title: Net -1 frameshifting on a noncanonical sequence in a herpes simplex virus drug-resistant mutant is stimulated by nonstop mRNA.
abstract: Ribosomal frameshifting entails slippage of the translational machinery during elongation. Frameshifting permits expression of more than one polypeptide from an otherwise monocistronic mRNA, and can restore expression of polypeptides in the face of frameshift mutations. A common mutation conferring acyclovir resistance in patients with herpes simplex virus disease deletes one cytosine from a run of six cytosines (C-chord) in the viral thymidine kinase (tk) gene. However, this mutation does not abolish TK activity, which is important for pathogenicity. To investigate how this mutant retains TK activity, we engineered and analyzed viruses expressing epitope-tagged TK. We found that the mutant's TK activity can be accounted for by low levels of full-length TK polypeptide produced by net -1 frameshifting during translation. The efficiency of frameshifting was relatively high, 3-5%, as the polypeptide from the reading frame generated by the deletion, which lacks stop codons (nonstop), was poorly expressed mainly because of inefficient protein synthesis. Stop codons introduced into this reading frame greatly increased its expression, but greatly decreased the level of full-length TK, indicating that frameshifting is strongly stimulated by a new mechanism, nonstop mRNA, which we hypothesize involves stalling of ribosomes on the polyA tail. Mutational studies indicated that frameshifting occurs on or near the C-chord, a region lacking a canonical slippery sequence. Nonstop stimulation of frameshifting also occurred when the C-chord was replaced with a canonical slippery sequence from HIV. This mechanism thus permits biologically and clinically relevant TK synthesis, and may occur more generally.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Dongli
authors.initials: D
authors.lastname: Pan
authors.forename: Donald M
authors.initials: DM
authors.lastname: Coen
mesh_terms: D000212:Acyclovir; D000818:Animals; D001345:Autoradiography; D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D016368:Frameshift Mutation / Q000235:genetics; D018965:Frameshifting, Ribosomal / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000472:pathogenicity; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D015513:Oncogene Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014709:Vero Cells
chemical_list: D017931:DNA Primers; C099763:NET1 protein, human; D015513:Oncogene Proteins; D013937:Thymidine Kinase; D000212:Acyclovir