Herpes simplex virus (HSV) reactivation from latency was investigated. Reactivation of thymidine kinasenegative HSV, which is defective for reactivation, was greatly enhanced by thymidine (TdR). The reactivationenhancing effect of TdR was blocked by dipyridamole (DPM), a known nucleoside transport inhibitor. DPM also inhibited wild-type HSV reactivation, suggesting potential antiviral use.In experimental models of herpes simplex virus (HSV) infection, expression of viral thymidine kinase (TK) has been shown to be important for viral latency. This was first suggested in studies of mice infected with TK-negative HSV. It was shown that in cases of acute infection, HSV replicated well in ocular tissues but not in trigeminal ganglia (TG) and that HSV reactivated poorly in ganglia during latency. Subsequently it was shown that establishment of latency was intact, i.e., latency-associated transcript (LAT) was readily detected in ganglion neurons, but reactivation was impaired (4, 5, 16). The defect of reactivation was explored in studies which showed that TKnegative HSV could in fact readily reactivate in ganglia if explant medium was supplemented with thymidine (TdR) (17). The present study extended this observation in three ways. First, it was shown that if a nucleoside transport inhibitor is added along with supplemental TdR, the effect of TdR on enhancing TK-negative HSV reactivation is blocked. This suggests the specific roles of TdR and phosphorylation by TK in the reactivation process. Second, it is shown that the nucleoside transport inhibitor also blocks wild-type HSV reactivation from latency. Lastly, supplemental TdR decreased the dipyridamole (DPM) block of wild-type HSV reactivation.Latent infection of TG and lumbar dorsal root ganglia (DRG) was established in randomly bred CD-1 mice (Charles River Laboratories, Wilmington, Mass.) by standard methods. In brief, mice were anesthetized (methoxyflurane), and corneal inoculation (5 l) or footpad inoculation (25 l) was performed (17). Inoculation was performed with either TK-positive wild-type HSV type 1 (HSV-1; strain KOS, 5 ϫ 10 8 PFU/ ml) or with mutant TK-negative HSV-1 (dlsactk, 4 ϫ 10 8 PFU/ml). The titers of the viruses were determined on Vero cells using standard methods. The KOS virus had been used previously (16,17). It readily established latency (i.e., LAT expression) and reactivated from latency in explants with a frequency of 90 to 100%. The dlsactk mutant strain was kindly provided by D. Coen (Harvard Medical School, Boston, Mass.). It was shown to express less than 1% of parental TK activity (4, 7). LAT expression during latency in mice inoculated with dlsactk was similar to that in mice with the TKpositive KOS strain, but reactivation from latency occurred at a frequency of 0 to 10% (4,7,17).After 28 to 30 days, mice were anesthetized (methoxyflurane) and exsanguinated by cardiac puncture. HSV inoculation of mice, as well as housing and eventual sacrifice, were done in accordance with institutional and federal guidelines. TG and DRG (from lumb...

The presence of herpes simplex virus (HSV) latency-associated transcript (LAT) was investigated in sensory ganglion neurons of mice after inoculation with thymidine kinase (TK) mutants of HSV. Ganglion serial sections were examined in order to quantitate numbers of LAT-positive neurons. After inoculation with TK-positive HSV, virus was isolated during latency from explants of most ganglia, and LAT was detected by in situ hybridization in 96% of ganglia. After inoculation with HSV TK mutants, virus was isolated from 0% of ganglia, but LAT was detected in 95 to 100% of ganglia. After inoculation of TK mutants of HSV, therefore, although latent infection as indicated by the isolation of virus from ganglion explants was not detected, the presence of LAT was common. These results suggest that the lack of reactivatable virus after inoculation of HSV TK mutants may be related to a role for HSV TK expression in the reactivation process.

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