Quantification and analysis of thymidine kinase expression from acyclovir-resistant G-string insertion and deletion mutants in herpes simplex virus-infected cells.
To be clinically relevant, drug-resistant mutants must both evade drug action and retain pathogenicity. Many acyclovir-resistant herpes simplex virus mutants from clinical isolates have one or two base insertions (G8 and G9) or one base deletion (G6) in a homopolymeric run of seven guanines (G string) in the gene encoding thymidine kinase (TK). Nevertheless, G8 and G9 mutants express detectable TK activity and can reactivate from latency in mice, a pathogenicity marker. On the basis of studies using cell-free systems, ribosomal frameshifting can explain this ability to express TK. To investigate frameshifting in infected cells, we constructed viruses that express epitope-tagged versions of wild-type and mutant TKs. We measured TK activity by plaque autoradiography and expression of frameshifted and unframeshifted TK polypeptides using a very sensitive immunoprecipitation-Western blotting method. The G6 mutant expressed ∼0.01% of wild-type levels of TK polypeptide. For the G9 mutant, consistent with previous results, much TK expression could be ascribed to reversion. For the G8 mutant, from these assays and pulse-labeling studies, we determined the ratio of synthesis of frameshifted to unframeshifted polypeptides to be 1:100. The effects of stop codons before or after the G string argue that frameshifting can initiate within the first six guanines. However, frameshifting efficiency was altered by stop codons downstream of the string in the 0 frame. The G8 mutant expressed only 0.1% of the wild-type level of full-length TK, considerably lower than estimated previously. Thus, remarkably low levels of TK are sufficient for reactivation from latency in mice.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
Dongli
D
Pan
Donald M
DM
Coen
D000212:Acyclovir / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D018389:Codon, Nonsense; D024882:Drug Resistance, Viral / Q000235:genetics; D016368:Frameshift Mutation; D017353:Gene Deletion; D015870:Gene Expression; D023061:Gene Order; D006801:Humans; D016254:Mutagenesis, Insertional; D009154:Mutation; D055550:Protein Stability; D018139:Simplexvirus / Q000235:genetics* / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism
D000998:Antiviral Agents; D018389:Codon, Nonsense; D013937:Thymidine Kinase; D000212:Acyclovir