During Herpes Simplex Virus Type 1 Infection of Rabbits, the Ability To Express the Latency-Associated Transcript Increases Latent-Phase Transcription of Lytic Genes

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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.

Section: Methodsmentioning

confidence: 99%

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

Flores

Nakayama

Whisnant

et al. 2013

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“…As the genome enters the latent phase in the mouse, an accumulation of the histone mark dimethyl H3 lysine 9 (diMe H3K9), historically known as a transcriptionally repressed marker, is observed on lytic genes, while enrichment of the transcriptionally active histone mark dimethyl H3 lysine 4 (diMe H3K4) decreases (24). Furthermore, once the virus has established latency, the LAT region is enriched in the transcriptionally active marks, acetylated histone H3 lysines 9 and 14 and diMe H3K4 in mice and rabbits, respectively, compared to the lytic genes encoding ICP27 and ICP0 (8,13,14). These studies suggest that the virus can associate with cellular enzymes responsible for catalyzing histone posttranslational modifications (PTMs) and that these PTMs on HSV-1 genes can reflect their transcriptional status.…”

mentioning

confidence: 99%

The Polycomb Group Protein Bmi1 Binds to the Herpes Simplex Virus 1 Latent Genome and Maintains Repressive Histone Marks during Latency

Kwiatkowski

Thompson

Bloom

2009

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141

171

The mechanism by which herpes simplex virus 1 (HSV-1) establishes latency in sensory neurons is largely unknown. Recent studies indicate that epigenetic modifications of the chromatin associated with the latent genome may play a key role in the transcriptional control of lytic genes during latency. In this study, we found both constitutive and facultative types of heterochromatin to be present on the latent HSV-1 genome. Deposition of the facultative marks trimethyl H3K27 and histone variant macroH2A varied at different sites on the genome, whereas the constitutive marker trimethyl H3K9 did not. In addition, we show that in the absence of the latency-associated transcript (LAT), the latent genome shows a dramatic increase in trimethyl H3K27, suggesting that expression of the LAT during latency may act to promote an appropriate heterochromatic state that represses lytic genes but is still poised for reactivation. Due to the presence of the mark trimethyl H3K27, we examined whether Polycomb group proteins, which methylate H3K27, were present on the HSV-1 genome during latency. Our data indicate that Bmi1, a member of the Polycomb repressive complex 1 (PRC1) maintenance complex, associates with specific sites in the genome, with the highest level of enrichment at the LAT enhancer. To our knowledge, these are the first data demonstrating that a virus can repress its gene transcription to enter latency by exploiting the mechanism of Polycomb-mediated repression.After primary infection by herpes simplex virus 1 (HSV-1) in mucosal epithelia, the virus enters sensory neurons, where it can persist as a transcriptionally silent extrachromosomal episome (19). The only abundant transcription that occurs during latency is from a diploid gene in the repeat long segment of the virus that produces the latency-associated transcript (LAT), an 8.3-to 8.5-kb noncoding RNA (20, 21). The LAT is detectably expressed in only one-third of neurons that contain latent HSV genomes (9, 16). Following a reactivation stimulus, some of the latent genomes can exit this state to actively express lytic transcripts and produce new virus. The mechanism by which the genome establishes and maintains this latent state is currently unknown, although it is hypothesized that the reversible nature of the latent phase involves the constant interplay between cellular and viral factors that either keep the viral genome repressed or allow for reactivation.Recent studies have shed light on epigenetic modifications to the HSV-1 genome during the establishment and maintenance of latency. As the genome enters the latent phase in the mouse, an accumulation of the histone mark dimethyl H3 lysine 9 (diMe H3K9), historically known as a transcriptionally repressed marker, is observed on lytic genes, while enrichment of the transcriptionally active histone mark dimethyl H3 lysine 4 (diMe H3K4) decreases (24). Furthermore, once the virus has established latency, the LAT region is enriched in the transcriptionally active marks, acetylated histone H3 lysines 9 and 14 and ...

“…HSV-1 genomes are regularly chromatinized during latency, and MCN digestions therefore release fragments of sizes characteristic of nucleosome DNA (20,65). As evaluated by chromatin immunoprecipitation (ChIP), histones bearing posttranslational modifications (PTMs) characteristic of facultative heterochromatin are associated with the transcriptionally restricted latent genomes of HSV-1 and HCMV (2,3,13,25,35,46,51,68,74,80). During reactivation from latency, however, there is a shift from PTMs associated with silencing to PTMs associated with transcription (2).…”

mentioning

confidence: 99%

During Lytic Infections, Herpes Simplex Virus Type 1 DNA Is in Complexes with the Properties of Unstable Nucleosomes

Lacasse

Schang

2010

The genomes of herpes simplex virus type 1 (HSV-1) are regularly chromatinized during latency such that their digestion with micrococcal nuclease (MCN) releases nucleosome-sized DNA fragments. In lytically infected cells, in contrast, MCN releases HSV-1 DNA in primarily heterogeneously sized fragments. Consistently, only a small percentage of this HSV-1 DNA coimmunoprecipitates with histones. Most current models propose that histones associate with HSV-1 DNA during lytic infections at low occupancy. However, histone modification or occupation is also proposed to regulate HSV-1 transcription. It remains unclear how the histones associated with a small percentage of HSV-1 DNA may regulate transcription globally. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. We evaluated the HSV-1 DNA-containing complexes at 5 h after (lytic) infection by biochemical fractionations. Nuclear HSV-1 DNA did not fractionate as protein-free HSV-1 DNA but as DNA in cellular nucleosomes. Moreover, MCN released HSV-1 DNA in complexes that fractionate as cellular mono-and dinucleosomes by centrifugation followed by sucrose gradients and size-exclusion chromatography. The HSV-1 DNA in such complexes was protected to heterogeneous sizes and was more accessible to MCN than DNA in most cellular chromatin. Using a modified MCN digestion to trap unstable digestion intermediates, HSV-1 DNA was quantitatively recovered in discrete mono-to polynucleosome sizes in complexes fractionating as cellular mono-to polynucleosomes. The HSV-1 DNAs in complexes fractionating as mono-to dinucleosomes were stabilized by cross-linking. Therefore, most HSV-1 DNA forms particularly unstable nucleosome-like complexes at 5 h of lytic infection.Herpes simplex virus type 1 (HSV-1) is a double-stranded DNA virus that transcribes and replicates in the nucleus. It establishes lytic infections in epithelial cells and latent infections in neurons in vivo. During lytic infections, all HSV-1 genes are expressed, and the genomes are replicated. During latent infections, HSV-1 gene expression is restricted, and the genomes are not replicated.Nuclear DNA is typically packaged into a nucleoprotein complex, chromatin. Chromatin is a chain of nucleosomes, 146 bp of DNA wrapped 1.75 turns around a histone octamer composed of two copies each of H2A, H2B, H3, and H4 (44). Linker histone H1 binds at entry and exit points and to linker DNA in between nucleosomes, promoting the compaction of chromatin into higher-order structures (31, 60, 61).The structure of chromatin is classically probed with nucleases (69) such as micrococcal (endo)nuclease (MCN). MCN preferentially cleaves exposed linker DNA between nucleosomes (6, 26). Mild MCN digestions first cleave linker DNA sparsely, releasing polynucleosome chains. Longer digestions eventually cleave the polynucleosomes into mononucleosomes. Under even more stringent digestion conditions, MCN eventually degrades even the DNA in mononucleosomes. Normal MCN digestions of regularly chromatinize...