DNA Damage Meets Neurotrophin Signaling: A Delicate Balancing AKT to Maintain Virus Latency.

In this issue of Molecular Cell, Hu et al. (2019) discover that coordinated regulation of AKT activity emanating from neurotrophic-factor stimulation and endogenous DNA damage maintains HSV latency. These studies provide novel insights into the role of AKT in integrating multiple signals to maintain neuronal homeostasis.

c-Jun signaling during initial HSV-1 infection modulates latency to enhance later reactivation in addition to directly promoting the progression to full reactivation.

Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates to permit transmission, which can result in clinical manifestations. Viral transactivators required for lytic infection are largely absent during latent infection, and therefore, HSV-1 relies on the co-option of neuronal host signaling pathways to initiate its gene expression. The activation of the neuronal c-Jun N-terminal kinase (JNK) cell stress pathway is central to initiating biphasic reactivation in response to multiple stimuli. However, how host factors work with JNK to stimulate the initial wave of gene expression (known as Phase I) or the progression to full Phase II reactivation remains unclear. Here, we found that c-Jun, the primary target downstream of neuronal JNK cell stress signaling, functions during reactivation but not during the JNK-mediated initiation of Phase I gene expression. Instead, c-Jun was required to transition from Phase I to full HSV-1 reactivation and was detected in viral replication compartments of reactivating neurons. Interestingly, we also identified a role for both c-Jun and enhanced neuronal stress during initial neuronal infection in promoting a more reactivation-competent form of HSV-1 latency. Therefore, c-Jun functions at multiple stages during the HSV latent infection of neurons to promote reactivation but not during the initial JNK-dependent Phase I. Importantly, by demonstrating that initial infection conditions can contribute to later reactivation abilities, this study highlights the potential for latently infected neurons to maintain a molecular scar of previous exposure to neuronal stressors.IMPORTANCEThe molecular mechanisms that regulate the reactivation of herpes simplex virus-1 (HSV-1) from latent infection are unknown. The host transcription and pioneer factor c-Jun is the main target of the JNK cell stress pathway that is known to be important in exit of HSV from latency. Surprisingly, we found that c-Jun does not act with JNK during exit from latency but instead promotes the transition to full reactivation. Moreover, c-Jun and enhanced neuronal stress during initial neuronal infection promoted a more reactivation-competent form of HSV-1 latency. c-Jun, therefore, functions at multiple stages during HSV-1 latent infection of neurons to promote reactivation. Importantly, this study contributes to a growing body of evidence that de novo HSV-1 infection conditions can modulate latent infection and impact future reactivation events, raising important questions on the clinical impact of stress during initial HSV-1 acquisition on future reactivation events and consequences.

The harms of promoting the lab leak hypothesis for SARS-CoV-2 origins without evidence.

Science is humanity's best insurance against threats from nature, but it is a fragile enterprise that must be nourished and protected. The preponderance of scientific evidence indicates a natural origin for SARS-CoV-2. Yet, the theory that SARS-CoV-2 was engineered in and escaped from a lab dominates media attention, even in the absence of strong evidence. We discuss how the resulting anti-science movement puts the research community, scientific research, and pandemic preparedness at risk.

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.

The Intersection of Innate Immune Pathways with the Latent Herpes Simplex Virus Genome.

Innate immune responses can impact different stages of viral life cycles. Herpes simplex virus latent infection of neurons and subsequent reactivation provide a unique context for immune responses to intersect with different stages of infection. Here, we discuss recent findings linking neuronal innate immune pathways with the modulation of latent infection, acting at the time of reactivation and during initial neuronal infection to have a long-term impact on the ability of the virus to reactivate.

HSV-1 miRNAs are post-transcriptionally edited in latently infected human ganglia.

IMPORTANCE: Herpes simplex virus 1 is an important human pathogen that has been intensively studied for many decades. Nevertheless, the molecular mechanisms regulating its establishment, maintenance, and reactivation from latency are poorly understood. Here, we show that HSV-1-encoded miR-H2 is post-transcriptionally edited in latently infected human tissues. Hyperediting of viral miRNAs increases the targeting potential of these miRNAs and may play an important role in regulating latency. We show that the edited miR-H2 can target ICP4, an essential viral protein. Interestingly, we found no evidence of hyperediting of its homolog, miR-H2, which is expressed by the closely related virus HSV-2. The discovery of post-translational modifications of viral miRNA in the latency phase suggests that these processes may also be important for other non-coding viral RNA in the latency phase, including the intron LAT, which in turn may be crucial for understanding the biology of this virus.

Ex Vivo Herpes Simplex Virus Reactivation Involves a Dual Leucine Zipper Kinase-Dependent Wave of Lytic Gene Expression That Is Independent of Histone Demethylase Activity and Viral Genome Synthesis.

Herpes simplex virus 1 (HSV-1) maintains a lifelong latent infection in neurons and periodically reactivates, resulting in the production of infectious virus. The exact cellular pathways that induce reactivation are not understood. In primary neuronal models of HSV latency, the cellular protein dual leucine zipper kinase (DLK) has been found to initiate a wave of viral gene expression known as phase I. Phase I occurs independently of both viral DNA replication and the activities of histone demethylase enzymes required to remove repressive heterochromatin modifications associated with the viral genome. In this study, we investigated whether phase I-like gene expression occurs in ganglia reactivated from infected mice. Using the combined trigger of explant-induced axotomy and inhibition of phosphatidylinositide 3-kinase (PI3K) signaling, we found that HSV lytic gene expression was induced rapidly from both sensory and sympathetic neurons. Ex vivo reactivation involved a wave of viral late gene expression that occurred independently of viral genome synthesis and histone demethylase activity and preceded the detection of infectious virus. Importantly, we found that DLK was required for the initial induction of lytic gene expression. These data confirm the essential role of DLK in inducing HSV-1 gene expression from the heterochromatin-associated genome and further demonstrate that HSV-1 gene expression during reactivation occurs via mechanisms that are distinct from lytic replication. IMPORTANCE Reactivation of herpes simplex virus from a latent infection is associated with clinical disease. To develop new therapeutics that prevent reactivation, it is important to understand how viral gene expression initiates following a reactivation stimulus. Dual leucine zipper kinase (DLK) is a cellular protein that has previously been found to be required for HSV reactivation from sympathetic neurons in vitro. Here, we show that DLK is essential for reactivation from sensory ganglia isolated from infected mice. Furthermore, we show that DLK-dependent gene expression ex vivo occurs via mechanisms that are distinct from production replication, namely, lytic gene expression that is independent of viral DNA replication and histone demethylase activity. The identification of a DLK-dependent wave of lytic gene expression from sensory ganglia will ultimately permit the development of novel therapeutics that target lytic gene expression and prevent the earliest stage of reactivation.

DLK-Dependent Biphasic Reactivation of Herpes Simplex Virus Latency Established in the Absence of Antivirals.

Understanding the molecular mechanisms of herpes simplex virus 1 (HSV-1) latent infection and reactivation in neurons requires the use of in vitro model systems. Establishing a quiescent infection in cultured neurons is problematic, as any infectious virus released can superinfect the cultures. Previous studies have used the viral DNA replication inhibitor acyclovir to prevent superinfection and promote latency establishment. Data from these previous models have shown that reactivation is biphasic, with an initial phase I expression of all classes of lytic genes, which occurs independently of histone demethylase activity and viral DNA replication but is dependent on the cell stress protein DLK. Here, we describe a new model system using HSV-1 Stayput-GFP, a reporter virus that is defective for cell-to-cell spread and establishes latent infections without the need for acyclovir. The establishment of a latent state requires a longer time frame than previous models using DNA replication inhibitors. This results in a decreased ability of the virus to reactivate using established inducers, and as such, a combination of reactivation triggers is required. Using this system, we demonstrate that biphasic reactivation occurs even when latency is established in the absence of acyclovir. Importantly, phase I lytic gene expression still occurs in a histone demethylase and viral DNA replication-independent manner and requires DLK activity. These data demonstrate that the two waves of viral gene expression following HSV-1 reactivation are independent of secondary infection and not unique to systems that require acyclovir to promote latency establishment. IMPORTANCE Herpes simplex virus-1 (HSV-1) enters a latent infection in neurons and periodically reactivates. Reactivation manifests as a variety of clinical symptoms. Studying latency and reactivation in vitro is invaluable, allowing the molecular mechanisms behind both processes to be targeted by therapeutics that reduce the clinical consequences. Here, we describe a novel in vitro model system using a cell-to-cell spread-defective HSV-1, known as Stayput-GFP, which allows for the study of latency and reactivation at the single neuron level. We anticipate this new model system will be an incredibly valuable tool for studying the establishment and reactivation of HSV-1 latent infection in vitro. Using this model, we find that initial reactivation events are dependent on cellular stress kinase DLK but independent of histone demethylase activity and viral DNA replication. Our data therefore further validate the essential role of DLK in mediating a wave of lytic gene expression unique to reactivation.

PML-NB-dependent type I interferon memory results in a restricted form of HSV latency.

Herpes simplex virus (HSV) establishes latent infection in long-lived neurons. During initial infection, neurons are exposed to multiple inflammatory cytokines but the effects of immune signaling on the nature of HSV latency are unknown. We show that initial infection of primary murine neurons in the presence of type I interferon (IFN) results in a form of latency that is restricted for reactivation. We also find that the subnuclear condensates, promyelocytic leukemia nuclear bodies (PML-NBs), are absent from primary sympathetic and sensory neurons but form with type I IFN treatment and persist even when IFN signaling resolves. HSV-1 genomes colocalize with PML-NBs throughout a latent infection of neurons only when type I IFN is present during initial infection. Depletion of PML prior to or following infection does not impact the establishment latency; however, it does rescue the ability of HSV to reactivate from IFN-treated neurons. This study demonstrates that viral genomes possess a memory of the IFN response during de novo infection, which results in differential subnuclear positioning and ultimately restricts the ability of genomes to reactivate.

Restarting Lytic Gene Transcription at the Onset of Herpes Simplex Virus Reactivation.

Herpes simplex virus (HSV) establishes a latent reservoir in neurons of human peripheral nerves. In this quiescent state, the viral genome persists as a circular, histone-associated episome, and transcription of viral lytic cycle genes is largely suppressed through epigenetic processes. Periodically, latent virus undergoes reactivation whereby lytic genes are activated and viral replication occurs. In this Gem, we review recent evidence that mechanisms governing the initial transcription of lytic genes are distinct from those of de novo infection and directly link reactivation to neuronal stress response pathways. We also discuss evidence that lytic cycle gene expression can be uncoupled from the full reactivation program, arguing for a less sharply bimodal definition of latency.

Physiological oxygen concentration during sympathetic primary neuron culture improves neuronal health and reduces HSV-1 reactivation.

Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates in response to a stimulus to permit transmission. In vitro models using primary neurons are invaluable to studying latent infection because they use bona fide neurons that have undergone differentiation and maturation in vivo. However, culture conditions in vitro should remain as close to those in vivo as possible. This is especially important when considering minimizing cell stress, as it is a well-known trigger of HSV reactivation. We recently developed an HSV-1 model system that requires neurons to be cultured for extended lengths of time. Therefore, we sought to refine culture conditions to optimize neuronal health and minimize secondary effects on latency and reactivation. Here, we demonstrate that culturing primary neurons under conditions closer to physiological oxygen concentrations (5% oxygen) results in cultures with features consistent with reduced stress. Furthermore, culture in these lower oxygen conditions diminishes the progression to full HSV-1 reactivation despite minimal impacts on latency establishment and earlier stages of HSV-1 reactivation. We anticipate that our findings will be useful for the broader microbiology community as they highlight the importance of considering physiological oxygen concentration in studying host-pathogen interactions.

Co-option of mitochondrial nucleic acid sensing pathways by HSV-1 UL12.5 for reactivation from latent Infection.

Although viruses subvert innate immune pathways for their replication, there is evidence they can also co-opt anti-viral responses for their benefit. The ubiquitous human pathogen, Herpes Simplex Virus-1 (HSV-1), encodes a protein (UL12.5) that induces the release of mitochondrial nucleic acid into the cytosol, which activates immune sensing pathways and reduces productive replication in non-neuronal cells. HSV-1 establishes latency in neurons and can reactivate to cause disease. We found that UL12.5 is required for HSV-1 reactivation in neurons and acts to directly promote viral lytic gene expression during initial exit from latency. Further, the direct activation of innate immune sensing pathways triggered HSV reactivation and compensated for a lack of UL12.5. Finally, we found that the induction of HSV-1 lytic genes during reactivation required intact RNA and DNA sensing pathways, demonstrating that HSV-1 can both respond to and active antiviral nucleic acid sensing pathways to reactivate from a latent infection.

Daxx mediated histone H3.3 deposition on HSV-1 DNA restricts genome decompaction and the progression of immediate-early transcription.

Herpesviruses are ubiquitous pathogens that cause a wide range of disease. Upon nuclear entry, their genomes associate with histones and chromatin modifying enzymes that regulate the progression of viral transcription and outcome of infection. While the composition and modification of viral chromatin has been extensively studied on bulk populations of infected cells by chromatin immunoprecipitation, this key regulatory process remains poorly defined at single-genome resolution. Here we use high-resolution quantitative imaging to investigate the spatial proximity of canonical and variant histones at individual Herpes Simplex Virus 1 (HSV-1) genomes within the first 90 minutes of infection. We identify significant population heterogeneity in the stable enrichment and spatial proximity of canonical histones (H2A, H2B, H3.1) at viral DNA (vDNA) relative to established promyelocytic leukaemia nuclear body (PML-NB) host factors that are actively recruited to viral genomes upon nuclear entry. We show the replication-independent histone H3.3/H4 chaperone Daxx to cooperate with PML to mediate the enrichment and spatial localization of variant histone H3.3 at vDNA that limits the rate of HSV-1 genome decompaction to restrict the progress of immediate-early (IE) transcription. This host response is counteracted by the viral ubiquitin ligase ICP0, which degrades PML to disperse Daxx and variant histone H3.3 from vDNA to stimulate the progression of viral genome expansion, IE transcription, and onset of HSV-1 replication. Our data support a model of intermediate and sequential histone assembly initiated by Daxx that limits the rate of HSV-1 genome decompaction independently of the stable enrichment of histones H2A and H2B at vDNA required to facilitate canonical nucleosome assembly. We identify HSV-1 genome decompaction upon nuclear infection to play a key role in the initiation and functional outcome of HSV-1 lytic infection, findings pertinent to the transcriptional regulation of many nuclear replicating herpesvirus pathogens.

Single-genome analysis reveals a 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. IMPORTANCE: Investigating the potential mechanisms of gene silencing for DNA viruses in different cell types is important to understand the differential outcomes of infection, particularly for viruses like herpesviruses that can undergo distinct types of infection in different cell types. In addition, investigating chromatin association with viral genomes informs on the mechanisms of epigenetic regulation of DNA processes. However, there is a growing appreciation for heterogeneity in the outcome of infection at the single cell, and even single viral genome, level. Here we describe a novel assay for quantifying viral genome foci with chromatin proteins and show that a portion of genomes are targeted for silencing by H3K27me2 and associate with the reader protein PHF20L1. This study raises important questions regarding the mechanism of H3K27me2-specific targeting to viral genomes, the contribution of epigenetic heterogeneity to herpesvirus infection, and the role of PHF20L1 in regulating the outcome of DNA virus infection.

c-Jun Signaling During Initial HSV-1 Infection Modulates Latency to Enhance Later Reactivation in addition to Directly Promoting the Progression to Full Reactivation.

Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and can periodically reactivate to permit transmission and clinical manifestations. Viral transactivators required for lytic infection are largely absent during latent infection and therefore HSV-1 relies on the co-option of neuronal host signaling pathways to initiate its gene expression. Activation of the neuronal c-Jun N-terminal kinase (JNK) cell stress pathway is central to initiating biphasic reactivation in response to multiple stimuli. However, how host factors work with JNK to stimulate the initial wave of gene expression (known as Phase I) or the progression to full, Phase II reactivation remains unclear. Here, we found that c-Jun, the primary target downstream of neuronal JNK cell stress signaling, functions during reactivation but not during the JNK-mediated initiation of Phase I gene expression. Instead, c-Jun was required for the transition from Phase I to full HSV-1 reactivation and was detected in viral replication compartments of reactivating neurons. Interestingly, we also identified a role for both c-Jun and enhanced neuronal stress during initial neuronal infection in promoting a more reactivation-competent form of HSV-1 latency. Therefore, c-Jun functions at multiple stages during HSV latent infection of neurons to promote reactivation. Importantly, by demonstrating that initial infection conditions can contribute to later reactivation abilities, this study highlights the potential for latently infected neurons to maintain a molecular scar of previous exposure to neuronal stressors.

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. In 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. This 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.

Herpes simplex virus VP16, but not ICP0, is required to reduce histone occupancy and enhance histone acetylation on viral genomes in U2OS osteosarcoma cells.

The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure.

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.