Latency-associated Transcript Research Articles

Patterns of accumulation of miRNAs encoded by herpes simplex virus during productive infection, latency, and on reactivation

The key events in herpes simplex virus (HSV) infections are (i) replication at a portal of entry into the body modeled by infection of cultured cells; (ii) establishment of a latent state characterized by a sole latency-associated transcript and microRNAs (miRNAs) modeled in murine peripheral ganglia 30 d after inoculation; and (iii) reactivation from the latent state modeled by excision and incubation of ganglia in medium containing anti-NGF antibody for a timespan of a single viral replicative cycle. In this report, we examine the pattern of synthesis and accumulation of 18 HSV-1 miRNAs in the three models. We report the following: (i) H2-3P, H3-3P, H4-3P, H5-3P, H6-3P, and H7-5P accumulated in ganglia harboring latent virus. All but H4-3P were readily detected in productively infected cells, and most likely they originate from three transcriptional units. (ii) H8-5P, H15, H17, H18, H26, and H27 accumulated during reactivation. Of this group, only H26 and H27 could be detected in productively infected cells. (iii) Of the 18 we have examined, only 10 miRNAs were found to accumulate above background levels in productively infected cells. The disparity in the accumulation of miRNAs in cell culture and during reactivation may reflect differences in the patterns of regulation of viral gene expression during productive infection and during reactivation from the latent state.

Mutations within the pathogenic region of herpes simplex virus 1 gK signal sequences alter cell surface expression and neurovirulence.

To investigate the role of the signal sequences of herpes simplex virus 1 (HSV-1) gK on virus replication and viral pathogenesis, we constructed recombinant viruses with or without mutations within the signal sequences of gK. These recombinant viruses expressed two additional copies of the mutated (MgK) or native (NgK) form of the gK gene in place of the latency-associated transcript with a myc epitope tag to facilitate detection at their 3' ends. The replication of MgK virus was similar to that of NgK both in vitro and in vivo, as well as in the trigeminal ganglia (TG) of latently infected mice. The levels of gB and gK transcripts in the corneas, TG, and brains of infected mice on days 3 and 5 postinfection were markedly virus and time dependent, as well as tissue specific. Mutation in the signal sequence of gK in MgK virus blocked cell surface expression of gK-myc in rabbit skin cells, increased 50% lethal dose, and decreased corneal scarring in ocularly infected mice compared to the NgK or revertant (RgK) virus. MgK and NgK viruses, and not the RgK virus, showed a reduced extent of explant reactivation at the lower dose of ocular infection but not at the higher dose. However, the time of reactivation was not affected by overexpression of the different forms of gK. Taken together, these results strongly suggest that the 8mer peptide (ITAYGLVL) within the signal sequence of gK promotes cell surface expression of gK in infected cells and ocular pathogenesis in infected mice. In this study, we show for the first time that mutations within the signal sequence of gK blocked cell surface expression of inserted recombinant gK in vitro. Furthermore, this blockage in cell surface expression was correlated with higher 50% lethal dose and less corneal scarring in vivo. Thus, these studies point to a key role for the 8mer within the signal sequence of gK in HSV-1-induced pathogenicity.

Biological Features of Herpes Simplex Virus Type 1 Latency in Mice According to Experimental Conditions and Type of Neurones

To compare the biological patterns of viral transcripts during herpes simplex virus type 1 (HSV1) latent infection according to experimental conditions. Two types of murine models of HSV1 infection were used: the corneal scarification model, often used in studies of HSV1 latency, and the oro-ocular murine model. Two strains of HSV1 were used for the inoculation: SC16, a wild type strain considered as highly neuroinvasive, and KOS, previously described as poorly neurovirulent. The amounts of viral genomes, and those of four types of viral transcripts (immediate-early, early and late, together with latency-associated transcripts [LATs]), were measured by quantitative PCR and RT-PCR in the main sites of HSV1 latent infection at 6 days, 1 and 3 months post inoculation, and the number of LAT-expressing neurons was assessed by in-situ hybridization on histological sections of trigeminal ganglia (TG). Using the SC16 strain of HSV1 in the oro-ocular model, immediate-early transcripts were still present at 1 month post inoculation (early stage of latent infection), but were not detected at 3 months (late stage of latent infection). In both cases, early and late viral genes transcripts were not detected, demonstrating the latent nature of the infection with this combination of experimental conditions. In contrast, such progress in the viral gene expression was not observed in the corneal scarification model, particularly when the KOS strain of HSV1 was used. These results highlight that the behavior of the virus in the nervous system depends on the method inoculation, and the viral strain. All these parameters are likely to influence the establishment of latent infection.

A 2.5-Kilobase Deletion Containing a Cluster of Nine MicroRNAs in the Latency-Associated-Transcript Locus of the Pseudorabies Virus Affects the Host Response of Porcine Trigeminal Ganglia during Established Latency

The alphaherpesvirus pseudorabies virus (PrV) establishes latency primarily in neurons of trigeminal ganglia when only the transcription of the latency-associated transcript (LAT) locus is detected. Eleven microRNAs (miRNAs) cluster within the LAT, suggesting a role in establishment and/or maintenance of latency. We generated a mutant (M) PrV deleted of nine miRNA genes which displayed properties that were almost identical to those of the parental PrV wild type (WT) during propagation in vitro. Fifteen pigs were experimentally infected with either WT or M virus or were mock infected. Similar levels of virus excretion and host antibody response were observed in all infected animals. At 62 days postinfection, trigeminal ganglia were excised and profiled by deep sequencing and quantitative RT-PCR. Latency was established in all infected animals without evidence of viral reactivation, demonstrating that miRNAs are not essential for this process. Lower levels of the large latency transcript (LLT) were found in ganglia infected by M PrV than in those infected by WT PrV. All PrV miRNAs were expressed, with highest expression observed for prv-miR-LLT1, prv-miR-LLT2 (in WT ganglia), and prv-miR-LLT10 (in both WT and M ganglia). No evidence of differentially expressed porcine miRNAs was found. Fifty-four porcine genes were differentially expressed between WT, M, and control ganglia. Both viruses triggered a strong host immune response, but in M ganglia gene upregulation was prevalent. Pathway analyses indicated that several biofunctions, including those related to cell-mediated immune response and the migration of dendritic cells, were impaired in M ganglia. These findings are consistent with a function of the LAT locus in the modulation of host response for maintaining a latent state. This study provides a thorough reference on the establishment of latency by PrV in its natural host, the pig. Our results corroborate the evidence obtained from the study of several LAT mutants of other alphaherpesviruses encoding miRNAs from their LAT regions. Neither PrV miRNA expression nor high LLT expression levels are essential to achieve latency in trigeminal ganglia. Once latency is established by PrV, the only remarkable differences are found in the pattern of host response. This indicates that, as in herpes simplex virus, LAT functions as an immune evasion locus.

Vaccine and oncogenic strains of gallid herpesvirus 2 contain specific subtype variations in the 5′ region of the latency-associated transcript that evolve in vitro and in vivo

Gallid herpesvirus 2 (GaHV-2) is the alphaherpesvirus responsible for Marek's disease (MD), a T-cell lymphoma of chickens. The virulence of the GaHV-2 field strain is steadily increasing, but MD is still controlled by the CVI988/Rispens vaccine. We tried to determine distinguishing traits of the CVI988/Rispens vaccine by focusing on the 5' end region of the latency-associated transcript (5'LAT). It includes a variable number of 60-bp tandem repeats depending on the GaHV-2 strain. By analyzing six batches of vaccine, we showed that CVI988/Rispens consisted of a population of 5'LAT molecular subtypes, all with deletions and lacking 60-bp tandem repeat motifs, with two major subtypes that probably constitute CVI988/Rispens markers. Serial passages in cell culture led to a substantial change in the frequency of CVI988/Rispens 5'LAT subtypes, with non-deleted subtypes harboring up to four 60-bp repeats emerging during the last few passages. Dynamic changes in the distribution of 5'LAT-deleted subtypes were also detected after infection of chickens. By contrast, the 5'LAT region of the oncogenic clonal RB-1B strain, which was investigated at every step from the isolation of the clonal bacmid RB-1B DNA to the isolation of the ovarian lymphoma cell line, consisted of non-deleted 5'LAT subtypes harboring at least two 60-bp repeats. Thus, vaccine and oncogenic GaHV-2 strains consist of specific populations of viral genomes that are constantly evolving in vivo and in vitro and providing potential markers for epidemiological surveys.

Small RNA cloning and sequencing strategy affects host and viral microRNA expression signatures

The establishment of the microRNA (miRNA) expression signatures is the basic element to investigate the role played by these regulatory molecules in the biology of an organism. Marek's disease virus 1 (MDV-1) is an avian herpesvirus that naturally infects chicken and induces T cells lymphomas. During latency, MDV-1, like other herpesviruses, expresses a limited subset of transcripts. These include three miRNA clusters. Several studies identified the expression of virus and host encoded miRNAs from MDV-1 infected cell cultures and chickens. But a high discrepancy was observed when miRNA cloning frequencies obtained from different cloning and sequencing protocols were compared. Thus, we analyzed the effect of small RNA library preparation and sequencing on the miRNA frequencies obtained from the same RNA samples collected during MDV-1 infection of chicken at different steps of the oncoviral pathogenesis. Qualitative and quantitative variations were found in the data, depending on the strategy used. One of the mature miRNA derived from the latency-associated-transcript (LAT), mdv1-miR-M7-5p, showed the highest variation. Its cloning frequency was 50% of the viral miRNA counts when a small scale sequencing approach was used. Its frequency was 100 times less abundant when determined through the deep sequencing approach. Northern blot analysis showed a better correlation with the miRNA frequencies found by the small scale sequencing approach. By analyzing the cellular miRNA repertoire, we also found a gap between the two sequencing approaches. Collectively, our study indicates that next-generation sequencing data considered alone are limited for assessing the absolute copy number of transcripts. Thus, the quantification of small RNA should be addressed by compiling data obtained by using different techniques such as microarrays, qRT-PCR and NB analysis in support of high throughput sequencing data. These observations should be considered when miRNA variations are studied prior addressing functional studies.

Transcription of the Herpes Simplex Virus 1 Genome during Productive and Quiescent Infection of Neuronal and Nonneuronal Cells

Herpes simplex virus 1 (HSV-1) can undergo a productive infection in nonneuronal and neuronal cells such that the genes of the virus are transcribed in an ordered cascade. HSV-1 can also establish a more quiescent or latent infection in peripheral neurons, where gene expression is substantially reduced relative to that in productive infection. HSV mutants defective in multiple immediate early (IE) gene functions are highly defective for later gene expression and model some aspects of latency in vivo. We compared the expression of wild-type (wt) virus and IE gene mutants in nonneuronal cells (MRC5) and adult murine trigeminal ganglion (TG) neurons using the Illumina platform for cDNA sequencing (RNA-seq). RNA-seq analysis of wild-type virus revealed that expression of the genome mostly followed the previously established kinetics, validating the method, while highlighting variations in gene expression within individual kinetic classes. The accumulation of immediate early transcripts differed between MRC5 cells and neurons, with a greater abundance in neurons. Analysis of a mutant defective in all five IE genes (d109) showed dysregulated genome-wide low-level transcription that was more highly attenuated in MRC5 cells than in TG neurons. Furthermore, a subset of genes in d109 was more abundantly expressed over time in neurons. While the majority of the viral genome became relatively quiescent, the latency-associated transcript was specifically upregulated. Unexpectedly, other genes within repeat regions of the genome, as well as the unique genes just adjacent the repeat regions, also remained relatively active in neurons. The relative permissiveness of TG neurons to viral gene expression near the joint region is likely significant during the establishment and reactivation of latency. During productive infection, the genes of HSV-1 are transcribed in an ordered cascade. HSV can also establish a more quiescent or latent infection in peripheral neurons. HSV mutants defective in multiple immediate early (IE) genes establish a quiescent infection that models aspects of latency in vivo. We simultaneously quantified the expression of all the HSV genes in nonneuronal and neuronal cells by RNA-seq analysis. The results for productive infection shed further light on the nature of genes and promoters of different kinetic classes. In quiescent infection, there was greater transcription across the genome in neurons than in nonneuronal cells. In particular, the transcription of the latency-associated transcript (LAT), IE genes, and genes in the unique regions adjacent to the repeats persisted in neurons. The relative activity of this region of the genome in the absence of viral activators suggests a more dynamic state for quiescent genomes persisting in neurons.

Coregulatory Interactions among CD8α Dendritic Cells, the Latency-Associated Transcript, and Programmed Death 1 Contribute to Higher Levels of Herpes Simplex Virus 1 Latency

The latency-associated transcript (LAT) of herpes simplex virus 1 (HSV-1), CD8α(+) dendritic cells (DCs), and programmed death 1 (PD-1) have all been implicated in the HSV-1 latency-reactivation cycle. It is not known, however, whether an interaction between LAT and CD8α(+) DCs regulates latency and T-cell exhaustion. To address this question, we used LAT-expressing [LAT(+)] and LAT-negative [LAT(-)] viruses. Depletion of DCs in mice ocularly infected with LAT(+) virus resulted in a reduction in the number of T cells expressing PD-1 in the trigeminal ganglia (TG), whereas depletion of DCs in mice similarly infected with LAT(-) virus did not alter PD-1 expression. CD8α(+) DCs, but not CD4(+) DCs, infected with LAT(+) virus had higher levels of ICP0, ICP4, thymidine kinase (TK), and PD-1 ligand 1 (PD-L1) transcripts than those infected with LAT(-) virus. Coculture of infected bone marrow (BM)-derived DCs from wild-type (WT) mice, but not infected DCs from CD8α(-/-) mice, with WT naive T cells contributed to an increase in PD-1 expression. Transfer of bone marrow from WT mice but not CD8α(-/-) mice to recipient Rag1(-/-) mice increased the number of latent viral genomes in reconstituted mice infected with the LAT(+) virus. Collectively, these data indicated that a reduction in latency correlated with a decline in the levels of CD8α(+) DCs and PD-1 expression. In summary, our results demonstrate an interaction among LAT, PD-1, and CD11c CD8α(+) cells that regulates latency in the TG of HSV-1-infected mice. Very little is known regarding the interrelationship of LAT, PD-1, and CD8α(+) DCs and how such interactions might contribute to relative numbers of latent viral genomes. We show here that (i) in both in vivo and in vitro studies, deficiency of CD8α(+) DCs significantly reduced T-cell exhaustion in the presence of LAT(+) virus but not LAT(-) virus; (ii) HSV-1 infectivity was significantly lower in LAT(-)-infected DCs than in their LAT(+)-infected counterparts; and (iii) adoptive transfer of bone marrow (BM) from WT but not CD8α(-/-) mice to recipient Rag1(-/-) mice restored latency to the level in WT mice following infection with LAT(+) virus. These studies point to a key role for CD8α(+) DCs in T-cell exhaustion in the presence of LAT, which leads to larger numbers of latent viral genomes. Thus, altering this negative function of CD8α(+) DCs can potentially be used to generate a more effective vaccine against HSV infection.

Inclusion of CD80 in HSV Targets the Recombinant Virus to PD-L1 on DCs and Allows Productive Infection and Robust Immune Responses

CD80 plays a critical role in stimulation of T cells and subsequent control of infection. To investigate the effect of CD80 on HSV-1 infection, we constructed a recombinant HSV-1 virus that expresses two copies of the CD80 gene in place of the latency associated transcript (LAT). This mutant virus (HSV-CD80) expressed high levels of CD80 and had similar virus replication kinetics as control viruses in rabbit skin cells. In contrast to parental virus, this CD80 expressing recombinant virus replicated efficiently in immature dendritic cells (DCs). Additionally, the susceptibility of immature DCs to HSV-CD80 infection was mediated by CD80 binding to PD-L1 on DCs. This interaction also contributed to a significant increase in T cell activation. Taken together, these results suggest that inclusion of CD80 as a vaccine adjuvant may promote increased vaccine efficacy by enhancing the immune response directly and also indirectly by targeting to DC.

Maintenance of large numbers of virus genomes in human cytomegalovirus-infected T98G glioblastoma cells.

After infection, human cytomegalovirus (HCMV) persists for life. Primary infections and reactivation of latent virus can both result in congenital infection, a leading cause of central nervous system birth defects. We previously reported long-term HCMV infection in the T98G glioblastoma cell line (1). HCMV infection has been further characterized in T98Gs, emphasizing the presence of HCMV DNA over an extended time frame. T98Gs were infected with either HCMV Towne or AD169-IE2-enhanced green fluorescent protein (eGFP) strains. Towne infections yielded mixed IE1 antigen-positive and -negative (Ag(+)/Ag(-)) populations. AD169-IE2-eGFP infections also yielded mixed populations, which were sorted to obtain an IE2(-) (Ag(-)) population. Viral gene expression over the course of infection was determined by immunofluorescent analysis (IFA) and reverse transcription-PCR (RT-PCR). The presence of HCMV genomes was determined by PCR, nested PCR (n-PCR), and fluorescence in situ hybridization (FISH). Compared to the HCMV latency model, THP-1, Towne-infected T98Gs expressed IE1 and latency-associated transcripts for longer periods, contained many more HCMV genomes during early passages, and carried genomes for a greatly extended period of passaging. Large numbers of HCMV genomes were also found in purified Ag(-) AD169-infected cells for the first several passages. Interestingly, latency transcripts were observed from very early times in the Towne-infected cells, even when IE1 was expressed at low levels. Although AD169-infected Ag(-) cells expressed no detectable levels of either IE1 or latency transcripts, they also maintained large numbers of genomes within the cell nuclei for several passages. These results identify HCMV-infected T98Gs as an attractive new model in the study of the long-term maintenance of virus genomes in the context of neural cell types. Our previous work showed that T98G glioblastoma cells were semipermissive to HCMV infection; virus trafficked to the nucleus, and yet only a proportion of cells stained positive for viral antigens, thus allowing continual subculturing and passaging. The cells eventually transitioned to a state where viral genomes were maintained without viral antigen expression or virion production. Here we report that during long-term T98G infection, large numbers of genomes were maintained within all of the cells' nuclei for the first several passages (through passage 4 [P4]), even in the presence of continual cellular division. Surprisingly, genomes were maintained, albeit at a lower level, through day 41. This is decidedly longer than in any other latency model system that has been described to date. We believe that this system offers a useful model to aid in unraveling the cellular components involved in viral genome maintenance (and presumably replication) in cells carrying long-term latent genomes in a neural context.

Characterization of Neuronal Populations in the Human Trigeminal Ganglion and Their Association with Latent Herpes Simplex Virus-1 Infection

Following primary infection Herpes simplex virus-1 (HSV-1) establishes lifelong latency in the neurons of human sensory ganglia. Upon reactivation HSV-1 can cause neurological diseases such as facial palsy, vestibular neuritis or encephalitis. Certain populations of sensory neurons have been shown to be more susceptible to latent infection in the animal model, but this has not been addressed in human tissue. In the present study, trigeminal ganglion (TG) neurons expressing six neuronal marker proteins were characterized, based on staining with antibodies against the GDNF family ligand receptor Ret, the high-affinity nerve growth factor receptor TrkA, neuronal nitric oxide synthase (nNOS), the antibody RT97 against 200kDa neurofilament, calcitonin gene-related peptide and peripherin. The frequencies of marker-positive neurons and their average neuronal sizes were assessed, with TrkA-positive (61.82%) neurons being the most abundant, and Ret-positive (26.93%) the least prevalent. Neurons positive with the antibody RT97 (1253 µm2) were the largest, and those stained against peripherin (884 µm2) were the smallest. Dual immunofluorescence revealed at least a 4.5% overlap for every tested marker combination, with overlap for the combinations TrkA/Ret, TrkA/RT97 and Ret/nNOS lower, and the overlap between Ret/CGRP being higher than would be expected by chance. With respect to latent HSV-1 infection, latency associated transcripts (LAT) were detected using in situ hybridization (ISH) in neurons expressing each of the marker proteins. In contrast to the mouse model, co-localization with neuronal markers Ret or CGRP mirrored the magnitude of these neuron populations, whereas for the other four neuronal markers fewer marker-positive cells were also LAT-ISH+. Ret and CGRP are both known to label neurons related to pain signaling.

Interactions between Herpesvirus Entry Mediator (TNFRSF14) and Latency-Associated Transcript during Herpes Simplex Virus 1 Latency

Herpesvirus entry mediator (HVEM) is one of several cell surface proteins herpes simplex virus (HSV) uses for attachment/entry. HVEM regulates cellular immune responses and can also increase cell survival. Interestingly, latency-associated transcript (LAT), the only viral gene consistently expressed during neuronal latency, enhances latency and reactivation by promoting cell survival and by helping the virus evade the host immune response. However, the mechanisms of these LAT activities are not well understood. We show here for the first time that one mechanism by which LAT enhances latency and reactivation appears to be by upregulating HVEM expression. HSV-1 latency/reactivation was significantly reduced in Hvem(-/-) mice, indicating that HVEM plays a significant role in HSV-1 latency/reactivation. Furthermore, LAT upregulated HVEM expression during latency in vivo and also when expressed in vitro in the absence of other viral factors. This study suggests a mechanism whereby LAT upregulates HVEM expression potentially through binding of two LAT small noncoding RNAs to the HVEM promoter and that the increased HVEM then leads to downregulation of immune responses in the latent microenvironment and increased survival of latently infected cells. Thus, one of the mechanisms by which LAT enhances latency/reactivation appears to be through increasing expression of HVEM.

Expression of the herpes simplex virus type 1 latency-associated transcripts does not influence latency establishment of virus mutants deficient for neuronal replication

Herpes simplex virus type 1 establishes latency within neurons of the trigeminal ganglion. During latency, viral gene expression is largely restricted to the latency-associated transcripts (LATs), which, whilst not essential for any aspect of latency, function to suppress lytic gene expression and enhance the survival of virus-infected neurons. The latent cell population comprises primary-order neurons infected directly from peripheral tissues and cells infected following further virus spread within the ganglion. In order to assess the role of LAT expression on latency establishment within first-order neurons, we infected ROSA26R reporter mice with Cre recombinase-expressing recombinant viruses harbouring deletion of the thymidine kinase lytic gene and/or the core LAT promoter. We found that LAT expression did not impact on latency establishment in viruses unable to replicate in neurons, and under these conditions, it was not required for the survival of neurons between 3 and 31 days post-infection.

Regulation of Human Cytomegalovirus Transcription in Latency: Beyond the Major Immediate-Early Promoter

Lytic infection of differentiated cell types with human cytomegalovirus (HCMV) results in the temporal expression of between 170–200 open reading frames (ORFs). A number of studies have demonstrated the temporal regulation of these ORFs and that this is orchestrated by both viral and cellular mechanisms associated with the co-ordinated recruitment of transcription complexes and, more recently, higher order chromatin structure. Importantly, HCMV, like all herpes viruses, establishes a lifelong latent infection of the host—one major site of latency being the undifferentiated haematopoietic progenitor cells resident in the bone marrow. Crucially, the establishment of latency is concomitant with the recruitment of cellular enzymes that promote extensive methylation of histones bound to the major immediate early promoter. As such, the repressive chromatin structure formed at the major immediate early promoter (MIEP) elicits inhibition of IE gene expression and is a major factor involved in maintenance of HCMV latency. However, it is becoming increasingly clear that a distinct subset of viral genes is also expressed during latency. In this review, we will discuss the mechanisms that control the expression of these latency-associated transcripts and illustrate that regulation of these latency-associated promoters is also subject to chromatin mediated regulation and that the instructive observations previously reported regarding the negative regulation of the MIEP during latency are paralleled in the regulation of latent gene expression.

Current State of Marek's Disease Virus MicroRNA Research

MicroRNA (miRNA) is a major family of small RNAs that posttranscriptionally regulate gene expression. Small RNA profiling studies have revealed that some viruses, particularly large DNA viruses, such as Marek's disease virus (MDV), encode their own set of miRNAs. There are currently 406 viral miRNAs in miRBase, of which 392 are encoded by herpesviruses. To date, 26 MDV-1 miRNAs, 36 MDV-2 miRNAs, and 28 herpesvirus of turkeys miRNAs have been identified. Interestingly, herpesvirus miRNAs appear to have spatial conservation, located in clusters within repeat regions, but lack sequence conservation. Two clusters of MDV-1 miRNA have been identified, one located near the MEQ gene and one within the latency-associated transcript (LAT). miRNA profiling studies have shown that MDV miRNA are differentially expressed between strains and stages of infection. For example, mdv1-miR-M4 and mdv1-miR-M2-3p are three- and sixfold higher, expressed, respectively, in vv+ strains compared to vv strains. A recent study found that deletion or seed region mutation of mdv1-miR-M4 reduces viral oncogenicity, suggesting a link between mdv1-mir-M4 and lymphoma development in MDV-infected birds. Taken together, current research suggests that viral miRNAs are a key component of MDV pathogenesis.

Small non-coding RNAs encoded within the herpes simplex virus type 1 latency associated transcript (LAT) cooperate with the retinoic acid inducible gene I (RIG-I) to induce beta-interferon promoter activity and promote cell survival

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected trigeminal ganglionic sensory neurons. Expression of the first 1.5kb of LAT coding sequences restores wild type reactivation to a LAT null HSV-1 mutant. The anti-apoptosis functions of the first 1.5kb of LAT coding sequences are important for wild type levels of reactivation from latency. Two small non-coding RNAs (sncRNAs) contained within the first 1.5kb of LAT coding sequences are expressed in trigeminal ganglia of latently infected mice, they cooperate to inhibit apoptosis, and reduce the efficiency of productive infection. In this study, we demonstrated that LAT sncRNA1 cooperates with the RNA sensor, retinoic acid inducible gene I (RIG-I), to stimulate IFN-β promoter activity and NF-κB dependent transcription in human or mouse cells. LAT sncRNA2 stimulated RIG-I induction of NF-κB dependent transcription in mouse neuroblastoma cells (Neuro-2A) but not human 293 cells. Since it is well established that NF-κB interferes with apoptosis, we tested whether the sncRNAs cooperated with RIG-I to inhibit apoptosis. In Neuro-2A cells, both sncRNAs cooperated with RIG-I to inhibit cold-shock induced apoptosis. Double stranded RNA (PolyI:C) stimulates RIG-I dependent signaling; but enhanced cold-shock induced apoptosis. PolyI:C, but not LAT sncRNAs, interfered with protein synthesis when cotransfected with RIG-I, which correlated with increased levels of cold-shock induced apoptosis. LAT sncRNA1 appeared to interact with RIG-I in transiently transfected cells suggesting this interaction stimulates RIG-I.

Mutational Inactivation of Herpes Simplex Virus 1 MicroRNAs Identifies Viral mRNA Targets and Reveals Phenotypic Effects in Culture

Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, expresses several viral microRNAs (miRNAs). These, along with the latency-associated transcript, represent the only viral RNAs detectable in latently infected neuronal cells. Here, for the first time, we analyze which HSV-1 miRNAs are loaded into the RNA-induced silencing complex (RISC), the key effector of miRNA function. Only 9 of the 17 reported HSV-1 miRNAs, i.e., miR-H1 to miR-H8 plus miR-H11, were found to actually load into the RISC. Surprisingly, this analysis also revealed that HSV-1 miRNAs loaded into the RISC with efficiencies that differed widely; <1% of the miR-H1-3p miRNA detectable in HSV-1-infected cells was loaded into the RISC. Analysis of HSV-1 mutants individually lacking the viral miR-H2, miR-H3, or miR-H4 miRNA revealed that loss of these miRNAs affected the rate of replication of HSV-1 in neuronal cells but not in fibroblasts. Analysis of mRNA and protein expression, as well as assays mapping viral miRNA binding sites in infected cells, showed that endogenous HSV-1 miR-H2 binds to viral ICP0 mRNA and inhibits its expression, while endogenous miR-H4 inhibits the expression of the viral ICP34.5 gene. In contrast, no viral mRNA target for miR-H3 could be detected, even though miR-H3, like miR-H4, is perfectly complementary to ICP34.5 mRNA. Together, these data demonstrate that endogenous HSV-1 miRNA expression can significantly alter viral replication in culture, and they also identify two viral mRNA targets for miR-H2 and miR-H4 that can partially explain this phenotype.

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.

The half-life of the HSV-1 1.5-kb LAT intron is similar to the half-life of the 2.0-kb LAT intron

Herpes simplex virus type 1 establishes a latent infection in the sensory neurons of the peripheral nervous system of humans. Although about 80 genes are expressed during the lytic cycle of the virus infection, essentially only one gene is expressed during the latent cycle. This gene is known as the latency-associated transcript (LAT), and it appears to play a role in the latency cycle through an anti-apoptotic function in the 5' end of the gene and miRNA encoded along the length of the transcript which downregulate some of the viral immediate-early gene products. The LAT gene is about 8.3 kb long and consists of two exons separated by an unusual intron. The intron between the exons consists of two nested introns. This arrangement of introns has been called a twintron. Furthermore, the larger (2 kb) intron has been shown to be very stable. In this study, we measure the stability of the shorter 1.5-kb nested intron and find its half-life is similar to the longer intron. This was achieved by deleting the 0.5-kb overlapping intron from a plasmid construct designed to express the LAT transcript from a tet-inducible promoter and measuring the half-life of the 1.5-kb intron in tissue culture cells. This finding supports the hypothesis that it is the common branch-point region of these nested introns that is responsible for their stability.

Kinetics of Facultative Heterochromatin and Polycomb Group Protein Association with the Herpes Simplex Viral Genome during Establishment of Latent Infection

ABSTRACTThe herpes simplex virus (HSV) genome is associated with heterochromatic histone modifications, including trimethylation of the lysine 27 residue of histone H3 (H3K27me3), during latent infection of neurons. Here we have examined the kinetics of general chromatin and H3K27me3 association with the viral genome during establishment of latent infection. Using both wild-type virus and a mutant virus that is unable to undergo replication in neurons, we found that histone H3 associates with viral gene promoters by 7 days postinfection (dpi). Levels of H3K27me3 were low at 7 dpi but increased dramatically by 14 dpi. Hence, general chromatin association and/or other factors may play a key role(s) in the initial silencing of lytic genes, and H3K27me3 may play a role in further suppression of the genome and/or the maintenance of latency. A component of Polycomb repressive complex 2 (PRC2), which mediates the addition of K27me3 to histone H3 (Suz12), was also recruited by 14 dpi. We have shown previously that the levels of H3K27me3 during latent infection are increased in the presence of the latency-associated transcript (LAT). However, the initial targeting of PRC2 was not found to be dependent on the LAT. We found that a component of the PRC1 complex (Bmi1), which binds to H3K27me3, was not enriched at promoters found previously to be enriched for H3K27me3. Our results are consistent with (i) chromatinization of viral DNA or other mechanisms causing the initial silencing of HSV lytic genes and (ii) facultative heterochromatin maintaining that silencing during latent infection of neurons.