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

ABSTRACT The 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

Single-genome analysis reveals heterogeneous association of the herpes simplex virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts

The fate of herpesvirus genomes following entry into different cell types is thought to regulate the outcome of infection. For the Herpes simplex virus 1 (HSV-1), latent infection of neurons is characterized by association with repressive heterochromatin marked with Polycomb silencing-associated lysine 27 methylation on histone H3 (H3K27me). However, whether H3K27 methylation plays a role in repressing lytic gene expression in non-neuronal cells is unclear. To address this gap in knowledge, and with consideration that the fate of the viral genome and outcome of HSV-1 infection could be heterogeneous, we developed an assay to quantify the abundance of histone modifications within single viral genome foci of infected fibroblasts. Using this approach, combined with bulk epigenetic techniques, we were unable to detect any role for H3K27me3 during HSV-1 lytic infection of fibroblasts. By contrast, we could detect the lesser studied H3K27me2 on a subpopulation of viral genomes, which was consistent with a role for H3K27 demethylases in promoting lytic gene expression. In addition, viral genomes co-localized with the H3K27me2 reader protein PHF20L1, and this association was enhanced by inhibition of the H3K27 demethylases UTX and JMJD3. Notably, targeting of H3K27me2 to viral genomes was enhanced following infection with a transcriptionally defective virus in the absence of Promyelocytic leukemia nuclear bodies. Collectively, these studies implicate a role for H3K27me2 in fibroblast-associated HSV genome silencing in a manner dependent on genome sub-nuclear localization and transcriptional activity

Neuronal hyperexcitability is a DLK-dependent trigger of Herpes Simplex Virus reactivation that can be induced by IL-1

Herpes simplex virus-1 (HSV-1) establishes a latent infection in neurons and periodically reactivates to cause disease. The stimuli that trigger HSV-1 reactivation have not been fully elucidated. We demonstrate HSV-1 reactivation from latently infected mouse neurons induced by forskolin requires neuronal excitation. Stimuli that directly induce neurons to become hyperexcitable also induced HSV-1 reactivation. Forskolin-induced reactivation was dependent on the neuronal pathway of DLK/JNK activation and included an initial wave of viral gene expression that was independent of histone demethylase activity and linked to histone phosphorylation. IL-1β is released under conditions of stress, fever and UV exposure of the epidermis; all known triggers of clinical HSV reactivation. We found that IL-1β induced histone phosphorylation and increased the excitation in sympathetic neurons. Importantly, IL-1β triggered HSV-1 reactivation, which was dependent on DLK and neuronal excitability. Thus, HSV-1 co-opts an innate immune pathway resulting from IL-1 stimulation of neurons to induce reactivation

Neuronal Stress Pathway Mediating a Histone Methyl/Phospho Switch Is Required for Herpes Simplex Virus Reactivation

Herpes simplex virus (HSV) reactivation from latent neuronal infection requires stimulation of lytic gene expression from promoters associated with repressive heterochromatin. Various neuronal stresses trigger reactivation, but how these stimuli activate silenced promoters remains unknown. We show that a neuronal pathway involving activation of c-Jun N-terminal kinase (JNK), common to many stress responses, is essential for initial HSV gene expression during reactivation. This JNK activation in neurons is mediated by dual leucine zipper kinase (DLK) and JNK-interacting protein 3 (JIP3), which direct JNK towards stress responses instead of other cellular functions. Surprisingly, JNK-mediated viral gene induction occurs independently of histone demethylases that remove repressive lysine modifications. Rather, JNK signaling results in a histone methyl/phospho switch on HSV lytic promoters, a mechanism permitting gene expression in the presence of repressive lysine methylation. JNK is present on viral promoters during reactivation, thereby linking a neuronal-specific stress pathway and HSV reactivation from latency

Virology—the path forward

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US

Mutations in SLC29A3, Encoding an Equilibrative Nucleoside Transporter ENT3, Cause a Familial Histiocytosis Syndrome (Faisalabad Histiocytosis) and Familial Rosai-Dorfman Disease

The histiocytoses are a heterogeneous group of disorders characterised by an excessive number of histiocytes. In most cases the pathophysiology is unclear and treatment is nonspecific. Faisalabad histiocytosis (FHC) (MIM 602782) has been classed as an autosomal recessively inherited form of histiocytosis with similarities to Rosai-Dorfman disease (RDD) (also known as sinus histiocytosis with massive lymphadenopathy (SHML)). To elucidate the molecular basis of FHC, we performed autozygosity mapping studies in a large consanguineous family and identified a novel locus at chromosome 10q22.1. Mutation analysis of candidate genes within the target interval identified biallelic germline mutations in SLC29A3 in the FHC kindred and in two families reported to have familial RDD. Analysis of SLC29A3 expression during mouse embryogenesis revealed widespread expression by e14.5 with prominent expression in the central nervous system, eye, inner ear, and epithelial tissues including the gastrointestinal tract. SLC29A3 encodes an intracellular equilibrative nucleoside transporter (hENT3) with affinity for adenosine. Recently germline mutations in SLC29A3 were also described in two rare autosomal recessive disorders with overlapping phenotypes: (a) H syndrome (MIM 612391) that is characterised by cutaneous hyperpigmentation and hypertrichosis, hepatomegaly, heart anomalies, hearing loss, and hypogonadism; and (b) PHID (pigmented hypertrichosis with insulin-dependent diabetes mellitus) syndrome. Our findings suggest that a variety of clinical diagnoses (H and PHID syndromes, FHC, and familial RDD) can be included in a new diagnostic category of SLC29A3 spectrum disorder

Time to Switch to Second-line Antiretroviral Therapy in Children With Human Immunodeficiency Virus in Europe and Thailand.

Background: Data on durability of first-line antiretroviral therapy (ART) in children with human immunodeficiency virus (HIV) are limited. We assessed time to switch to second-line therapy in 16 European countries and Thailand. Methods: Children aged <18 years initiating combination ART (≥2 nucleoside reverse transcriptase inhibitors [NRTIs] plus nonnucleoside reverse transcriptase inhibitor [NNRTI] or boosted protease inhibitor [PI]) were included. Switch to second-line was defined as (i) change across drug class (PI to NNRTI or vice versa) or within PI class plus change of ≥1 NRTI; (ii) change from single to dual PI; or (iii) addition of a new drug class. Cumulative incidence of switch was calculated with death and loss to follow-up as competing risks. Results: Of 3668 children included, median age at ART initiation was 6.1 (interquartile range (IQR), 1.7-10.5) years. Initial regimens were 32% PI based, 34% nevirapine (NVP) based, and 33% efavirenz based. Median duration of follow-up was 5.4 (IQR, 2.9-8.3) years. Cumulative incidence of switch at 5 years was 21% (95% confidence interval, 20%-23%), with significant regional variations. Median time to switch was 30 (IQR, 16-58) months; two-thirds of switches were related to treatment failure. In multivariable analysis, older age, severe immunosuppression and higher viral load (VL) at ART start, and NVP-based initial regimens were associated with increased risk of switch. Conclusions: One in 5 children switched to a second-line regimen by 5 years of ART, with two-thirds failure related. Advanced HIV, older age, and NVP-based regimens were associated with increased risk of switch

Herpes Simplex Virus ICP0 Promotes both Histone Removal and Acetylation on Viral DNA during Lytic Infection▿

During lytic infection, the genome of herpes simplex virus 1 (HSV-1) is associated with limited levels of histones but does not form a regular repeating nucleosomal structure. However, the previous observation that chromatin remodeling factors are recruited into viral replication compartments indicates that chromatin remodeling plays a role in HSV-1 gene expression and DNA replication. In this study we demonstrate the presence of histone H3 on HSV-1 DNA early in infection at levels equivalent to those found on a cellular gene. The proportion of viral DNA associated with histone H3 decreases at later times postinfection, independently of either viral DNA replication or transcription. We demonstrate that an immediate-early protein, infected cell protein 0 (ICP0), is required for both a reduction in the proportion of HSV-1 DNA associating with histone H3 and an increase in histone acetylation. This study provides evidence that ICP0 directly alters the chromatin structure of the HSV-1 genome during lytic infection, and this system will serve as a useful model for the reduction of histone load in higher eukaryotes

De Novo Polycomb Recruitment: Lessons from Latent Herpesviruses

The Human Herpesviruses persist in the form of a latent infection in specialized cell types. During latency, the herpesvirus genomes associate with cellular histone proteins and the viral lytic genes assemble into transcriptionally repressive heterochromatin. Although there is divergence in the nature of heterochromatin on latent herpesvirus genomes, in general, the genomes assemble into forms of heterochromatin that can convert to euchromatin to permit gene expression and therefore reactivation. This reversible form of heterochromatin is known as facultative heterochromatin and is most commonly characterized by polycomb silencing. Polycomb silencing is prevalent on the cellular genome and plays a role in developmentally regulated and imprinted genes, as well as X chromosome inactivation. As herpesviruses initially enter the cell in an un-chromatinized state, they provide an optimal system to study how de novo facultative heterochromatin is targeted to regions of DNA and how it contributes to silencing. Here, we describe how polycomb-mediated silencing potentially assembles onto herpesvirus genomes, synergizing what is known about herpesvirus latency with facultative heterochromatin targeting to the cellular genome. A greater understanding of polycomb silencing of herpesviruses will inform on the mechanism of persistence and reactivation of these pathogenic human viruses and provide clues regarding how de novo facultative heterochromatin forms on the cellular genome

Selective Uptake of Small RNA Molecules in the Virion of Murine Gammaherpesvirus 68▿

Noncoding RNAs are a feature of many herpesvirus genomes. They include microRNAs, whose function is the subject of intense investigation, in addition to longer RNA molecules such as the Epstein-Barr virus-encoded RNAs and herpesvirus saimiri U RNAs, which have been known for some time but whose function is still not well defined. Murine gammaherpesvirus 68 (MHV-68) encodes eight viral tRNA-like molecules (vtRNAs) of unknown function. Investigating the kinetics of expression of the vtRNAs, we observed that they were present directly after infection with the virus. This strongly suggested that vtRNAs were part of the virion structure, which was confirmed by their detection within various purified, RNase-treated preparations. Although both viral and cellular mRNAs were also detected within the MHV-68 virion, the major RNA species present were small RNAs of around 70 nucleotides in length. Interestingly, incorporation of viral mRNA was not related to the relative abundance in infected cells, as M11 mRNA, which is present at low abundance, was found in virions. MHV-76, which lacks the genes encoding the vtRNAs, also incorporated small RNA molecules within the virion, suggesting a requirement for these molecules for virion maturation. In productively infected cells the vtRNAs localized predominantly within the cytoplasm, although they also exhibited a globular pattern of nuclear staining. Their presence in the cytoplasm is consistent with interaction with virion components prior to maturation of virus particles. The significance of these findings for virion architecture and function is discussed