The mechanism by which LAT sequences influence establishment of and reactivation from latency is not well understood. Previous studies indicated that HSV LAT-encoded miRNAs could target ICP34.5, ICP0, and ICP4 in in vitro
). Here we studied a recently identified miRNA, HSV-2 miR-H6, encoded by the sequences upstream of the HSV-2 LAT promoter in an antisense direction relative to the LAT. miR-H6 was detected in guinea pig DRG latently and acutely infected with HSV-2 and in mouse TG latently infected with HSV-2. Deletion of the HSV-2 LAT promoter not only abolished the LAT-encoded miRNAs, as previously found (19
), but also abolished miR-H6 expression in vivo
(), initially suggesting that miR-H6 might play a role in phenotypes previously attributed to the LAT, based on a study of LAT promoter deletion mutants. A mutant virus with SV40 polyadenylation signal sequences inserted upstream of miR-H6 had significantly reduced expression of miR-H6 in infected-cell cultures and infected guinea pigs and mice. However, the recurrence phenotype of the NotPolyA mutant was similar to those of its rescuant and the wild-type virus, indicating that downregulation of miR-H6 is not able to change the wild-type recurrence phenotype.
Although HSV-1 and HSV-2 are different viral species, the viruses share a similar genomic organization and similar sequences in many regions. Viral miRNAs from HSV-1 and HSV-2 are generally conserved in location, although there are significant sequence differences, including in the critical seed sequences. Both viruses express miRNAs targeting ICP34.5 (HSV-2 miR-I and miR-II and HSV-1 miR-H4 and miR-H3, which have some sequence homology) and a miRNA targeting ICP0 (HSV-2 miR-III and HSV-1 miR-H2, which are nonhomologous but similarly located). HSV-1 miR-H6 and HSV-2 miR-H6 do not possess sufficient sequence homology to have permitted prediction of the HSV-2 miR-H6 sequence based solely on the HSV-1 miR-H6 sequence. One interesting additional difference between HSV-1 and HSV-2 miRNA expression is that the relative abundance of each miRNA is different for each virus. In HSV-2, miR-I is the most abundant latently expressed miRNA, while in HSV-1, miR-H2 is the most abundant. The levels of viral miRNAs likely affect the extent of their function, suggesting that HSV-2 may use its miRNAs primarily to target ICP34.5, while for HSV-1, it may be relatively more important to target ICP0.
The HSV-1 LAT exon 1 region is critical for efficient reactivation of HSV-1 in vivo
). The 5′ end of LAT exon 1 is significantly enriched in acetyl-histone H3 (K9, K14) (11
), which is often associated with active promoters. Recently, Bertke et al. reported that the 5′ LAT exon region is important for HSV viral species-specific phenotypes (2
). In the present study, a deletion encompassing the first 238 bp of LAT exon 1 and the LAT promoter abolished HSV-2 miR-H6 expression in vivo
, suggesting that the LAT promoter and part of the LAT exon 1 region are critical for miR-H6 transcription and likely contain the promoter and transcription initiation site for the miR-H6 primary transcript. Deletion of the LAT promoter and partial LAT exon 1 region did not significantly affect miR-H6 expression in infected nonneuronal cell cultures. We previously reported that LAT-encoded miRNAs, including miR-I, -II, and -III could be detected in ΔLAT-infected cell cultures but not in ΔLAT-infected guinea pigs (19
). These results suggest that both the LAT sense-strand miRNAs and miR-H6 promoter are expressed differently in infected DRG in vivo
than in infected epithelial cell cultures in vitro
. These results further imply that the function of the LAT-associated miRNAs in vivo
might be quite difficult to evaluate in vitro
in infected-cell cultures.
The functions of many LAT-encoded miRNAs are likely conserved. For example, miR-I and -II and their homologs in HSV-1 silence ICP34.5 efficiently (17
; also unpublished data), and miR-III and its HSV-1 homolog, miR-H2, are suggested to target ICP0 (20
). However, HSV-2 miR-H6 and HSV-1 miR-H6 may have different functions. Umbach et al. reported that HSV-1 miR-H6 could target HSV-1 ICP4 in an in vitro
). The seed sequence (2 to 8 nt) between HSV-1 miR-H6 and HSV-2 miR-H6 is quite different. In addition, HSV-1 and HSV-2 ICP4 sequences share less than 90% homology. These sequence differences imply that if miR-H6 targets ICP4 in both HSV-1 and HSV-2, both the miRNA seed sequence and the ICP4 target sequence would likely be different for each virus. Although Jurak et al. predicted two target sites on ICP4 for HSV-2 miR-H6 (9
), we were not able to observe a significant downregulation of ICP4 by HSV-2 miR-H6 through cotransfection of ICP4 expression plasmid and miR-H6 or pretransfection of miR-H6 following infection with HSV-2.
Because the NotPolyA mutant virus had a normal recurrence phenotype and displayed the wild-type HSV-2 phenotype of preferential establishment of latency in KH10-positive neurons, it seems unlikely that HSV-2 miR-H6 plays a critical role in viral latency and reactivation. A potential role for miR-H6 in the observed neuroattenuation of the NotPolyA virus () may still be considered, although the mechanism underlying this observation is unclear. HSV-2 miR-H6 might influence viral targets other than ICP4 and ICP0, cellular targets, or could affect ICP4 expression in vivo (although no in vitro effect could be identified). Although the mutation in the NotPolyA virus nearly eliminated latent miR-H6 expression (), we observed low-level miR-H6 expression in infected-cell cultures, likely due to incomplete polyadenylation by HSV during lytic infection (). Thus, we cannot rule out the possibility that trace amounts of miR-H6 could be expressed during viral replication during recurrence from infected animals. We speculate that the very low level of miR-H6 in one of seven guinea pig DRG latently infected with NotPolyA may have been due to recurrence on the day when the ganglia were prepared (). However, the residual miR-H6 expressed by NotPolyA was at levels approximately 2 log units below that expressed by the rescuant virus (), and thus significantly below the physiological level.
Although LAT-associated miRNAs likely contribute to the phenotype attributed to the LAT, little is known about the extent of their contribution or the role of each individual miRNA. The present study is the first to study the recurrence phenotype of a virus with a mutation reducing expression of a miRNA without reducing LAT expression (i.e., the NotPolyA virus). ΔLAT, a mutant in which the LAT promoter and part of the LAT exon 1 sequence has been deleted, has a phenotype of greatly reduced recurrence frequency in the guinea pig model. However, in infected guinea pigs and mice, the mutation in ΔLAT substantially reduced expression of all of the LAT-encoded miRNAs and the upstream miR-H6. Thus, the phenotype of ΔLAT cannot be directly attributed to an effect on miR-H6 expression but could be a collective effect of all of the HSV-2 LAT-associated miRNAs. Further in vivo studies of HSV gene-encoded miRNAs are needed to elucidate the molecular functions of LAT-associated miRNAs and LAT.