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...

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.

Sensory ganglia latently infected with herpes simplex virus (HSV) were transplanted beneath the renal capsule of syngeneic recipients, and the latent infection remaining was investigated. HSV latency-associated transcript (LAT) expression and reactivation of HSV after explant of transplanted dorsal root ganglia were monitored as markers of latency. Two to four weeks after transplantation, both indicated evidence of HSV latency in transplants. At those times, infectious virus was not detected in direct ganglion homogenates. In addition, viral antigen and infected cell polypeptide 4 RNA were not detected. Taken together, the results suggested that HSV latent infection rather than persistent infection was present in transplants. From these results, two explanations seemed possible: latency was maintained in transplanted neurons, or alternatively, latency developed after transplantation, in neurons not previously latently infected. The latter was considered putative secondary latency and was investigated in three ways. First, evidence of reactivation which might serve as a source for secondary latency was evaluated. Reactivation of HSV in transplants was evident from HSV antigen expression (52% of transplants) and the presence of cell-free virus (38% of transplants) 3 to 5 days after transplantation. Second, putative secondary latency was investigated in recipients immunized with HSV prior to receiving latently infected ganglia. Reactivation was not detected 3 to 5 days after transplantation in immunized recipients, and LAT expression was rare in these recipients after 3 to 4 weeks. Lastly, the possibility of secondary latency was investigated by comparing results obtained with standard HSV and with reactivation-defective thymidine kinase-negative (TK-) HSV. Defective reactivation of TK- HSV was demonstrated by immunohistochemistry and by the inability to isolate infectious virus. Donor dorsal root ganglia latently infected with TK+ HSV showed many LAT-positive neurons 2 or more weeks after transplantation (average, 26 per transplant). However, LAT expression was undetectable or minimal > 2 weeks after transplantation in donor ganglia latently infected with TK- HSV (average, 0.2 per transplant). Impaired reactivation of TK- HSV-infected donor ganglia after transplantation, therefore, was correlated with subsequent limited LAT expression. From these results, the occurrence of secondary latency was concluded for ganglia latently infected with TK+ HSV and transplanted beneath the kidney capsule. In vivo reactivation in this transplant model may provide a more useful means to investigate HSV reactivation than in usual in vitro explant models and may complement other in vivo reactivation models. The occurrence of secondary latency was unique. The inhibition of secondary latency by the immune system may provide an avenue to evaluate immunological control of HSV latency.