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Latency-associated transcript (LAT) sequences regulate herpes simplex virus (HSV) latency and reactivation from sensory neurons. We found a HSV-2 LAT-related microRNA (miRNA) designated miR-I in transfected and infected cells in vitro and in acutely and latently infected ganglia of guinea pigs in vivo. miR-I is also expressed in human sacral dorsal root ganglia latently infected with HSV-2. miR-I is expressed under the LAT promoter in vivo in infected sensory ganglia. We also predicted and identified a HSV-1 LAT exon-2 viral miRNA in a location similar to miR-I, implying a conserved mechanism in these closely related viruses. In transfected and infected cells, miR-I reduces expression of ICP34.5, a key viral neurovirulence factor. We hypothesize that miR-I may modulate the outcome of viral infection in the peripheral nervous system by functioning as a molecular switch for ICP34.5 expression.HSV ͉ latency-associated transcript ͉ latency ͉ reactivation ͉ human H erpes simplex virus 1 and 2 (HSV-1 and HSV-2) are closely related human herpes viruses. HSV-1 and HSV-2 establish lifelong incurable latency in and reactivate preferentially from trigeminal ganglia and dorsal root ganglia to cause oro-facial and genital herpes, respectively. Although infections are usually mild, these viruses can cause severe disease including encephalitis and neonatal herpes, and HSV-2 infection is a risk factor for HIV acquisition. The only readily detectable viral transcript during latency of both HSV-1 and HSV-2 is the noncoding latency-associated transcript (LAT), which is transcribed from within the long repeats of the viral genome ( Fig. 1) (1). A Ϸ8-kb primary LAT is spliced, yielding a stable Ϸ2-kb LAT intron (2). Deletion of the LAT promoter in both HSV-1 and HSV-2 reduces the efficiency of reactivation (3-5), and substitution of HSV-1 LAT for native HSV-2 LAT sequences confers an HSV-1 reactivation phenotype (6). The HSV-1 LAT is currently believed to act in part by increasing the establishment or maintenance of latency, likely via an effect on the survival of acutely infected neurons (7), which may be mediated by inhibition of apoptosis in infected neurons (8). The molecular function of HSV-2 LAT remains largely unknown.miRNAs are a family of 21Ϸ24-nt noncoding RNAs that regulate gene expression based on sequence similarity to their targets. Mammalian viruses including EBV, Kaposi's sarcoma-associated herpesvirus, human cytomegalovirus, and SV40 encode viral miRNAs (9). Viral encoded miRNAs were predicted for HSV-1 (10, 11) and also for HSV-2 (10). However, no miRNAs have been identified in HSV-2 or HSV-1 LAT sequences. Results HSV-2 LAT Exon 2 Encodes a miRNA.To find miRNAs within the HSV-2 LAT region, a plasmid containing the LAT sequence and its promoter (pSSK) and a mutant plasmid expressing LAT under control of the CMV-IE promoter (pCMV-SSK) were transfected into 293 cells. Small RNAs isolated from the transfected cells were cloned and sequenced. A 22-23-nt HSV-2 RNA sequence (designated HSV-2 miR-I) appeared at a frequenc...

Infection of the mouse trigeminal ganglia (TG) is the most commonly used model for the study of herpes simplex virus type 1 (HSV-1) latency. Its popularity is caused, at least in part, by the perception that latent infection can be studied in this system in the absence of spontaneous viral reactivation. However, this perception has never been rigorously tested. To carefully study this issue, the eyes of Swiss-Webster mice were inoculated with HSV-1 (KOS), and 37-47 days later the TG were dissected, serial-sectioned, and probed for HSV-1 ICP4, thymidine kinase, glycoprotein C, and latency-associated transcript RNA by in situ hybridization. Serial sections of additional latently infected TG were probed with HSV-1-specific polyclonal antisera. Analysis of thousands of probed sections revealed abundant expression of viral transcripts, viral protein, and viral DNA replication in about 1 neuron per 10 TG tested. These same neurons were surrounded by a focal white cell infiltrate, indicating the presence of an antigenic stimulus. We conclude that productive cycle viral genes are abundantly expressed in rare neurons of latently infected murine TG and that these events are promptly recognized by an active local immune response. In the absence of detectable infectious virus in these ganglia, we propose the term ''spontaneous molecular reactivation'' to describe this ongoing process.H erpes simplex virus type 1 (HSV-1) infections of the skin or eye lead to invasion of the trigeminal ganglia (TG), where the virus follows one of two pathways (1-3). In some infected neurons, the virus replicates and destroys the cell. This lytic cycle path involves regulated viral gene expression in which immediate-early genes are required for the efficient expression of early and late genes. In other neurons, a latent infection is established in which lytic cycle genes are not expressed and infectious virus is not produced. Latent infection of neurons is characterized by the expression of the latency-associated transcripts (LAT), which are encoded in a single region of the HSV-1 genome and which accumulate to high levels in the nucleus of the host cell (4-10). In situ hybridization (ISH) studies of latently infected sensory ganglia have revealed numerous neurons expressing LAT but no other viral RNAs (4, 5, 7, 9-15). Thus, for many years a latent neuron has been defined in molecular terms as a neuron that is positive for LAT and negative for other viral RNAs.In humans, HSV-1 intermittently reactivates from the latent state, with peripheral shedding of infectious virus. Shedding of infectious virus is similarly observed in rabbit ocular models of HSV infection, both spontaneously and in response to local or systemic stimuli (16,17). In contrast, latent HSV-1 infection in the mouse seems to be more tightly regulated, and attempts to identify infectious virus or viral antigen in murine sensory ganglia after resolution of primary infection (indicative of a spontaneous reactivation localized to the sensory ganglion) have been unsuccessful (2,7,9,...

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We recently identified an acutely and latently expressed viral microRNA (miRNA), miR-I, encoded by herpes simplex virus 2 (HSV-2) latency-associated transcript (LAT) through small RNA cloning and two miRNAs encoded by HSV-1 LAT through prediction. We now report the use of high-throughput sequencing technology to identify two additional relatively less-abundant viral miRNAs, miR-II and miR-III, encoded by HSV-2 LAT exon 2. miR-II includes two miRNAs, miR-II-5p and miR-II-3p, which are processed from the same miRNA precursor. miR-II and miR-III map antisense to the 5 untranslated region of ICP34.5 and to the coding region of ICP0 exon 3, respectively. These novel miRNAs are conserved in different HSV-2 strains, and their presence in infected-and transfected-cell cultures was confirmed by Northern hybridization. All three HSV-2 LAT-encoded miRNAs map to genome locations similar to those of three out of four identified HSV-1 LAT-encoded miRNAs, but the sequences of these miRNAs are not conserved. The expression of LAT-encoded miRNAs is negatively regulated by ICP4, the major viral transactivator. We further show that, similar to miR-I, miR-II is able to efficiently silence the expression of ICP34.5, a key viral neurovirulence factor, and that miR-III is able to silence the expression of ICP0, a key viral transactivator. All these data suggest that LAT sequences likely contribute to HSV latency and reactivation through tight control of these LAT-encoded miRNAs and their viral targets.Herpes simplex virus 1 (HSV-1) and HSV-2 are closely related herpesviruses. HSV-1 typically infects the facial region and establishes a lifelong latent infection in sensory neurons of the trigeminal ganglia, while HSV-2 typically infects the genital region and establishes a lifelong latent infection in sensory neurons of the sacral dorsal root ganglia. Periodically, either virus may reactivate to cause symptomatic or asymptomatic recurrences in the area served by these sensory neurons. HSV-2 and HSV-1 have similar latent transcription patterns, in which the latency-associated transcript (LAT) is transcribed from within the genomic long repeats. In contrast to other viral promoters, the LAT promoter is highly active during latency, and LAT is the only viral gene product that is readily detectable during latency (39). HSV-1 LAT expression is inhibited by ICP4, the major viral transactivator required for most post-␣ gene expression (9, 12, 24), through an ICP4 binding site near the LAT transcription initiation site (14). The LAT introns (ϳ2.2 kb in HSV-2 and ϳ2 kb and 1.4 kb in HSV-1), which overlap the ICP0 transcript in an antisense direction, are much more abundant and stable than the ϳ8.5-kbp primary LAT transcript (13), which overlaps both the ICP0 and ICP34.5 transcripts in an antisense direction. ICP0 can transactivate a number of viral and host genes and is essential for HSV reactivation (4,5,18,19). ICP34.5, a key viral neurovirulence factor, is a protein kinase R inhibitor and is required for efficient viral replication in neurons...

Abnormal expression of major histocompatibility complex (MHC) class I and class II in various tissues is associated with autoimmune disease. Autoimmune responses can be triggered by viral infections or tissue injuries. We show that the ability of a virus or a tissue injury to increase MHC gene expression is duplicated by any fragment of double-stranded (ds) DNA or dsRNA introduced into the cytoplasm of nonimmune cells. Activation is sequenceindependent, is induced by ds polynucleotides as small as 25 bp in length, and is not duplicated by single-stranded polynucleotides. In addition to causing abnormal MHC expression, the ds nucleic acids increase the expression of genes necessary for antigen processing and presentation: proteasome proteins (e.g., LMP2), transporters of antigen peptides; invariant chain, HLA-DM, and the costimulatory molecule B7.1. The mechanism is different from and additive to that of ␥-interferon (␥IFN), i.e., ds polynucleotides increase class I much more than class II, whereas ␥IFN increases class II more than class I. The ds nucleic acids also induce or activate Stat1, Stat3, mitogen-activated protein kinase, NF-B, the class II transactivator, RFX5, and the IFN regulatory factor 1 differently from ␥IFN. CpG residues are not responsible for this effect, and the action of the ds polynucleotides could be shown in a variety of cell types in addition to thyrocytes. We suggest that this phenomenon is a plausible mechanism that might explain how viral infection of tissues or tissue injury triggers autoimmune disease; it is potentially relevant to host immune responses induced during gene therapy.

Herpes simplex virus type 1 (HSV-1) and HSV-2 cause very similar acute infections but differ in their abilities to reactivate from trigeminal and dorsal root ganglia. To investigate differences in patterns of viral infection, we colabeled murine sensory ganglia for evidence of HSV infection and for the sensory neuron marker A5 or KH10. During acute infection, 7 to 10% of HSV-1 or HSV-2 antigen-positive neurons were A5 positive and 13 to 16% were KH10 positive, suggesting that both viruses reach each type of neuron in a manner proportional to their representation in uninfected ganglia. In murine trigeminal ganglia harvested during HSV latency, 25% of HSV-1 latency-associated transcript (LAT)-and 4% of HSV-2 LAT-expressing neurons were A5 positive, while 12% of HSV-1 LAT-and 42% of HSV-2 LAT-expressing neurons were KH10 positive. A similar difference was observed in murine dorsal root ganglia. These differences could not be attributed to differences in LAT expression levels in A5-versus KH10-positive neurons. Thus, HSV-1 demonstrated a preference for the establishment of latency in A5-positive neurons, while HSV-2 demonstrated a preference for the establishment of latency in KH10-positive neurons. A chimeric HSV-2 mutant that expresses the HSV-1 LAT exhibited an HSV-1 phenotype, preferentially establishing latency in A5-positive neurons. These data imply that the HSV-1 and HSV-2 LAT regions influence the ability of virus to establish latency in different neuronal subtypes. That the same chimeric virus has a characteristic HSV-1 reactivation phenotype further suggests that LATinfluenced establishment of latency in specific neuronal subtypes could be an important part of the mechanism by which LAT influences viral reactivation phenotypes.Primary infection of mice with herpes simplex virus type 1 (HSV-1) and HSV-2 is characterized by viral replication at the site of inoculation, followed by retrograde axonal transport of the virus to corresponding sensory ganglia where infection follows two very different pathways (14,19,28,33). In some neurons, the virus expresses productive cycle genes, replicates, and causes host cell death, whereas in other neurons, the virus establishes a latent infection characterized by limited viral transcription except for the latency-associated transcripts (LATs), which accumulate to high copy number in the nuclei of latently infected cells (29). The LATs code from the long repeat region of the viral genome and run antisense to the immediate-early transactivator ICP0, the protein kinase R inhibitor ICP34.5, the 3Ј end of the immediate-early transactivator ICP4 and the AL gene (24). A unique feature of the major 2-kb LAT is that it is a stable intron, spliced from a much less stable primary transcript (5). Studies from multiple labs suggest that the LAT region of the viral genome plays an important role in both the establishment (25, 31) and the reactivation of latent infection (13). The mechanisms responsible for this are poorly defined. Hypotheses (reviewed in reference 3) include that the...