Biology subjects

Dochnal, S. A.

Publications and source records attributed to Dochnal, S. A..

4 recordsLinked to original sources

bioRxiv2023

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. ImportanceInvestigating 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 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.

microbiology↗

bioRxiv2023

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. ImportanceThe molecular mechanisms that regulate the reactivation of HSV-1 from latent infection are unknown. In addition, studies on the mechanisms of reactivation can be complicated by factors that act during latency establishment to ultimately impact reactivation. Here we focused on the host transcription and pioneer factor c-Jun because it is the main target of the JNK cell stress pathway 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 latent infection of neurons to promote reactivation and serves as a relevant therapeutic target for attenuating reactivation and related clinical consequences. 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.

microbiology↗

bioRxiv2023

HSV-1 miRNAs are posttranscriptionaly edited in latently infected human ganglia

Viruses use miRNAs to enable efficient replication, control host defense mechanisms, and regulate latent infection. Herpes simplex virus 1 (HSV-1) expresses multiple miRNAs, whose functions are largely unknown. The evolutionary conservation of many HSV-1 miRNAs in the closely related HSV-2 suggests their functional importance. miRNAs, similar to other transcripts, can undergo various posttranscriptional modifications that may affect their biogenesis, stability and targeting. To investigate whether editing occurs in HSV-1 miRNAs, we sequenced samples from latently infected human ganglia. We show that one of the six HSV-1 miRNAs (miR-H2 to -H8) that define HSV-1 latency, miR-H2, exhibits A-to-I hyperediting within the miRNA seed sequence. We observed the same specific miR-H2 hyperediting phenomenon in miRNAs isolated from the ganglia of latently infected mice and, to a lesser extent, during productive infection in cultured cells. Curiously, we found no evidence of editing of the encoded HSV-2 homolog in latently infected mice or in cultured cells. The efficient loading of the edited miRNAs onto the RISC complex, indicates their ability to function as miRNAs. To investigate the potential of the edited miRNA to alter mRNA targeting, we predicted the host and viral targets for the modified miRNAs. Nucleotide substitution in the seed region significantly increased the number of potential host and viral targets. Most notably, ICP4, an essential viral protein, was predicted to be an additional target. Using transfection assays, we demonstrated that edited miRNAs have the potential to regulate ICP4 in addition to the previously identified target ICP0. Our study identifies a specific hyperedited HSV-1 mRNA, miR-H2, and highlights how the virus can use a single miRNA to target multiple transcripts during persistent, latent infection. ImportanceHerpes simplex virus 1 is an important human pathogen and 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 (miR-H2-e) can target ICP4, an essential viral protein. Interestingly, we found no evidence of hyperdating of its homolog, miR-H2, which is expressed by the closely related virus HSV-2. The discovery of posttranslational 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.

microbiology↗

bioRxiv2022

DLK-dependent biphasic reactivation of herpes simplex virus latency established in the absence of antivirals

Understanding the molecular mechanism of herpes simplex virus type 1 (HSV-1) latent infection and reactivation in neurons requires the use of model systems. However, establishing a quiescent infection in cultured neurons is problematic as infectious virus released from any productively infected neuron can superinfect the cultures. Here, we describe a new reporter virus, HSV-1 Stayput-GFP, that is defective for cell-to-cell spread and can be used to model latency and reactivation at the single neuron level. Importantly, quiescent infection of neurons with Stayput-GFP can be established without the use of a viral DNA replication inhibitor. The establishment of a quiescent state requires a longer time frame than previous models of HSV latency using DNA replication inhibitors. This results in a decreased ability of the virus to reactivate, and the use of multiple reactivation triggers is required. Using this system, we demonstrate that an initial Phase I wave of lytic gene expression occurs independently of histone demethylase activity and viral DNA replication but is dependent on the neuronal cell stress protein, DLK. Progression into the later, Phase II wave of reactivation, characterized by detectable late viral protein synthesis and a requirement for histone demethylase activity, is also observed with the Stayput-GFP model system. These data demonstrate that the two waves of viral gene expression following HSV-1 reactivation are independent of secondary infection and occur when latent infections are established in the absence of a viral DNA replication inhibitor. ImportanceHerpes 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 demonstrate the essential role of DLK in mediating a wave of lytic gene expression unique to reactivation.

microbiology↗