Primary ganglionic infection with HSV-1 is characterized by two very different patterns of viral gene expression. In some neurons, there is abundant expression of lytic cycle genes, resulting in the production of infectious virus, whereas in other neurons, a latent infection is established (14
). This pattern is not random; primary sensory neurons identified by MAb KH10 are very permissive for productive infection with HSV-1, whereas neurons identified by MAb A5 serve as the principal reservoir of latent infection (34
). In the current study, we demonstrate that HSV-2 follows a very different pattern of ganglionic infection, with KH10-positive neurons serving as the principal reservoir of latent infection in both the TG and lumbar-sacral DRG. These data suggest that HSV-1 and HSV-2 take advantage of two different biological niches within the peripheral nervous system: niches that go beyond the simple boundaries of “above versus below” the waist.
It is almost certain that the different lactoseries glycoconjugates recognized by MAbs A5 and KH10 (4
) have little to do with the differences in permissiveness of the neurons recognized by these markers. It is more likely that the expression of these markers simply correlates with differences in cellular protein expression that enable different neuronal populations to differentially support or suppress the expression of key viral genes, thus leading to different outcomes of infection. As suggested by the immunostaining patterns in Table , A5- and KH10-positive neurons largely represent two different classes of intensively studied nociceptors, small-diameter BSL-IB4
-positive and BSL-IB4
-negative ganglionic neurons; neurons with differential responsiveness to nerve growth factor and glial cell line-derived neurotrophic factor (20
). It is therefore tempting to speculate that neurotrophic factor-responsive signaling pathways may play a role in regulating the establishment and maintenance of HSV latent infection. These considerations highlight the importance of studying the interaction of HSV with different sensory neuronal subtypes, and not just ganglia as whole, in understanding viral latency and reactivation.
The ability of acyclovir to reduce the tropism of HSV-1 latent infection for A5-positive neurons implies that the preferential establishment of latency in these cells is dependent upon viral replication. One possibility is that type-specific sequences enhance viral spread to A5-positive neurons during establishment of latency and that acyclovir inhibits this spread. Alternatively, by inhibiting viral DNA replication, acyclovir might tip the course of infection in replication-permissive neurons toward latency, thus reducing the relative latent viral load in A5-positive neurons.
The HSV LAT is the only available reliable marker of latently infected neurons. While neurons may contain viral DNA as detected by PCR-based techniques, it is unclear whether viral DNA detected in this manner represents reactivation-competent latent virus or simply fragments of the viral genome. While it is not known whether viral reactivation is restricted to LAT-producing neurons, animal data showing the altered recurrence phenotypes of viruses with impaired capacity to produce LAT supports the conclusion that LAT production is a biologically relevant marker of viral latency (10
The different distributions of latent HSV-1 and HSV-2 in murine sensory ganglia cannot simply be a consequence of differential LAT accumulation in A5-positive and KH10-positive neurons. First, we found no significant difference in HSV-1 or HSV-2 LAT signal intensity among A5- and KH10-positive neurons. Second, studies of HSV-1 LAT transgene expression in murine trigeminal ganglia revealed no evidence of differential LAT accumulation in A5- and KH10-positive neurons. Third, the treatment of mice with acyclovir, a drug that inhibits productive viral infection but should not affect the accumulation of LAT, shifted the distribution of LAT-positive, latently infected neurons closer to the distribution of A5- and KH10-positive neurons in uninfected ganglia. Fourth, we previously demonstrated that the ratio of LAT-expressing A5-positive and KH10-positive neurons was independent of the sensitivity of the assay used to measure LAT (34
), an observation that would not be expected if there were different levels of LAT expression in these neuronal populations. And fifth, following ocular inoculation with a TK deletion virus where latency is the only possible outcome of infection, the distribution of viral latency in A5-positive and KH10-positive neurons, as assayed by LAT expression, closely matches the relative distribution of these neurons in the TG; a result that demonstrates similar accumulation of LAT in A5- and KH10-positive neurons if the virus has equal opportunity to establish latency in these two neuronal populations (Y. Imai et al., personal communication).
In the course of these studies, we found that a chimeric HSV-2 mutant that expresses the HSV-1 LAT (HSV-2 333/LAT1) establishes latency primarily in A5-positive neurons, similar to HSV-1. This indicates that the difference in the latency phenotype of the two viruses can be mapped to a small portion of the long repeat region of the viral genome, the same region that codes for the stable LAT intron and which conveys the phenotype of virus type-associated, site-specific recurrence (35
). This viral function might be associated with any or all of the substituted sequences, including the LAT promoter, the primary 5′ end sequences or the sequences coding for the LAT intron. It might also be associated with sequences coding in an antisense direction relative to LAT, such as those recently described for AL (24
) or with the overlapping 3′ end of the ICP0 coding region, although the close homology of the HSV-1 and HSV-2 ICP0 3′ amino acid sequences makes this unlikely.
Our findings are particularly interesting in light of our previous report of KOS 62, an HSV-1 LAT deletion virus that expresses the lacZ gene under control of the HSV-1 LAT promoter. Although containing a 1.6-kb LAT region deletion, this virus preferentially established latency (as assayed by β-galactosidase expression) in A5 neurons, similar to that with the wild-type virus. Together with the current findings, this suggests that critical sequences for neuron subtype-specific establishment of latency may reside in regions not shared by the 1.6-kb deletion in KOS 62 and the 2.8-kb substitution in HSV-2 333/LAT1. Thus, sequences in the HSV-1 LAT promoter, the LAT 5′ exon, or in the 3′ half of the LAT intron seem likely to be important for this phenotype. It also is possible that these findings are influenced by other differences between the HSV-1 backbone of KOS 62 and the HSV-2 backbone of HSV-2 333/LAT1.
Implication of the LAT region of the viral genome in neuronal type-specific establishment of latency is intriguing since it appears to play an important role in both the establishment (25
) and reactivation of latent infection (13
), and more recent studies suggest that LAT influences neuronal survival, possibly via an effect on apoptosis (1
). None of the leading hypotheses of LAT function serve to explain the type-specific role of LAT sequences either in viral reactivation phenotype or in establishment of latency. Thus, further investigation into the mechanism of LAT action is clearly warranted. Our findings are consistent with a potential role for LAT sequences in selectively promoting latent infection in specific types of sensory neurons via an as-yet-undefined mechanism.
The ability of LAT region sequences to direct latent infection to specific sensory neuronal subtypes and the corresponding differences in the abilities of each virus to recur from different anatomical locations (16
) imply that HSV derives an advantage in recurrence frequency from establishing latency in different neurons in each anatomical location, A5-positive neurons in trigeminal ganglia and KH10-positive neurons in lumbar-sacral ganglia. The increased prevalence of KH10-positive neurons in lumbar-sacral dorsal root ganglia suggests one mechanism by which this phenotype could influence type-specific recurrence patterns.