A net +1 frameshift permits synthesis of thymidine kinase from a drug-resistant herpes simplex virus mutant.

Abstract

Clinical resistance to antiviral drugs requies that a virus evade drug therapy yet retain pathogenicity. Thymidine kinase (TK)-negative mutants ofherpes simplex virus are resistant to the drug, acyclovir, but are attenuated for pathogenicity in animal models. However, numerous cases of clinical resistance to acyclovir have been associated with viruses that were reported to express no TK activity. We studied an acyclovir-resistant clinical mutant that contains a single-base insertion in its tk gene, predicting the synthesis of a truncated TK polypeptide with no TK activity. Nevertheless, the mutant retained some TK activity and the ability to reactivate from latent infections of mouse trigeminal ganglia. The mutant expressed both the predicted truncated polypeptide and a low level of a polypeptide that comigrated with full-length TK on polyacrylamide gels and reacted with anti-TK antiserum, providing evidence for a frameshifting mechanism. In vitro transcription and tanslation ofmutant tkgenes, incuding constructs in which reporter epitopes could be expressed only if frameshiffing occurred, also gave rise to truncated and full-length polypeptides. Reverse transcriptase-polymerase chain reaction analysis coupled with open reading fram cloning failed to detect alterations in tktrspts that could account for the synthesis offull-length polypeptide. Thus, synthesis of full-length TK was due to an unusual net +1 frmeshlft during translation, a phenomenon hitherto confined in eukaryotic cells to certain RNA viruses and retrotransposons. Utilization of cellular frameshifting mechanisms may permit an otherwise TK-negative virus to exhibit clinical acyclovir resistance.Herpes simplex virus (HSV) is an important human pathogen, especially in patients with AIDS. A major advance in antiviral therapy has been the use of acyclovir to treat HSV infections, but acyclovir resistance is a problem of increasing clinical significance in immunocompromised patients (1). Clinical resistance implies that HSV can mutate to evade drug therapy yet retain pathogenicity. Because the sensitivity of HSV to acyclovir is due largely to the viral thymidine kinase (TK), which activates the drug (2), HSV tk mutations can confer acyclovir resistance (3). Indeed, there have been numerous cases ofpatients who suffered severe HSV disease despite acyclovir therapy and shed acyclovir-resistant viruses that were reported to express no detectable TK activity and/or full-length TK polypeptides (4-13).Although TK is not essential for viral replication in cell culture, it is important for viral pathogenesis in animal models. In particular, TK-mutants fail to reactivate from latent infections of mouse sensory ganglia (14-16), due to the tk mutation (16). Thus, the association of virus that appears TK-with severe HSV disease has been puzzling. One possible resolution of this paradox is that the clinical isolates expressed low levels of TK that were not detected. There are HSV mutants that are severely impaired for TK activity and thus acyclovir-resistant...

Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

“…Instead, it is driven by specific mRNA structure features such as rare codons [44][45][46][47][48] or mononucleotide repeat of 6 to 9 guanine nucleotides (termed G-string). 31,42,[49][50][51][52] The efficiency of incidental frameshift is usually low (i.e., <5%) although the efficiency of frame shifts at hungry codons can reach as high as 50%. 48 One of the most important examples of incidental frameshift is the frameshift on G-strings of 6 to 9 guanine nucleotides.…”

“…48 One of the most important examples of incidental frameshift is the frameshift on G-strings of 6 to 9 guanine nucleotides. 31,42,[49][50][51][52] Frameshifts on G-strings have been extensively studied owing to their importance in the drug resistance mechanism of ACV. 31,49-52 As mentioned earlier, HSV mutants evade the anti-viral activity of ACV primarily by inserting a G in the G7 string in their TK gene.…”

Ribosomal frameshifting on viral RNAs

“…However, in the bacteriophage examples studied, frameshifting is involved in the production of virion structural components. In certain yeast retrotransposons (see Pande et al, 1995 and references therein) and in a recently identified acyclovir-resistant isolate of herpes simplex virus type 1 (Hwang et al, 1994), + 1 ribosomal frameshifts have been documented. The available evidence suggests that + 1 frameshift sites differ both structurally and mechanistically from -1 frameshift sites and will not be discussed further in this review.…”

Human DNA tumor viruses generate alternative reading frame proteins through repeat sequence recoding

“…Both viral frameshifted proteins are detectable when heterologously expressed, and LANA1 ARF is present in naturally KSHV-infected PEL cell lines. The only previous examples of frameshift products reported in DNA viruses have resulted from DNA mutations in some herpesvirus simplex type 2 and EBV strain isolates in the thymidine kinase (TK) (29) and LF3 early genes (30), respectively.…”