Reactivation of thymidine kinase-defective herpes simplex virus is enhanced by nucleoside

Abstract

Herpes simplex virus (HSV) mutants defective for thymidine kinase expression (TK ؊ ) have been reported to establish latent infection of sensory ganglia of mice, in that HSV latency-associated transcript is expressed, but to be defective for reactivation. In the present study, the mechanism of defective reactivation by TK ؊ HSV was investigated. Latent infection established by each of three reactivation-defective HSV type 1 mutants was studied. Reactivation in explant culture was markedly enhanced by the addition of thymidine (dTdR) to the explant culture medium. Without added dTdR, reactivation occurred in 0 of 32 ganglia, while when dTdR (200 M) was present, reactivation occurred in 32 of 37 ganglia (86%). Reactivation was minimal or did not occur after treatment with other nucleosides; specificity for dTdR would suggest the importance of dTdR nucleotide levels rather than more general nucleotide pool imbalance. Enhanced reactivation by dTdR was dose dependent and was blocked by acyclovir. While some degree of inhibition of TK ؊ HSV by acyclovir may be expected, the complete block of dTdR-enhanced reactivation was unexpected. This result may suggest that HSV is particularly vulnerable during initial reactivation events. The mechanism of dTdR-enhanced reactivation of TK ؊ HSV was further evaluated during in vivo infection by TK ؊ HSV. For mice infected with TK ؊ HSV, virus was undetectable in ganglia 3 days later. However, for mice infected with TK ؊ HSV and treated with dTdR, virus was readily detected (2.8 ؋ 10 3 PFU per ganglion). This result suggested that in vivo treatment with dTdR enhanced replication of TK ؊ HSV in ganglion neurons. In turn, this suggests that in latently infected ganglia, dTdR-enhanced reactivation of TK ؊ HSV occurred as a result of viral replication in neurons following initial reactivation events.

Competition and complementation between thymidine kinase-negative and wild-type herpes simplex virus during co-infection of mouse trigeminal ganglia

“…Co-infection may prolong the replication of TK 2 mutant in the eye, perhaps by providing TK activity in trans, although this increase may not always be detectable. The concept of TK activity being provided in trans has been suggested previously (Coen et al, 1989b;Tenser et al, 1996), and our finding that ganglionic cells were infected dually with both TK 2 and TK + viruses addresses this possibility.…”

Interferons Regulate the Phenotype of Wild-type and Mutant Herpes Simplex Viruses In Vivo

“…How, then, is a tk mutant able to replicate in the nervous system of IFNR −/− mice as shown in this study? One clue comes from the observations that tk mutants can be made to reactivate by addition of thymidine to the culture medium of latently infected ganglia and that IFN-α can significantly reduce steady state levels of thymidine and thymidine metabolites in HSV-infected cells ( 58 , 59 ). Therefore, it is possible that the lack of IFN-α/β receptors in the knockout mice leads to higher intracellular levels of thymidine in the nervous system, thereby allowing a tk -deleted virus to replicate to detectable and significant levels.…”

Human Thymidine Kinase Can Functionally Replace Herpes Simplex Virus Type 1 Thymidine Kinase for Viral Replication in Mouse Sensory Ganglia and Reactivation from Latency upon Explant

“…However, the levels of these enzymes in neurons are not known. Tenser et al (54) previously observed that supraphysiologial concentrations of thymidine but not uridine, dU, or dC could overcome the reactivation defects of TK Ϫ viruses. Perhaps this led to synthesis of TMP from mitochondrial TK or residual cytosolic TK that could then be phosphorylated by cellular TMPK.…”