineinserting Sup + tRNA from yeast (Cremer et al, 1979). The HSV-1 TK gene, which is not essential for virus growth in tissue culture, has three operational ATG translational start signals resulting in the synthesis of one major (M,.…”

“…Initially, the amber TK4 mutant of HSV-1 strain Cl 101 (Cremer et al, 1979;Haarr et al, 1985) was used to test the suppressor tRNA system. TK4 has a single base change within codon 44 of the thymidine kinase (TK) gene changing a glutamine codon (CAG) to amber (UAG) (Haarr et al, 1985).…”