Herpesviruses are characterized by their ability to maintain life-long latent infections in their animal hosts. However, the mechanisms that allow establishment and maintenance of the latent state remain poorly understood. Herpes simplex virus 1 (HSV-1) establishes latency in neurons of sensory ganglia, where the only abundant viral gene product is a non-coding RNA, the latency associated transcript (LAT)1,2. Here, we show that LAT functions as a primary microRNA (miRNA) precursor that encodes four distinct miRNAs in HSV-1 infected cells. One of these miRNAs, miR-H2-3p, is transcribed antisense to ICP0, a viral immediate-early transcriptional activator thought to play a key role in productive HSV-1 replication and reactivation from latency3. miR-H2-3p is indeed able to reduce ICP0 protein expression, but does not significantly affect ICP0 mRNA levels. We also identified a fifth HSV-1 miRNA in latently infected trigeminal ganglia, miR-H6, which derives from a previously unknown transcript distinct from LAT. miR-H6 displays extended seed complementarity to the mRNA encoding a second HSV-1 transcription factor, ICP4, and inhibits expression of ICP4, which is required for expression of most HSV-1 genes during productive infection4. These results may explain the reported ability of LAT to promote latency5-9. Thus, HSV-1 expresses at least two primary miRNA precursors in latently infected neurons that may facilitate the establishment and maintenance of viral latency by post-transcriptionally regulating viral gene expression.

The closely related microRNA (miRNA) and RNAi pathways have emerged as important regulators of virus–host cell interactions. Although both pathways are relatively well conserved all the way from plants to invertebrates to mammals, there are important differences between these systems. A more complete understanding of these differences will be required to fully appreciate the relationship between these diverse host organisms and the various viruses that infect them. Insights derived from this research will facilitate a better understanding of viral pathogenesis and the host innate immune response to viral infection.

Analysis of cells infected by a wide range of herpesviruses has identified numerous virally encoded microRNAs (miRNAs), and several reports suggest that these viral miRNAs are likely to play key roles in several aspects of the herpesvirus life cycle. Here we report the first analysis of human ganglia for the presence of virally encoded miRNAs. Deep sequencing of human trigeminal ganglia latently infected with two pathogenic alphaherpesviruses, herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV), confirmed the expression of five HSV-1 miRNAs, miR-H2 through miR-H6, which had previously been observed in mice latently infected with HSV-1. In addition, two novel HSV-1 miRNAs, termed miR-H7 and miR-H8, were also identified. Like four of the previously reported HSV-1 miRNAs, miR-H7 and miR-H8 are encoded within the second exon of the HSV-1 latency-associated transcript. Although VZV genomic DNA was readily detectable in the three human trigeminal ganglia analyzed, we failed to detect any VZV miRNAs, suggesting that VZV, unlike other herpesviruses examined so far, may not express viral miRNAs in latently infected cells.MicroRNAs (miRNAs) are a family of ϳ22-nucleotide (nt) noncoding RNAs that are capable of binding to specific target mRNAs and inhibiting their expression (reviewed in reference 1). They are typically derived from one arm of RNA stemloops found within noncoding regions of capped and polyadenylated transcripts (4, 26). Successive cleavage of these hairpin structures by the RNase III enzymes Drosha in the nucleus (25) and Dicer in the cytoplasm (7, 20) generates a miRNA duplex structure of ϳ20 bp with 2-nt 3Ј overhangs. One arm of this duplex is then loaded into the RNA-induced silencing complex (RISC), where it is used as a guide to target complementary transcripts for inhibition (19,28). In mammalian cells, miRNAs usually guide the RISC to imperfectly complementary target sites, resulting in the translational arrest of bound mRNAs and a modest but detectable mRNA destabilization (12,31,43).Due to their small size and nonimmunogenic nature, miRNAs appear ideally suited for use as regulatory molecules by viruses, and indeed, a number of human DNA viruses, including many herpesviruses, have now been reported to encode miRNAs (39). Herpesviruses can be divided into three subfamilies, the alpha-, beta-, and gammaherpesviruses, based on replication characteristics, genomic organization, and preferred latency sites. Members of all three subfamilies have been found to encode miRNAs, ranging from a low of 3 in the alphaherpesvirus herpes simplex virus 2 (HSV-2) (37, 38) to a high of 25 in Epstein-Barr virus (EBV) (5,17,33,46). The fact that all herpesviruses examined to date express miRNAs suggests that miRNAs play important roles in the herpesvirus life cycle, and several studies have in fact demonstrated the downregulation of cellular and/or viral mRNA targets by herpesvirus miRNAs (reviewed in reference 16).HSV-1 and varicella-zoster virus (VZV) are pathogenic human viruses both of which belong to ...