Previously in our laboratory, a chimeric HSV-2 expressing the LAT from HSV-1 failed to reactivate efficiently in the guinea pig genital model of HSV infection (38
). One goal of the present study was to determine whether the LAT promoter or the LAT sequence provides the essential elements for type-specific reactivation of HSV-2. The HSV LAT region substituted in our previous chimera was divided into the promoter region (from NotI to PvuI sites) and the sequence region (from PvuI to XhoI sites), and additional chimeric HSV-2 viruses were constructed. HSV2-LAT-P1 is HSV-2 that expresses the native HSV-2 LAT sequence under the control of the HSV-1 LAT promoter. HSV2-LAT-S1 is HSV-2 that expresses HSV-1 LAT sequences, including the 5′ exon and most of the LAT intron, under the control of the native HSV-2 LAT promoter. HSV2-LAT-P1 reactivated efficiently in the guinea pig genital model of infection, although the acute infection was attenuated in both lesion severity and urinary tract dysfunction. The chimeric HSV2-LAT-S1 virus and its rescuant produced acute infections with lesion severities similar to those for wild-type HSV-2 as well as for wild-type HSV-1. However, HSV2-LAT-S1 reactivated inefficiently in the guinea pig genital model of infection, with a reactivation frequency similar to that of wild-type HSV-1, and a wild-type recurrence phenotype was restored with its rescuant, HSV2-LAT-S1-R. Thus, the LAT sequence including the 5′ exon and intron, rather than the LAT promoter, provides the essential elements for efficient genital reactivation of HSV-2. This correlates well with studies on HSV-1, which imply that the region between the promoter and the LAT intron contains the reactivation-critical region (3
) and enhancer activities (2
) for HSV-1. Since this region of HSV-1 has known enhancer activity, we hypothesize that enhancer activity within this region in both HSV-1 and HSV-2 may play a role in determining site-specific recurrence phenotypes.
Infections with the HSV2-LAT-S1 chimera were characterized by a dramatic increase in virulence and mortality relative to those with wild-type HSV-2, the rescuant HSV2-LAT-S1-R, HSV2-LAT-P1, and wild-type HSV-1. During acute disease, a greater percentage of animals infected with HSV2-LAT-S1 than with HSV-2 developed urinary tract dysfunction, suggestive of more pronounced autonomic nervous system involvement. During latency, HSV-1 and HSV2-LAT-S1 did not reactivate efficiently. During its rare recurrences, HSV-1 produced a single vesicular lesion lasting 1 to 2 days, similar in character to recurrences produced by HSV-2, HSV2-LAT-P1, and HSV2-LAT-S1-R. However, in guinea pigs infected with the HSV2-LAT-S1 chimera, recurrent disease often progressed from a single lesion to multiple lesions coinciding with neurological symptoms, followed by rapid deterioration and death. The most likely explanation for this progression of symptoms is that the HSV2-LAT-S1 chimera was able to spread more efficiently through the nervous system via a change in neurotropism, mediated by its HSV-1 LAT sequences. In a previous report on HSV-1 LAT region deletion mutants, virulence either increased or decreased depending on the size of the deletion and the species infected (31
), which suggests that the LAT region has multiple functional elements and that specific neuronal factors may interact with LAT sequences in HSV-1 to regulate viral replication. Fatal recurrences were not observed with the previously tested HSV-2 333/LAT1 chimeric virus, which contained both the HSV-1 LAT promoter and sequence, implying that an interaction of the HSV-2 LAT promoter and HSV-1 LAT sequence may have permitted unusual spread of the HSV2-LAT-S1 virus, contributing to its increased virulence. Alternatively, a cis
-acting regulatory element located at the mutation junction site may have been disrupted, permitting unusual replicative characteristics. Although the LAT promoters are fairly well conserved, there is little discernible similarity between the HSV-1 and HSV-2 LAT regions downstream of the promoter. This region in both HSV-1 and HSV-2 is densely populated by putative transcription factor binding sites and enhancer elements, which likely function cooperatively to modulate the activity of the LAT region. LAT sequences could play a role in down-regulation of viral replication, which may fail when non-type-specific sequences are introduced or when specific sequences are deleted, or these LAT sequences could facilitate growth in different types of neurons, which could also give rise to an unusual neurotropic phenotype.
The acute infection caused by HSV2-LAT-P1 was attenuated in both lesion severity and urinary tract dysfunction. However, since HSV2-LAT-P1 demonstrated a wild-type reactivation phenotype, a rescuant of this chimeric virus was not constructed. Any secondary mutation that may have occurred during the construction of the chimera did not affect the reactivation phenotype, although construction of a rescuant would be necessary to ascertain whether the modest differences observed during acute infection with HSV2-LAT-P1 compared to that with HSV-2 were attributable to the LAT promoter substitution.
HSV-1 spread to the lumbar spinal cord more efficiently than HSV-2, while all of the HSV-2-based viruses spread more efficiently than HSV-1 to the sacral spinal cord. The autonomic nervous system innervates the genitalia and bladder through both sympathetic and parasympathetic fibers extending from the paracervical ganglia to the pelvic organs. Presynaptic parasympathetic fibers extend from the sacral spinal cord to the paracervical ganglia, while sympathetic fibers originate in the lumbar cord (13
). While HSV-1 has been shown to replicate and establish latency in sympathetic neurons (10
), HSV-2 appears to be limited in its ability to replicate or establish latency in sympathetic neurons (28
). HSV-1 and HSV-2 are both able to gain access to parasympathetic (5
) and sensory nerve fibers. It is thus possible that the difference between lumbar and sacral quantities of HSV-1 and HSV-2 DNA could be related to different autonomic pathways for reaching the cord, potentially more likely to be sympathetic for HSV-1 and parasympathetic for HSV-2. During recurrences, HSV-2 viral DNA quantities increased in both the sacral cord and the DRG compared with levels present during latent infection (compare Fig. ), suggesting that HSV-2 viral replication may occur in both the sacral cord and the DRG during reactivation. HSV-1 exhibited increases in viral DNA in both the lumbar cord and the DRG during recurrences, suggesting that HSV-1 may also replicate in the lumbar spinal cord as well as the DRG during reactivation. However, it is unclear whether the increased DNA in the spinal cord during symptomatic recurrences is the result of viral spread from the DRG or reactivation originating in neurons of the spinal cord. Since genital reactivation of viruses with greater quantities of viral DNA in the sacral cord was more efficient, this also raises the possibility that some proportion of HSV reactivation may occur through autonomic pathways, which may provide further insight into type-specific differences in reactivation.
Substitution of HSV-1 LAT sequences into the HSV-2 LAT region did not eliminate the HSV-2 preference for the sacral spinal cord but did alter the quantity of DNA found in the sacral cord and the DRG during recurrences. The more virulent sequence chimera, HSV2-LAT-S1, had higher levels of viral DNA in the sacral cord and lower levels in the DRG than the wild-type HSV-2 during recurrences. Thus, the LAT sequence is not the major factor that permits HSV-2-based viruses to more efficiently reach the sacral spinal cord, but it does influence this process, potentially via a mechanism involving spread through or replication in autonomic neurons. The increased efficiency of HSV2-LAT-S1 in reaching and replicating in the sacral spinal cord further supports the conclusion that LAT region sequences are involved in neurotropism, or the ability of the virus to infect and replicate within specific neuronal subtypes.
Consistent with previous experiments, HSV-1 failed to reactivate efficiently in the guinea pig genital model. Wild-type HSV-2, HSV2-LAT-P1, and HSV2-LAT-S1-R reactivated efficiently, and these viruses also showed relatively higher DNA levels in the sacral spinal cord than HSV-1. This suggests that efficient replication in the sacral spinal cord and the ability to appropriately down-regulate viral replication are correlated with efficient genital reactivation during latency. Although the HSV2-LAT-S1 chimera efficiently reached the sacral spinal cord, its reduced-recurrence phenotype and high degree of virulence after reactivation may have been due to an inability to down-regulate the productive cycle upon reactivation, thus destroying its host instead of reestablishing a latent state from which it could again reactivate.
HSV-1 and HSV-2, while similar in many ways, exhibit significant differences in patterns of latency and reactivation, manifested clinically by site-specific recurrence patterns and differences in CNS manifestations. In the present study, we show that type-specific recurrence of HSV-2 depends on the presence of appropriate LAT region sequences downstream of the promoter. This same region of the LAT sequence appears to influence neurotropism within the CNS as well as the autonomic nervous system, which in turn influences reactivation as well as the pathogenesis of the virus, suggesting that some portion of the LAT phenotype may be due to an effect on viral replication and/or establishment of latency in different types of neurons.