Evidence that the herpes simplex virus type 1 ICP0 protein does not initiate reactivation from latency in vivo.

The stress-induced host cell factors initiating the expression of the herpes simplex virus lytic cycle from the latent viral genome are not known. Previous studies have focused on the effect of specific viral proteins on reactivation, i.e., the production of detectable infectious virus. However, identification of the viral protein(s) through which host cell factors transduce entry into the lytic cycle and analysis of the promoter(s) of this (these) first protein(s) will provide clues to the identity of the stress-induced host cell factors important for reactivation. In this report, we present the first strategy developed for this type of analysis and use this strategy to test the established hypothesis that the herpes simplex virus ICP0 protein initiates reactivation from the latent state. To this end, ICP0 null and promoter mutants were analyzed for the abilities (i) to exit latency and produce lytic-phase viral proteins (initiate reactivation) and (ii) to produce infectious viral progeny (reactivate) in explant and in vivo. Infection conditions were manipulated so that approximately equal numbers of latent infections were established by the parental strains, the mutants, and their genomically restored counterparts, eliminating disparate latent pool sizes as a complicating factor. Following hyperthermic stress (HS), which induces reactivation in vivo, equivalent numbers of neurons exited latency (as evidenced by the expression of lytic-phase viral proteins) in ganglia latently infected with either the ICP0 null mutant dl1403 or the parental strain. In contrast, infectious virus was detected in the ganglia of mice latently infected with the parental strain but not with ICP0 null mutant dl1403 or FXE. These data demonstrate that the role of ICP0 in the process of reactivation is not as a component of the switch from latency to lytic-phase gene expression; rather, ICP0 is required after entry into the lytic cycle has occurred. Similar analyses were carried out with the DeltaTfi mutant, which contains a 350-bp deletion in the ICP0 promoter, and the genomically restored isolate, DeltaTfiR. The numbers of latently infected neurons exiting latency were not different for DeltaTfi and DeltaTfiR. However, DeltaTfi did not reactivate in vivo, whereas DeltaTfiR reactivated in approximately 38% of the mice. In addition, ICP0 was detected in DeltaTfiR-infected neurons exiting latency but was not detected in those neurons exiting latency infected with DeltaTfi. We conclude that while ICP0 is important and perhaps essential for infectious virus production during reactivation in vivo, this protein is not required and appears to play no major role in the initiation of reactivation in vivo.

Herpes simplex virus DNA synthesis is not a decisive regulatory event in the initiation of lytic viral protein expression in neurons in vivo during primary infection or reactivation from latency.

The herpes simplex virus genome can enter a repressed transcriptional state (latency) in sensory neurons of the host nervous system. Although reduced permissiveness of the neuronal environment is widely accepted as a causal factor, the molecular pathway(s) directing and maintaining the viral genome in the latent state remains undefined. Over the past decade, the field has been strongly influenced by the observations of Kosz-Vnenchak et al., which have been interpreted to indicate that, in sensory neurons in vivo, a critical level of viral DNA synthesis within the neuron is required for sufficient viral immediate-early (IE) and early (E) gene expression (M. Kosz-Vnenchak, J. Jacobson, D. M. Coen, and D. M. Knipe, J. Virol. 67:5383-5393, 1993). The levels of IE and E genes are, in turn, thought to regulate the decision to enter the lytic cycle or latency. We have reexamined this issue using new strategies for in situ detection and quantification of viral gene expression in whole tissues. Our results using thymidine kinase-null and rescued mutants as well as wild-type strains in conjunction with viral DNA synthesis blockers demonstrate that (i) despite inhibition of viral DNA replication, many neurons express lytic viral proteins, including IE proteins, during acute infection in the ganglion; (ii) at early times postinoculation, the number of neurons expressing viral proteins in the ganglion is not reduced by inhibition of viral DNA replication; and (iii) following a reactivation stimulus, the numbers of neurons and apparent levels of lytic viral proteins, including IE proteins, are not reduced by inhibition of viral DNA replication. We conclude that viral DNA replication in the neuron per se does not regulate IE gene expression or entry into the lytic cycle.

Comparison of herpes simplex virus reactivation in ganglia in vivo and in explants demonstrates quantitative and qualitative differences.

The in vivo ganglionic environment directs the latent herpes simplex virus transcriptional program. Since stress-driven perturbations in sensory neurons are thought to play a critical role in the transition from latency to reactivation, a primary concern in the selection of a valid model of the molecular interactions leading to reactivation is the faithful recapitulation of these environments. In this study reactivation of latently infected ganglia excised and cultured in vitro (explanted) is compared to reactivation occurring in latently infected ganglia in vivo following hyperthermic stress. Three notable points emerged. (i). Neurons in explanted ganglia exhibited marked morphological changes within 2 to 3 h postexplant. DNA fragmentation in neuronal nuclei was detected at 3 h, and atypical expression of cell cycle- and stress-regulated proteins such as geminin, cdk2, cdk4, and cytochrome c became apparent at 2 to 48 h. These changes were associated with axotomy and explant and not with the initiation or progression of reactivation and were not observed in ganglia following in vivo hyperthermic stress. (ii). Despite these differences, during the first 22 h primary reactivation events were restricted to a very small number of neurons in vivo and in explanted ganglia. This suggests that at any given time only a few latently infected neurons are competent to reactivate or that the probability of reactivation occurring in any particular neuron is very low. Importantly, the marked changes detected in explanted ganglia were not correlated with increased reactivation, demonstrating that these changes were not associated with the reactivation process per se. (iii). Secondary spread of virus was evident in explanted ganglia within 36 h, an event not observed in vivo. We conclude that explant reactivation may provide an ancillary system for selected studies of the early events in reactivation. However, clear signs of neuronal degeneration within 2 to 3 h postexplant indicate that these ganglia are undergoing major physiological changes not associated with the reactivation process. This ongoing neurodegeneration could alter even the early virus-host interactions in reactivation, and thus caution in the extrapolation of results obtained in explants to the in vivo interactions initiating reactivation is warranted.

Analysis of herpes simplex virus ICP0 promoter function in sensory neurons during acute infection, establishment of latency, and reactivation in vivo.

We have begun an analysis of the functional architecture of the ICP0 promoter in neurons in vivo with the ultimate goal of determining how this gene is regulated during reactivation in vivo. Promoter/reporter mutants in which the Escherichia coli beta-galactosidase (beta-Gal) gene was driven by various permutations of the ICP0 promoter were employed to permit the analysis of promoter function without the added complications that would arise due to inappropriate regulation of ICP0 protein levels. A whole-ganglion immunohistochemical staining procedure (N. M. Sawtell, J. Virol. 77:4127-4138, 2003) was used for direct comparisons of the expression of the promoter/reporter gene to expression of the native protein in the same cell. In this way, the expression of the putative wild-type promoter could be validated and results for mutant promoters could be compared to expression of the native gene. We found that a DNA fragment from bp -562 through the methionine start codon of the ICP0 gene contained all sequences required for properly regulated ICP0 expression in diverse cell types (including sensory neurons of the trigeminal ganglia [TG]) in vitro and in vivo, as indicated by colocalization of ICP0 and beta-Gal. Truncation of the ICP0 promoter to bp -145 or -129 resulted in the loss of immediate-early (alpha) kinetics. The truncated promoters expressed high levels of the reporter gene with leaky late (gamma1) kinetics in vitro and in some cell types in vivo. Unexpectedly, the truncated promoters did not express in TG neurons. Thus, TAATGARAT or other sequences upstream of bp -145 in the ICP0 promoter are required for basal expression of ICP0 in neurons but are not required for basal expression in other cells in vivo. There was a >95% concordance between reporter and native protein expression detected with the 562-bp promoter in neurons during the acute stage. However, this was not the case during reactivation from latency in vivo, as nearly twice as many neurons contained detectable beta-Gal as contained detectable ICP0. This same 562-bp promoter/reporter cassette, when placed in the context of a latency-associated transcript (LAT) null mutant, resulted in >95% concordance of expression of beta-Gal and ICP0 during reactivation in vivo. These last results strongly suggest that there is a posttranscriptional constraint on the expression of ICP0 protein during reactivation from latency and that this constraint is mediated by LAT.

Quantitative analysis of herpes simplex virus reactivation in vivo demonstrates that reactivation in the nervous system is not inhibited at early times postinoculation.

Recent studies utilizing an ex vivo mouse model of herpes simplex virus (HSV) reactivation have led to the hypothesis that, under physiologic conditions inducing viral reactivation, the immune cells within the infected ganglion block the viral replication cycle and maintain the viral genome in a latent state. One prediction from the ex vivo study is that reactivation in ganglia in vivo would be inhibited at early times postinoculation, when the numbers of inflammatory cells in the ganglia are greatest. To distinguish between an effect of the immune infiltrates on (i) infectious virus produced and/or recovered in the ganglion and (ii) the number of neurons undergoing lytic transcriptional activity (reactivating), an assay to quantify the number of neurons expressing lytic viral protein in ganglia in vivo was developed. Infectious virus and HSV protein-positive neurons were quantified from days 9 through 240 postinoculation in latently infected trigeminal ganglia before and at 22 h after hyperthermic-stress-induced reactivation. Significant increases in the amount of virus and the number of positive neurons were detected poststress in ganglia at all times examined. Unexpectedly, the greatest levels of reactivation occurred at the times examined most proximal to inoculation. Acyclovir was utilized to stop residual acute-phase virus production, and this treatment did not reduce the level of reactivation on day 14. Thus, the virus measured after induction was a product of reactivation. These data indicate that, in contrast to observations in the ex vivo model, immune cells in the ganglia during the resolution of acute infection do not inhibit reactivation of the virus in ganglia in vivo.

Preparation of single cells from solid tissues for analysis by PCR.

This unit details a protocol for the separation of solid tissues into single cell suspensions for subsequent analysis of nucleic acids and protein. Tissues are fixed in situ by perfusion, which terminates cell processes and thus changes that would accompany dissociating the living tissue. The procedure is particularly useful when the cell type of interest represents a minor population relative to other cells types in the tissue. Once separated, individual cells or groups of a particular cell type can then be analyzed using PCR strategies. The procedure can also be adapted to allow the quantification of the number of cells within a tissue containing specific nucleic acid sequences, for example, a particular viral DNA or RNA sequence.

Effect of transgenic overexpression of NR2B on NMDA receptor function and synaptic plasticity in visual cortex.

The NMDA receptor (NMDAR) is a heteromer comprised of NR1 and NR2 subunits. Mice that overexpress the NR2B subunit exhibit enhanced hippocampal LTP, prolonged NMDAR currents, and improved memory ( Tang et al., 1999). In the current study, we explored visual cortex plasticity and NMDAR function in NR2B overexpressing transgenic mice. Unlike the hippocampus, in vitro synaptic plasticity of the visual cortex was unaltered by NR2B overexpression. Consistent with the plasticity findings, NMDAR excitatory postsynaptic current (EPSC) durations from layer 2/3 pyramidal cells were similar in wild-type (wt) and transgenic (tg) mice. Furthermore, temporal summation of NMDAR EPSCs to 10, 20, and 40 Hz stimulation did not differ between cells from wt and tg mice. Finally, although in situ studies clearly demonstrate overexpression of NR2B mRNA in visual cortex, we failed to observe a significant elevation in the synaptic expression of NR2B protein. We conclude that the synaptic ratio of NR2B over NR2A in the NMDA receptor complex in the visual cortex is not significantly influenced by the transgene overexpression. These data suggest that mRNA availability is not a limiting factor for the synthesis of NR2B protein in the visual cortex, and support the hypothesis that levels of NR2A, rather than NR2B, normally determine the subunit composition of NMDARs in visual cortex.

Early intervention with high-dose acyclovir treatment during primary herpes simplex virus infection reduces latency and subsequent reactivation in the nervous system in vivo.

There remains a lack of agreement on the effect of antiviral therapy on herpes simplex virus (HSV) latency and subsequent reactivation. To gain insight into this important issue, a single-cell polymerase chain reaction assay was used to quantify the effects of high-dose acyclovir on latent infection in a mouse model. Treatment with 50 mg/kg of acyclovir every 8 h reduced the number of latently infected neurons by >90% when treatment was begun before 24 h after infection and by 80% and 70% when begun at 48 or 72 h after infection, respectively. The biologic significance of these reductions was evaluated by using a well-established in vivo reactivation model. The number of animals in which virus reactivated was reduced significantly, even when acyclovir therapy was delayed until 72 h after infection, a time when animals had developed lesions. These findings indicate that potent antiviral therapy during early primary HSV infection can reduce the magnitude of the latent infection, such that a significant decrease in reactivation is observed.

Herpes simplex virus type 1 latency-associated transcript gene promotes neuronal survival.

A complex interaction has evolved between the host's peripheral nervous system (PNS) and herpes simplex virus type 1 (HSV-1). Sensory neurons are permissive for viral replication, yet the virus can also enter a latent state in these cells. The interplay of viral and neuronal signals that regulate the switch between the viral lytic and latent states is not understood. The latency-associated transcript (LAT) regulates the establishment of the latent state and is required for >65% of the latent infections established by HSV-1 (R. L. Thompson and N. M. Sawtell, J. Virol. 71:5432-5440, 1997). To further investigate how LAT functions, a 1.9-kb deletion that includes the entire LAT promoter and 827 bp of the 5' end of the primary LAT mRNA was introduced into strain 17syn+. The wild-type parent, three independently derived deletion mutants, and two independently derived genomically rescued variants of the mutants were analyzed in a mouse ocular model. The number of latent sites established in trigeminal ganglion (TG) neurons was determined using a single-cell quantitative PCR assay for the viral genome on purified TG neurons. It was found that the LAT null mutants established ~75% fewer latent infections than the number established by the parental strain or rescued variant. The reduced establishment phenotype of LAT null mutants was due at least in part to a dramatic increase in the loss of TG neurons in animals infected with the LAT mutants. Over half of the neurons in the TG were destroyed following infection with the LAT mutants, and this was significantly more than were lost following infection with wild type. This is the first demonstration that the HSV LAT locus prevents the destruction of sensory neurons. The death of these neurons did not appear to be the result of increased apoptosis as measured by a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay. Animals latently infected with the LAT null mutants reactivated less frequently in vivo and this was consistent with the reduction in the number of neurons in which latency was established. Thus, one function of the LAT gene is to protect sensory neurons and enhance the establishment of latency in the PNS.

Replication of herpes simplex virus type 1 within trigeminal ganglia is required for high frequency but not high viral genome copy number latency.

The replication properties of a thymidine kinase-negative (TK(-)) mutant of herpes simplex virus type 1 (HSV-1) were exploited to examine the relative contributions of replication at the body surface and within trigeminal ganglia (TG) on the establishment of latent infections. The replication of a TK(-) mutant, 17/tBTK(-), was reduced by approximately 12-fold on the mouse cornea compared to the rescued isolate 17/tBRTK(+), and no replication of 17/tBTK(-) in the TG of these mice was detected. About 1.8% of the TG neurons of mice infected with 17/tBTK(-) harbored the latent viral genome compared to 23% of those infected with 17/tBRTK(+). In addition, the latent sites established by the TK(-) mutant contained fewer copies of the HSV-1 genome (average, 2.3/neuron versus 28/neuron). On the snout, sustained robust replication of 17tBTK(-) in the absence of significant replication within the TG resulted in a modest increase in the number of latent sites. Importantly, these latently infected neurons displayed a wild-type latent-genome copy number profile, with some neurons containing hundreds of copies of the TK(-) mutant genome. As expected, the replication of the TK(-) mutant appeared to be blocked prior to DNA replication in most ganglionic neurons in that (i) virus replication was severely restricted in ganglia, (ii) the number of neurons expressing HSV proteins was reduced 30-fold compared to the rescued isolate, (iii) cell-to-cell spread of virus was not detected within ganglia, and (iv) the proportion of infected neurons expressing late proteins was reduced by 89% compared to the rescued strain. These results demonstrate that the viral TK gene is required for the efficient establishment of latency. This requirement appears to be primarily for efficient replication within the ganglion, which leads to a sixfold increase in the number of latent sites established. Further, latent sites with high genome copy number can be established in the absence of significant virus genome replication in neurons. This suggests that neurons can be infected by many HSV virions and still enter the latent state.

Induction of NMDA receptor-dependent long-term depression in visual cortex does not require metabotropic glutamate receptors.

We tested the role of group I mGluRs in the induction of long-term depression (LTD) in the visual cortex, using the novel mGluR antagonist LY341495 and mice lacking mGluR5, the predominant phosphoinositide (PI)-linked mGluR in the visual cortex. We find that LY341495 is a potent blocker of glutamate-stimulated PI hydrolysis in visual cortical synaptoneurosomes, and that it effectively antagonizes the actions of the mGluR agonist 1S, 3R-aminocyclopentane-1,3-dicarboxylic acid (ACPD) on synaptic transmission in visual cortical slices. However, LY341495 has no effect on the induction of LTD by low-frequency stimulation. Furthermore, mice lacking mGluR5 show normal NMDA receptor-dependent LTD. These results indicate that group I mGluR activation is not required for the induction of NMDA receptor-dependent LTD in the visual cortex.

A temporal analysis of acyclovir inhibition of induced herpes simplex virus type 1 In vivo reactivation in the mouse trigeminal ganglia.

It is generally assumed that reactivation of latent herpes simplex virus occurs through initiation of lytic viral gene transcription from the latent viral genome. Thus, antiviral compounds such as acyclovir, whose activation is dependent upon viral thymidine kinase, should be effective in preventing the initial production of infectious virus associated with reactivation. To test this concept, the ability of acyclovir to prevent the production of infectious virus was determined in the murine hyperthermic stress (HS) model of in vivo reactivation. Acyclovir treatment after HS blocked the production of infectious virus within the ganglia. Efficacy was dependent upon the timing of the first post-HS dose and the length of exposure to acyclovir. A single dose administered 6-9 h after HS resulted in >90% reduction in reactivation. Acyclovir administered 12 h after HS resulted in 75% reduction, but there was no effect if treatment was delayed for 18 h after HS.

Brain-derived neurotrophic factor alters the synaptic modification threshold in visual cortex.

The effects of brain-derived neurotrophic factor (BDNF) were investigated on synaptic transmission and two forms of activity-dependent synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD), in visual cortex slices prepared from young (P21 -28) rats. The slices treated for 2-5 h in BDNF showed no difference from control slices when a 'strong' tetanus was used (theta-burst stimulation) to elicit a maximal level of LTP but displayed significantly greater synaptic potentiation in response to a 'weak' (20 Hz) tetanus. The BDNF-treated slices also showed significantly less LTD in response to a 1 Hz tetanus. Thus, BDNF treatment alters the relationship between stimulation frequency and synaptic plasticity in the visual cortex, shifting the modification threshold to the left. The effects of BDNF on LTP and LTD induction may be attributed to the significant enhancement of synaptic responses that was observed during conditioning stimulation. These data suggest that one role of BDNF during development of the visual cortex may be to modulate the properties of synaptic plasticity, enhancing synaptic strengthening and reducing synaptic weakening processes which contribute to the formation of specific synaptic connections.

The probability of in vivo reactivation of herpes simplex virus type 1 increases with the number of latently infected neurons in the ganglia.

The purpose of this study was to define the relationship between herpes simplex virus (HSV) latency and in vivo ganglionic reactivation. Groups of mice with numbers of latently infected neurons ranging from 1.9 to 24% were generated by varying the input titer of wild-type HSV type 1 strain 17syn+. Reactivation of the virus in mice from each group was induced by hyperthermic stress. The number of animals that exhibited virus reactivation was positively correlated with the number of latently infected neurons in the ganglia over the entire range examined (r = 0.9852, P < 0. 0001 [Pearson correlation]).

The latent herpes simplex virus type 1 genome copy number in individual neurons is virus strain specific and correlates with reactivation.

The viral genetic elements that determine the in vivo reactivation efficiencies of fully replication competent wild-type herpes simplex virus (HSV) strains have not been identified. Among the common laboratory strains, KOS reactivates in vivo at a lower efficiency than either strain 17syn+ or strain McKrae. An important first step in understanding the molecular basis for this observation is to distinguish between viral genetic factors that regulate the establishment of latency from those that directly regulate reactivation. Reported here are experiments performed to determine whether the reduced reactivation of KOS was associated with a reduced ability to establish or maintain latent infections. For comparative purposes, latent infections were quantified by (i) quantitative PCR on DNA extracted from whole ganglia, (ii) the number of latency-associated transcript (LAT) promoter-positive neurons, using KOS and 17syn+ LAT promoter-beta-galactosidase reporter mutants, and (iii) contextual analysis of DNA. Mice latently infected with 17syn+-based strains contained more HSV type 1 (HSV-1) DNA in their ganglia than those infected with KOS strains, but this difference was not statistically significant. The number of latently infected neurons also did not differ significantly between ganglia latently infected with either the low- or high-reactivator strains. In addition to the number of latent sites, the number of viral genome copies within the individual latently infected neurons has recently been demonstrated to be variable. Interestingly, neurons latently infected with KOS contained significantly fewer viral genome copies than those infected with either 17syn+ or McKrae. Thus, the HSV-1 genome copy number profile is viral strain specific and positively correlates with the ability to reactivate in vivo. This is the first demonstration that the number of HSV genome copies within individual latently infected neurons is regulated by viral genetic factors. These findings suggest that the latent genome copy number may be an important parameter for subsequent induced reactivation in vivo.

Effects of the metabotropic glutamate receptor antagonist MCPG on phosphoinositide turnover and synaptic plasticity in visual cortex.

The neurotransmitter glutamate, in addition to activating ligand-gated ion channels, also stimulates phosphoinositide (PI) hydrolysis in neurons by activating a group of G-protein-coupled metabotropic glutamate receptors (mGluRs). A role for mGluRs in synaptic plasticity originally was hypothesized based on the observation that the developmental decline in glutamate-stimulated PI turnover is well correlated with the decline in experience-dependent synaptic plasticity in visual cortex. Over the past few years, the compound alpha-methyl-4-carboxyphenylglycine (MCPG) has been widely used to test the role of PI-coupled mGluRs in a number of types of synaptic plasticity, including long-term potentiation (LTP), long-term depression (LTD), ocular dominance plasticity in visual cortex, and the neural plasticity underlying learning and memory. The conclusions of most of these studies were based on the assumption that MCPG blocks the actions of glutamate at PI-coupled mGluRs in the cerebral cortex. Here we show that this assumption is not valid in visual cortex. Although MCPG does antagonize the actions of the synthetic mGluR agonist 1S, 3R-aminocyclopentane-1,3-dicarboxylic acid, it fails to block PI turnover and changes in spike adaptation stimulated by glutamate, the endogenous mGluR ligand. In addition, we find that MCPG fails to block the NMDA receptor-dependent forms of LTP, LTD, and depotentiation in visual cortex.

Comprehensive quantification of herpes simplex virus latency at the single-cell level.

To date, characterization of latently infected tissue with respect to the number of cells in the tissue harboring the viral genome and the number of viral genomes contained within individual latently infected cells has not been possible. This level of cellular quantification is a critical step in determining (i) viral or host cell factors which function in the establishment and maintenance of latency, (ii) the relationship between latency burden and reactivation, and (iii) the effectiveness of vaccines or antivirals in reducing or preventing the establishment of latent infections. Presented here is a novel approach for the quantitative analysis of nucleic acids within the individual cells comprising complex solid tissues. One unique feature is that the analysis reflects the nucleic acids within the individual cells as they were in the context of the intact tissue-hence the name CXA, for contextual analysis. Trigeminal ganglia latently infected with herpes simplex virus (HSV) were analyzed by CXA of viral DNA. Both the type and the number of cells harboring the viral genome as well as the number of viral genomes within the individual latently infected cells were determined. Here it is demonstrated that (i) the long-term repository of HSV-1 DNA in the ganglion is the neuron, (ii) the viral-genome copy number within individual latently infected neurons is variable, ranging over 3 orders of magnitude from <10 to >1,000, (iii) there is a direct correlation between increasing viral input titer and the number of neurons in which latency is established in the ganglion, (iv) increasing viral input titer results in more neurons with greater numbers of viral-genome copies, (v) treatment with acyclovir (ACV) during acute infection reduces the number of latently infected ganglionic neurons 20-fold, and (vi) ACV treatment results in uniformly low (<10)-copy-number latency. This report represents the first comprehensive quantification of HSV latency at the level of single cells. Beyond viral latency, CXA has the potential to advance many studies in which rare cellular events occur in the background of a complex solid tissue mass, including microbial pathogenesis, tumorigenesis, and analysis of gene transfer.

The herpes simplex virus type 1 latency-associated transcript gene regulates the establishment of latency.

Herpes simplex virus type 1 establishes latent infections in sensory neurons. During latency only one locus, the latency-associated transcript (LAT), is abundantly transcribed. Several lines of evidence suggest that this locus is required for the efficient reactivation from latency in experimental models. However, it is not yet clear whether this is a direct effect on the reactivation process per se or, as we have suggested, an indirect effect resulting from a decreased efficiency of establishment of latent infections. In this report wild-type and genetically engineered viral mutants were analyzed in a mouse model using a recently developed approach to precisely quantify latently infected neurons. It was found that strain KOS/M established latent infections, as defined by the presence of the viral genome, in about 30% of the neurons. Thirty-three percent of the mice with this latent viral burden reactivated in vivo following hyperthermic stress. In contrast, mutants in which either the basal LAT promoter or the 5' end of the LAT gene was deleted established latency in only 10% of trigeminal neurons (P < 0.00001), and these mice were impaired for reactivation. Repair of the locus resulted in wild-type levels of establishment and reactivation, mapping this function to the LAT region. Finer mapping demonstrated that a 2.3-kb fragment that contains the major LAT transcripts was sufficient to promote efficient establishment and subsequent reactivation when expressed in the context of a foreign gene. Hyperthermic stress applied during the first 3 days postinfection resulted in greatly increased numbers of neurons harboring the latent viral genome. This approach was found to increase the level of establishment of LAT-null mutants to that normally achieved by wild-type KOS/M. These establishment-repaired mice reactivated with wild-type efficiency. Thus, the LAT gene serves to increase the number of neurons in which latency is established, and no direct role for the LAT locus in reactivation could be demonstrated.