Translational Compensation of a Frameshift Mutation Affecting Herpes Simplex Virus Thymidine Kinase Is Sufficient To Permit Reactivation from Latency

Abstract

Herpes simplex virus thymidine kinase is important for reactivation of virus from its latent state and is a target for the antiviral drug acyclovir. Most acyclovir-resistant isolates have mutations in the thymidine kinase gene; however, how these mutations confer clinically relevant resistance is unclear. Reactivation from explanted mouse ganglia was previously observed with a patient-derived drug-resistant isolate carrying a single guanine insertion within a run of guanines in the thymidine kinase gene. Despite this mutation, low levels of active enzyme were synthesized following an unusual ribosomal frameshift. Here we report that a virus, generated from a pretherapy isolate from the same patient, engineered to lack thymidine kinase activity, was competent for reactivation. This suggested that the clinical isolate contains alleles of other genes that permit reactivation in the absence of thymidine kinase. Therefore, to establish whether thymidine kinase synthesized via a ribosomal frameshift was sufficient for reactivation under conditions where reactivation requires this enzyme, we introduced the mutation into the well-characterized strain KOS. This mutant virus reactivated from latency, albeit less efficiently than KOS. Plaque autoradiography revealed three phenotypes of reactivating viruses: uniformly low thymidine kinase activity, mixed high and low activity, and uniformly high activity. We generated a recombinant thymidine kinase-null virus from a reactivating virus expressing uniformly low activity. This virus did not reactivate, confirming that mutations in other genes that would influence reactivation had not arisen. Therefore, in strains that require thymidine kinase for reactivation from latency, low levels of enzyme synthesized via a ribosomal frameshift can suffice.

Quantification and Analysis of Thymidine Kinase Expression from Acyclovir-Resistant G-String Insertion and Deletion Mutants in Herpes Simplex Virus-Infected Cells

“…Nevertheless, there is considerable evidence that much of the TK activity of G8 mutants is a result of ribosomal frameshifting (12,16,20). Plaque autoradiography provides no evidence for reversion of G8 mutants in cell culture (14,17). Full-length TK expression has been observed in G8 mutant-infected cells and has been recapitulated in an in vitro rabbit reticulocyte lysate translation system, where it was shown to be due to translational rather than other mechanisms (20).…”

“…Plaque autoradiography was performed by modifying a previously described protocol (14). The modifications were that the dishes were seeded with 4 ϫ 10 5 143B cells 24 h prior to addition of an inoculum of 200 PFU of virus/dish and that radiolabeling was carried out for 20 h in the presence of 5 Ci/ml of [ 3 H]thymidine.…”

“…However, G8 and G9 mutants are not truly TK negative (reviewed in reference 13). Both G8 and G9 exhibit low but higher-than-background TK activities, as seen by plaque autoradiography (14,16,17), which can suffice to permit some reactivation of the viruses from latent infection in mice, a marker of pathogenicity (14). In contrast, isogenic truly TK-negative viruses fail to reactivate from latency (4,7,10,29,30).…”

“…Reversion can also account for some of the ability of G8 mutants to reactivate from latency (14,25,26). Nevertheless, there is considerable evidence that much of the TK activity of G8 mutants is a result of ribosomal frameshifting (12,16,20).…”

Alternative Translational Products and Cryptic T Cell Epitopes: Expecting the Unexpected

“…In fact, the site of insertion was a guanine-rich sequence in the mRNA suitable as a frameshifting signal, resulting in realignment of the reading frame (50) and production of normal TK protein at 1-2% of wild-type translation (49). These levels were sufficient for HSV reactivation from latency but not high enough for virus susceptibility to acyclovir treatment (51,52). Taking advantage of this system, a model conventional influenza nucleoprotein epitope in tandem with the OVA SIINFEKL peptide was engineered downstream of the TK element (48).…”