During an initial infection, herpes simplex virus 1 (HSV-1) and HSV-2 establish latency in the sensory ganglia innervating the peripheral site of inoculation. In response to various stimuli, virus can reactivate from the sensory neurons to cause recurrent disease at or near the original site of inoculation. Although these viruses are very similar, there are notable differences in their abilities to reactivate in a manner specific to each viral species. HSV-1 reactivates most efficiently from the trigeminal ganglia to cause recurrent orofacial herpes, while HSV-2 reactivates more efficiently from the lumbosacral dorsal root ganglia (DRG) to cause recurrent genital herpes (18
The latency-associated transcript (LAT) is the most abundant latently transcribed RNA in both HSV-1 and HSV-2. Stable LAT introns are spliced from less stable primary transcripts (Fig. ) (10
). HSV-1 and HSV-2 LAT promoter mutants that express no detectable LAT during latency are impaired for reactivation in several animal models (5
). The HSV-2 LAT intron expressed transgenically in mice has no influence on HSV infection or the reactivation phenotype (31
), and recombinant HSV-1 viruses with deletions in the intron behave like wild-type virus (9
). The mutation of the splice branch points destabilizes the accumulation of the HSV-1 LAT intron but does not influence reactivation (22
FIG. 1. Virus construction. Genomic and endonuclease restriction sites are shown relative to HSV-2 strain HG52. The HSV genome consists of unique long and short (UL and US) regions flanked by internal and terminal repeats (IRL/IRS and TRL/TRS). The primary LAT (more ...)
Studies using recombinant viruses with deletions in the LAT region showed that sequences in LAT exon 1 are required for the efficient reactivation of HSV-1 (4
) and HSV-2 (3
). The LAT exon 1 region also provides enhancer functions for the continued long-term expression of HSV-1 LAT during latency (2
), and the corresponding region of HSV-2 also influences LAT expression levels (34
). Deletions within HSV-1 LAT exon 1 alter virulence differently in mice and rabbits (23
), suggesting that this region responds to cellular factors to exert an effect on virulence. Thus, LAT exon 1 sequences may confer regulatory influence over reactivation and virulence.
Primary sensory neurons are a diverse population of cells that can be classified according to cellular morphology, physiological response properties, and patterns of gene expression. Although all neuronal populations in the trigeminal ganglia and DRG are capable of supporting productive HSV infection, some populations are more likely than others to harbor latent virus, as identified by dual fluorescent staining for HSV LAT and specific surface markers identified by monoclonal antibodies A5 (specific for a population of neurons expressing Galβ1-4GlcNAc-R epitopes) and KH10 (recognizing a different population of neurons that express Galα1-3Galβ1-4Nac-R epitopes). Most A5+ neurons are immunoreactive for the calcitonin gene-related peptide and the high-affinity nerve growth factor receptor (TrkA), whereas KH10+ neurons colabel with the lectin BSL-IB4, identifying them as a population of small-diameter neurons responsive to glial cell-derived neurotrophic factor. Neurons latently infected with HSV-1 that express the LAT are more likely to be identified by monoclonal antibody A5, while LAT expression during HSV-2 latent infection is more associated with neurons identified by monoclonal antibody KH10 (19
). In these previous studies, no differences were observed in the ability of A5+ or KH10+ cells to express the HSV-1 or HSV-2 LAT; thus, these findings indicated viral species-specific differences in the ability of the virus to become latent and express LAT at detectable levels in these neuronal subpopulations.
We previously reported on the chimeric HSV-2 viruses HSV2-LAT1, HSV2-LAT-P1, and HSV2-LAT-S1, which contain HSV-1 LAT sequences in place of homologous HSV-2 sequences (Fig. ). HSV2-LAT1, which contains HSV-1 LAT promoter and downstream sequences homologous to an HSV-2 NotI-XhoI subsequence, displayed an HSV-1 recurrence phenotype in guinea pig genital and rabbit eye models (33
) and was found to express LAT in A5+ neurons, also an HSV-1 phenotype (19
). HSV2-LAT-P1, containing HSV-1 LAT promoter sequences (in a NotI-PvuI fragment), reactivated efficiently in the guinea pig genital model (an HSV-2 phenotype), while HSV2-LAT-S1, which contained HSV-1 LAT sequences downstream of the promoter (homologous to an HSV-2 PvuI-XhoI fragment), was impaired for reactivation in guinea pigs, implying that sequences within the LAT exon 1 and intron (and not the LAT promoter) were responsible for the efficient reactivation of HSV-2 in the guinea pig genital model (3
In the present study, to further define the region of LAT that is critical for HSV-2 species-specific latency phenotypes, an additional chimeric virus was constructed and characterized. The region of HSV-2 from the LAT TATA box to the 5′ splice site of the LAT intron (LAT exon 1) was replaced by the corresponding region of HSV-1 and designated HSV2-LAT-E1 (for exon from HSV-1). A rescuant, HSV2-LAT-E1R, also was constructed to restore the native HSV-2 sequences. After the in vitro characterization of HSV2-LAT-E1, the chimeric virus and its rescuant were tested for reactivation in the guinea pig genital model of HSV infection. Tissues also were evaluated for the distribution of viral DNA during acute and latent infection to determine if LAT exon 1 influenced viral spread and the establishment of latency. To evaluate a potential mechanism for any differences, viral transcript expression levels were quantified for LAT and ICP0 (a viral transactivator essential for reactivation). Latently infected LAT-expressing neurons from HSV2-LAT-E1, HSV2-LAT-P1, HSV2-LAT-S1, and their rescuants (where available) also were evaluated for the expression of A5 and KH10 neuronal markers to characterize the subpopulation of neurons in which LAT expression-competent virus established latency.