HSV-1 Latency-associated Transcript Research Articles

The importance of IFNα2A (Roferon-A) in HSV-1 latency and T cell exhaustion in ocularly infected mice.

Published studies have generated compelling results indicating that type I IFN modulates function of HSV-1 latency-associated transcript (LAT). One member of type I IFN is IFNα2A also called Roferon-A). IFNα2A has been used in monotherapy or in combination therapy with other drugs to treat viral infections and different kinds of cancer in humans. The goal of this study was to determine whether the absence of IFNα2A affects primary and latent infections in ocularly infected mice. Therefore, we generated a mouse strain lacking IFNα2A expression (IFNα2A-/-). Ocular HSV-1 replication, IFN and immune cell expressions on days 3 and 5 post infection (PI), as well as eye disease, survival, latency-reactivation, and T cell exhaustion were evaluated in ocularly infected IFNα2A-/- and wild type (WT) control mice. Absence of IFNα2A did not affect other members of the IFNα family but it affected IFNβ and IFNγ expressions as well as some immune cells on day 5 PI compared to WT mice. Viral replication in the eye, eye disease, and survival amongst ocularly infected IFNα2A-/- mice were similar to that of WT infected mice. The absence of IFNα2A significantly reduced the levels of latency and T cell exhaustion but not time of reactivation compared with control mice. Our results suggest that blocking IFNα2A expression may be a useful tool in reducing latency and the subsequent side effects associated with higher levels of latency.

HSV-1 latency-associated transcript miR-H3 and miR-H4 target STXBP1 and GABBR2 genes.

During latent infection, the HSV-1 virus generates only a single transcript, LAT, which encodes six miRNAs. The GABAergic pathway signaling system is an essential cell signaling pathway influenced by various therapeutic targets and some brain disorders, such as epilepsy. This study found that miRNAs encoding LAT might target the STXBP1 and GABBR2 genes, which are among the significant genes in the GABAergic pathway. Bioinformatic analysis utilizing TargetScan version 5.2 and the RNA22 tools uncovered miRNAs encoding LAT that can influence STXBP1 and GABBR2 transcripts. To evaluate the targeting effect of candidate microRNAs encoding LAT, namely, miR-H3 and miR-H4, LAT constructs were transfected into HEK 293T cells. The expression levels of microRNAs encoding LAT, as well as STXBP1 and GABBR2, were assayed by real-time PCR. Finally, the targeting potential of STXBP1 and GABBR2 3'UTR by LAT-encoded microRNAs was evaluated by the luciferase assay. In the current study, the bioinformatic tool TargetScan demonstrated that miR-H3 has the potential to target the transcripts of the STXBP1 and GABBR2 genes, whereas miR-H4 solely targeted GABBR2. On the other hand, the bioinformatic tool RNA22 validated the potential targeting of STXBP1 and GABBR2 by miR-H3 and miR-H4. Our findings showed that overexpression of miR-H4, miR-H3, or LAT significantly decreased STXBP1 gene expression by an average of 0.0593-fold, 0.237-fold, and 0.84-fold, respectively. Similarly, overexpression of miR-H3 or miR-H4 decreased GABBR2 expression by an average of 0.055- or 0.687-fold, respectively. Notably, targeting the GABBR2 3'UTR with the LAT transcript had no detectable effect. The evaluation of the targeting potential of STXBP1 and GABBR2 3'UTR by microRNAs encoded by LAT was conducted with a luciferase assay. Our results showed that miR-H3 overexpression reduces Renilla expression in psiCHECK2 plasmids with STXBP1 or GABBR2 3'UTR genes by 0.62- and 0.55-fold, respectively. miR-H4 reduced Renilla gene expression regulated by GABBR2's 3'UTR plasmid but had no effect on the Renilla gene expression regulated by STXBP1's 3'UTR. When the LAT transcript was overexpressed, there was a decrease in Renilla expression by 0.44-fold because of the regulation of STXBP1's 3'UTR. However, there was no significant effect observed through the control of GABBR2's 3'UTR.

Small Noncoding RNA (sncRNA1) within the Latency-Associated Transcript Modulates Herpes Simplex Virus 1 Virulence and the Host Immune Response during Acute but Not Latent Infection.

The HSV-1 latency-associated transcript (LAT) locus contains two small noncoding RNA (sncRNA) sequences (sncRNA1 and sncRNA2) that are not microRNAs (miRNAs). We recently reported that sncRNA1 is more important for in vitro activation of the herpesvirus entry mediator than sncRNA2, but its in vivo function is not known. To determine the role, if any, of sncRNA1 during herpes simplex virus 1 (HSV-1) infection in vivo, we deleted the 62-bp sncRNA1 sequence in HSV-1 strain McKrae using dLAT2903 (LAT-minus) virus, creating ΔsncRNA1 recombinant virus. Deletion of the sncRNA1 in ΔsncRNA1 virus was confirmed by complete sequencing of ΔsncRNA1 virus and its parental virus (i.e., McKrae). Replication of ΔsncRNA1 virus in tissue culture or in the eyes of infected mice was similar to that of HSV-1 strain McKrae and dLAT2903 viruses. However, the absence of sncRNA1 significantly reduced the levels of ICP0, ICP4, and gB but not LAT transcripts in infected rabbit skin cells in vitro. In contrast, the absence of sncRNA1 did reduce LAT expression in trigeminal ganglia (TG), but not in corneas, by day 5 postinfection (p.i.) in infected mice. Levels of eye disease in mice infected with ΔsncRNA1 or McKrae virus were similar, and despite reduced LAT levels in TG during acute ΔsncRNA1 infection, McKrae and ΔsncRNA1 viruses did not affect latency or reactivation on day 28 p.i. However, mice infected with ΔsncRNA1 virus were more susceptible to ocular infection than their wild-type (WT) counterparts. Expression of host immune response genes in corneas and TG of infected mice during primary infection showed reduced expression of beta interferon (IFNβ) and IFNγ and altered activation of key innate immune pathways, such as the JAK-STAT pathway in ΔsncRNA1 virus compared with parental WT virus. Our results reveal novel functions for sncRNA1 in upregulating the host immune response and suggest that sncRNA1 has a protective role during primary ocular HSV-1 infection. IMPORTANCE HSV-1 latency-associated transcript (LAT) plays a major role in establishing latency and reactivation; however, the mechanism by which LAT controls these processes is largely unknown. In this study, we sought to establish the role of the small noncoding RNA1 (sncRNA1) encoded within LAT during HSV-1 ocular infection. Our results suggest that sncRNA1 has a protective role during acute ocular infection by modulating the innate immune response to infection.

Herpes Simplex Virus 1 MicroRNA miR-H8 Is Dispensable for Latency and Reactivation In Vivo.

The regulatory functions of 10 individual viral microRNAs (miRNAs) that are abundantly expressed from the herpes simplex virus 1 (HSV-1) latency-associated transcript (LAT) region remain largely unknown. Here, we focus on HSV-1 miRNA miR-H8, which is within the LAT 3p exon, antisense to the first intron of ICP0, and has previously been shown to target a host glycosylphosphatidylinositol (GPI)-anchoring pathway. However, the functions of this miRNA have not been assessed in the context of the viral genome during infection. Therefore, we constructed a recombinant virus lacking miR-H8 (17dmiR-H8) and compared it to the parental wild-type and rescue viruses to characterize phenotypic differences. In rabbit skin cells, 17dmiR-H8 exhibited only subtle reductions in viral yields. In contrast, we found significant decreases in both viral yields (8-fold) and DNA replication (9.9-fold) in murine neuroblastoma cells, while 17dmiR-H8 exhibited a 3.6-fold increase in DNA replication in differentiated human neuronal cells (Lund human mesencephalic [LUHMES] cells). These cell culture phenotypes suggested potential host- and/or neuron-specific roles for miR-H8 in acute viral replication. To assess whether miR-H8 plays a role in HSV latency or reactivation, we used a human in vitro reactivation model as well as mouse and rabbit reactivation models. In the LUHMES cell-induced reactivation model, there was no difference in viral yields at 48 h postreactivation. In the murine dorsal root ganglion explant and rabbit ocular adrenergic reactivation models, the deletion of miR-H8 had no detectable effect on genome loads during latency or reactivation. These results indicate that miR-H8 is dispensable for the establishment of HSV-1 latency and reactivation.IMPORTANCE Herpesviruses have a remarkable ability to sustain lifelong infections by evading host immune responses, establishing a latent reservoir, and maintaining the ability to reactivate the lytic cascade to transmit the virus to the next host. The HSV-1 latency-associated transcript region is known to regulate many aspects of HSV-1 latency and reactivation, although the mechanisms for these functions remain unknown. To this end, we characterize an HSV-1 recombinant containing a deletion of a LAT-encoded miRNA, miR-H8, and demonstrate that it plays no detectable role in the establishment of latency or reactivation in differentiated human neurons (LUHMES cells) and mouse and rabbit models. Therefore, this study allows us to exclude miR-H8 from phenotypes previously attributed to the LAT region. Elucidating the genetic elements of HSV-1 responsible for establishment, maintenance, and reactivation from latency may lead to novel strategies for combating persistent herpesvirus infections.

The Latency-Associated Transcript Inhibits Apoptosis via Downregulation of Components of the Type I Interferon Pathway during Latent Herpes Simplex Virus 1 Ocular Infection.

The herpes simplex virus (HSV-1) latency-associated transcript (LAT) has been shown to inhibit apoptosis via inhibiting activation of proapoptotic caspases. However, the mechanism of LAT control of apoptosis is unclear, because LAT is not known to encode a functional protein, and the LAT transcript is found largely in the nucleus. We hypothesized that LAT inhibits apoptosis by regulating expression of genes that control apoptosis. Consequently, we sought to establish the molecular mechanism of antiapoptosis functions of LAT at a transcriptional level during latent HSV-1 ocular infection in mice. Our results suggest the following. (i) LAT likely inhibits apoptosis via upregulation of several components of the type I interferon (IFN) pathway. (ii) LAT does not inhibit apoptosis via the caspase cascade at a transcriptional level or via downregulating Toll-like receptors (TLRs). (iii) The mechanism of LAT antiapoptotic effect is distinct from that of the baculovirus inhibitor of apoptosis (cpIAP) because replacement of LAT with the cpIAP gene resulted in a different gene expression pattern than in either LAT+ or LAT- viruses. (iv) Replacement of LAT with the cpIAP gene does not cause upregulation of CD8 or markers of T cell exhaustion despite their having similar levels of latency, further supporting that LAT and cpIAP function via distinct mechanisms.IMPORTANCE The HSV-1 latency reactivation cycle is the cause of significant human pathology. The HSV-1 latency-associated transcript (LAT) functions by regulating latency and reactivation, in part by inhibiting apoptosis. However, the mechanism of this process is unknown. Here we show that LAT likely controls apoptosis via downregulation of several components in the JAK-STAT pathway. Furthermore, we provide evidence that immune exhaustion is not caused by the antiapoptotic activity of the LAT.

In Vivo Knockdown of the Herpes Simplex Virus 1 Latency-Associated Transcript Reduces Reactivation from Latency.

During herpes simplex virus (HSV) latency, most viral genes are silenced, with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long noncoding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilized an adeno-associated virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition, these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness.IMPORTANCE Herpes simplex virus (HSV) establishes a lifelong infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency, with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This work shows that the LAT RNA is important for reactivation and suggests the potential of this treatment as a therapy for treating HSV infections.

Herpes Simplex Virus 2 Latency-Associated Transcript (LAT) Region Mutations Do Not Identify a Role for LAT-Associated MicroRNAs in Viral Reactivation in Guinea Pig Genital Models

Despite the long-standing observation that herpes simplex virus (HSV) latency-associated transcript (LAT) promoter deletion viruses show impaired recurrence phenotypes in relevant animal models, the mechanism by which these sequences exert this phenotypic effect is unknown. We constructed and evaluated four mutant HSV-2 isolates with targeted mutations in the LAT promoter and LAT-associated microRNAs (miRNAs) affecting (i) the LAT TATA box; (ii) the LAT ICP4-binding site; (iii) miRNA I (miR-I) and miR-II (miR-I/II), which both target ICP34.5; and (iv) miR-III, which targets ICP0. While the LAT TATA box mutant caused milder acute infections than wild-type (WT) virus, there was no difference in the recurrence phenotype between these viruses. LAT and miRNA expression during latency was not impaired by this mutation, suggesting that other promoter elements may be more important for latent HSV-2 LAT expression. Mutation of the LAT ICP4-binding site also did not cause an in vivo phenotypic difference between mutant and WT viruses. Acute infection and reactivation from latency of the miR-I/II mutant were similar to those of its rescuant, although the acute infection was slightly reduced in severity relative to that caused by the wild-type virus. The miR-III mutant also exhibited WT phenotypes in acute and recurrent phases of infection. While they do not rule out an effect of these elements in human latency or reactivation, these findings do not identify a specific role for LAT or LAT-associated miRNAs in the HSV-2 LAT promoter deletion phenotype in guinea pigs. Thus, other sequences in this region may play a more important role in the long-studied LAT-associated phenotype in animals.IMPORTANCE While it has been known for several decades that specific HSV-1 and HSV-2 sequences near the LAT promoter are required for efficient viral reactivation in animal models, the mechanism is still not known. We constructed four mutant viruses with the goal of identifying critical sequence elements and of specifically testing the hypothesis that microRNAs that are expressed during latency play a role. Determination that specific LAT promoter sequences and miRNA sequences do not influence viral reactivation of HSV-2 helps to narrow down the search for the mechanism by which the virus controls its latency and recurrence phenotype.

A Tribute to Professor Steven L. Wechsler (1948–2016): The Man and the Scientist

ABSTRACTProfessor Steven L. Wechsler, a world-renowned eye researcher and virologist, passed away unexpectedly on June 12, 2016 at the age of 68. Many scientists came to know Professor Wechsler as a gifted researcher in the field of ocular Herpes Simplex Virus (HSV-1) latency, reactivation, and pathogenesis. Professor Wechsler published over 150 peer-reviewed scientific papers during his career, pushing forward the frontiers of his field eye research. His colleagues would say, ‘Steve literally wrote the book on herpes latency and reactivation.’ He was the first to show that the HSV-1 latency-associated transcript (LAT) is essential for the HSV-1 high spontaneous reactivation phenotype and that LAT has anti-apoptosis activity. This discovery of LAT’s anti-apoptosis activity, which is a key factor in how the LAT gene enhances reactivation, was published in Science in 2000 and created a new paradigm that greatly increased understanding of HSV-1 latency and reactivation. In collaboration with Professor Lbachir BenMohamed, an immunologist, they later demonstrated that LAT also acts as an immune evasion gene. He was a caring scientist who truly enjoyed working and sharing his experience and expertise with young researchers. He will be remembered as a significant pillar within scientific and ocular herpes research communities worldwide. Professor Wechsler’s dedication to science, his compassionate character, and wonderful sense of humor were exemplary. We, who were his friends and colleagues, will mourn his passing deeply.

Anti-apoptotic function of herpes simplex virus -2 latency-associated transcript RL1 sequence and screening of its encoded microRNAs.

The latency-associated transcript (LAT) gene of herpes simplex virus (HSV)-2 is the only detectable viral gene expressed during latent infection in neurons. LAT inhibits apoptosis and maintains latency by promoting the survival of infected neurons. However, whether LAT functions during HSV-2 infection via its encoded RNAs or via its encoded proteins remain unknown. Increasing evidence has indicated that LAT is likely to functionally promote the establishment of latent infection via LAT-encoded microRNAs (miRNAs). To explore whether the RL1 fragment of the five adjacent miRNAs has an effect on cell apoptosis, then provide supporting evidence to elucidate the potential role of these miRNAs and to aid screening of their cellular targets. A number of techniques, including MTT assay, flow cytometry and DNA ladder analysis, were used to verify the role of the RL1 fragment and the contribution of the individual miRNAs to the anti-apoptotic effect. Five miRNAs (miR-H3, miR-H4-3p, miR-H4-5p, miR-H24 and miR-H19) were detected by quantitative PCR in PC12 cells stably expressing RL1 after pEGFP-RL1 plasmid transfection invitro. The data indicated that expression of HSV-2 LAT RL1 seems to provide protection against apoptosis of PC12 cells induced by ActD. Antisense miRNAs specifically inhibiting these five miRNAs could efficiently reduce their expression. Transfection of antisense-miR-H3, antisense-miR-H4-5p and antisense-miR-H19 into PC12 cells stably expressing RL1 were able to partly reverse the anti-apoptotic effect of these miRNAs. These findings indicate that the apoptotic role of the RL1 fragment is likely to be related to overexpression of miR-H3, miR-H4-5p and miR-H19 in PC12 cells.

Different Mechanisms Regulate Productive Herpes Simplex Virus 1 (HSV-1) and HSV-2 Infections in Adult Trigeminal Neurons

Herpes simplex virus 1 (HSV-1) and HSV-2 establish latency in different neuronal subtypes (A5+ and KH10+) in murine trigeminal ganglia, results which correlate with restricted productive infection in these neurons in vitro. HSV-2 latency-associated transcript (LAT) contains a cis-acting regulatory element near the transcription start site that promotes productive infection in A5+ neurons and a second element in exon 1 that inhibits productive infection in KH10+ neurons. HSV-1 contains no such regulatory sequences, demonstrating different mechanisms for regulating productive HSV infection in neurons.

A5-Positive Primary Sensory Neurons Are Nonpermissive for Productive Infection with Herpes Simplex Virus 1 In Vitro

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) establish latency and express the latency-associated transcript (LAT) preferentially in different murine sensory neuron populations, with most HSV-1 LAT expression in A5(+) neurons and most HSV-2 LAT expression in KH10(+) neurons. To study the mechanisms regulating the establishment of HSV latency in specific subtypes of neurons, cultured dissociated adult murine trigeminal ganglion (TG) neurons were assessed for relative permissiveness for productive infection. In contrast to that for neonatal TG, the relative distribution of A5(+) and KH10(+) neurons in cultured adult TG was similar to that seen in vivo. Productive infection with HSV was restricted, and only 45% of cultured neurons could be productively infected with either HSV-1 or HSV-2. A5(+) neurons supported productive infection with HSV-2 but were selectively nonpermissive for productive infection with HSV-1, a phenomenon that was not due to restricted viral entry or DNA uncoating, since HSV-1 expressing β-galactosidase under the control of the neurofilament promoter was detected in ∼90% of cultured neurons, with no preference for any neuronal subtype. Infection with HSV-1 reporter viruses expressing enhanced green fluorescent protein (EGFP) from immediate early (IE), early, and late gene promoters indicated that the block to productive infection occurred before IE gene expression. Trichostatin A treatment of quiescently infected neurons induced productive infection preferentially from non-A5(+) neurons, demonstrating that the nonpermissive neuronal subtype is also nonpermissive for reactivation. Thus, HSV-1 is capable of entering the majority of sensory neurons in vitro; productive infection occurs within a subset of these neurons; and this differential distribution of productive infection is determined at or before the expression of the viral IE genes.

The Role of LAT in Increased CD8 + T Cell Exhaustion in Trigeminal Ganglia of Mice Latently Infected with Herpes Simplex Virus 1

Herpes simplex virus (HSV) infection is a classic example of latent viral infection in humans and experimental animal models. The HSV-1 latency-associated transcript (LAT) plays a major role in the HSV-1 latency reactivation cycle and thus in recurrent disease. Whether the presence of LAT leads to generation of dysfunctional T cell responses in the trigeminal ganglia (TG) of latently infected mice is not known. To address this issue, we used LAT-positive [LAT(+)] and LAT-deficient [LAT(-)] viruses to evaluate the effect of LAT on CD8 T cell exhaustion in TG of latently infected mice. The amount of latency as determined by quantitative reverse transcription-PCR (qRT-PCR) of viral DNA in total TG extracts was 3-fold higher with LAT(+) than with LAT(-) virus. LAT expression and increased latency correlated with increased mRNA levels of CD8, PD-1, and Tim-3. PD-1 is both a marker for exhaustion and a primary factor leading to exhaustion, and Tim-3 can also contribute to exhaustion. These results suggested that LAT(+) TG contain both more CD8(+) T cells and more CD8(+) T cells expressing the exhaustion markers PD-1 and Tim-3. This was confirmed by flow cytometry analyses of expression of CD3/CD8/PD-1/Tim-3, HSV-1, CD8(+) T cell pentamer (specific for a peptide derived from residues 498 to 505 of glycoprotein B [gB(498-505)]), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α). The functional significance of PD-1 and its ligands in HSV-1 latency was demonstrated by the significantly reduced amount of HSV-1 latency in PD-1- and PD-L1-deficient mice. Together, these results may suggest that both PD-1 and Tim-3 are mediators of CD8(+) T cell exhaustion and latency in HSV-1 infection.

Investigation of the Mechanism by Which Herpes Simplex Virus Type 1 LAT Sequences Modulate Preferential Establishment of Latent Infection in Mouse Trigeminal Ganglia

We previously demonstrated that herpes simplex virus type 1 (HSV-1) preferentially establishes latent infection in monoclonal antibody (MAb) A5-positive ganglionic neurons and that a 2.8-kb portion of the HSV-1 genome, corresponding to the 5' end of the LAT (latency-associated transcript) coding region, is responsible for this phenotype (38, 65). In the current study we carried out further genetic mapping of this latency phenotype and investigated some of the mechanisms that might be responsible. Studies with the chimeric virus HSV-1 17syn+/LAT2, an HSV-1 virus engineered to express HSV-2 LAT, demonstrated that this virus exhibited an HSV-2 latency phenotype, preferentially establishing latency in MAb KH10-positive neurons. This result is complementary to that previously described for the chimeric virus HSV-2 333/LAT1 and indicate that the HSV-1 latency phenotype can be changed to that of HSV-2 by substitution of a 2.8-kb piece of complementary viral DNA. Sequential studies in which we evaluated the pattern of HSV-1 latent infection of the mouse trigeminal ganglion following ocular inoculation with viruses with deletions of functional thymidine kinase, glycoprotein E, ICP0, and US9 protein demonstrate that preferential establishment of HSV-1 latent infection in A5-positive neurons is not a consequence of (i) differential access of HSV-1 to A5-positive neurons,(ii) differential cell-to-cell spread of HSV-1 to A5-positive neurons, (iii) differential "round-trip" spread of HSV-1 to A5-positive neurons, or (iv) expression of ICP0. Additional mapping studies with the HSV-1 LAT deletion viruses dLAT371, 17DeltaSty, and 17Delta348 indicate that most of the LAT 5' exon is not required for HSV-1 to preferentially establish latent infection in A5-positive neurons.

Cell type specific accumulation of the major latency-associated transcript (LAT) of herpes simplex virus type 2 in LAT transgenic mice

We performed in situ hybridization to determine the cell type specific accumulation of the intron of the latency-associated transcript (LAT) in tissues in HSV-2 LAT transgenic mice in which LAT expression is driven by its native promoter. We identified LAT in multiple cell types in most tissues analyzed from HSV-2 LAT transgenic mice. While weak to moderate signals were seen in brain and spinal cord neurons, epithelial cells, and muscle cells, the strongest signals were detected in neurons from dorsal root and trigeminal ganglia. About 70–86% of neurons in these ganglia were LAT-positive with varying signal intensities, while cells surrounding the neurons were LAT-negative. The frequency of A5 or KH10-positive neurons was similar in LAT-positive and total neurons. These data indicate that HSV-2 LAT promoter activity is not restricted to neurons and that LAT accumulation in ganglionic neurons is likely regulated by cell-specific factors.

Novel Less-Abundant Viral MicroRNAs Encoded by Herpes Simplex Virus 2 Latency-Associated Transcript and Their Roles in Regulating ICP34.5 and ICP0 mRNAs

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.

An acutely and latently expressed herpes simplex virus 2 viral microRNA inhibits expression of ICP34.5, a viral neurovirulence factor

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.

Herpes Simplex Virus Latency-Associated Transcript Sequence Downstream of the Promoter Influences Type-Specific Reactivation and Viral Neurotropism

Herpes simplex virus (HSV) establishes latency in sensory nerve ganglia during acute infection and may later periodically reactivate to cause recurrent disease. HSV type 1 (HSV-1) reactivates more efficiently than HSV-2 from trigeminal ganglia while HSV-2 reactivates more efficiently than HSV-1 from lumbosacral dorsal root ganglia (DRG) to cause recurrent orofacial and genital herpes, respectively. In a previous study, a chimeric HSV-2 that expressed the latency-associated transcript (LAT) from HSV-1 reactivated similarly to wild-type HSV-1, suggesting that the LAT influences the type-specific reactivation phenotype of HSV-2. To further define the LAT region essential for type-specific reactivation, we constructed additional chimeric HSV-2 viruses by replacing the HSV-2 LAT promoter (HSV2-LAT-P1) or 2.5 kb of the HSV-2 LAT sequence (HSV2-LAT-S1) with the corresponding regions from HSV-1. HSV2-LAT-S1 was impaired for reactivation in the guinea pig genital model, while its rescuant and HSV2-LAT-P1 reactivated with a wild-type HSV-2 phenotype. Moreover, recurrences of HSV-2-LAT-S1 were frequently fatal, in contrast to the relatively mild recurrences of the other viruses. During recurrences, HSV2-LAT-S1 DNA increased more in the sacral cord compared to its rescuant or HSV-2. Thus, the LAT sequence region, not the LAT promoter region, provides essential elements for type-specific reactivation of HSV-2 and also plays a role in viral neurotropism. HSV-1 DNA, as quantified by real-time PCR, was more abundant in the lumbar spinal cord, while HSV-2 DNA was more abundant in the sacral spinal cord, which may provide insights into the mechanism for type-specific reactivation and different patterns of central nervous system infection of HSV-1 and HSV-2.

Herpes Simplex Virus Type 2 (HSV-2) Establishes Latent Infection in a Different Population of Ganglionic Neurons than HSV-1: Role of Latency-Associated Transcripts

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 LAT-influenced establishment of latency in specific neuronal subtypes could be an important part of the mechanism by which LAT influences viral reactivation phenotypes.

The bovine herpesvirus-1 LR ORF2 is critical for this gene's ability to restore the high wild-type reactivation phenotype to a herpes simplex virus-1 LAT null mutant.

During neuronal latency of herpes simplex virus (HSV)-1, the latency-associated transcript (LAT) is the only viral gene readily detectable. LAT is required for the high-level reactivation phenotype in animal models. LAT's anti-apoptotic activity was recently demonstrated by our group and it was proposed that LAT's anti-apoptotic function is involved in enhancing the reactivation phenotype. Recently, using chimeric virus CJLAT, it was shown that the reactivation phenotype of LAT(-) mutant dLAT2903 can be restored to wild-type levels by inserting the bovine herpes virus (BHV)-1 latency-related (LR) gene into the LAT locus of this HSV-1 LAT deletion mutant. Although transcription of the LR gene, like LAT, inhibits apoptosis, LR appears to be multifunctional. To investigate whether the LR gene's anti-apoptotic function was responsible for restoring the high-reactivation phenotype, a mutated BHV-1 LR gene was inserted into the LAT locus of HSV-1 generating the chimeric virus CJLATmut. This mutation consists of three stop codons inserted just after the ATG of the first LR open reading frame (ORF2). In plasmids and in a BHV-1 mutant, this mutation eliminated the LR gene's anti-apoptotic activity, strongly suggesting that ORF2 encodes a protein responsible for LR's anti-apoptotic activity. Reactivation of the CJLATmut virus, in both rabbits and mice, was significantly lower than in wild-type McKrae virus (P=0.0001 and P=0.0003, respectively) and CJLAT virus, containing wild-type LR in place of LAT (P<0.0001) and was similar to LAT(-) dLAT2903 (P=0.8 and P=0.7, respectively). Thus, disruption of BHV-1 LR ORF2 eliminated the high-reactivation phenotype.

Establishment and maintenance of HSV latent infection is mediated through correct splicing of the LAT primary transcript

To study the effect of the splicing of HSV-1 latency-associated transcript (LAT) on viral latency, we constructed two mutant viruses (FHλ+ and FHλ−) in which the 168-bp HpaI– HpaI fragment within the 2-kb LAT intron was replaced by a 447-bp bacteriophage lambda sequence. The lambda DNA was inserted in opposite orientations in FHλ+ and FHλ−. The mutation in FHλ+ disrupted the splicing of LAT primary transcript and altered both LAT exon and intron, whereas the mutation in FHλ− virus preserved the wild-type splice sites and the wild-type exon. Quantitative PCR analysis revealed that during latency there was a reduction in the number of viral genomes in mouse trigeminal ganglia infected with FHλ+ but not in those infected with FHλ−. The decrease in the latent genome numbers was not due to a defect in viral replication during the acute stage of infection. Furthermore, trigeminal ganglia from mice latently infected with FHλ+ displayed a slower reactivation kinetics compared to those infected with the parental strain. To elucidate the mechanism, we examined the antiapoptotic properties of these LAT constructs. A plasmid containing the pHλ+ construct was found to be less protective for cells against apoptosis than plasmid containing the wild-type or pHλ− constuct. These results suggest that the splicing of LAT primary transcript, and thus the correctly spliced exon product, play an important role in promoting the establishment and/or maintenance of viral latency.