Ex Vivo Herpes Simplex Virus Reactivation Involves a DLK-Dependent Wave of Lytic Gene Expression that is Independent of Histone Demethylase Activity and Viral Genome Synthesis

Whitford, Abigail L; Clinton, Corinne A; Kennedy, Evory; Dochnall, Sara A; Suzich, Jon B.; Cliffe, Anna R.

doi:10.1101/2022.02.25.481951

Cited by 3 publications

(2 citation statements)

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“…HSV-1 gene expression is highly variable within a population of infected cells (28,47). Additionally, heterogeneity in latent HSV-1 infection of neurons has been investigated (9,29,48,49), and variable states of HSV-1 gnome condensation (50,51) and gene activity (52) directly influence the capacity for viral reactivation. Expanding temporal observation of dual reporter HSV-1 infected cells using innovative single-cell culturing techniques will provide unmatched insight into HSV-1 gene expression, replication, and pathogenesis (53).…”

Section: Discussionmentioning

confidence: 99%

A dual-fluorescent recombinant for live observation of Herpes simplex-type 1 infection outcomes

Domanico,

Dunn,

Kobiler

et al. 2023

Preprint

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Critical stages of lytic Herpes simplex type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 also persists in neuronal tissues via a non-replicative, transcriptionally repressed infection called latency. Understanding the regulation of lytic and latent transcriptional profiles provides focused insight into HSV-1 infection and disease. We sought a fluorescence-based approach to observe temporal progression of HSV-1 infection at the single-cell level. We constructed and characterized a novel HSV-1 recombinant that reports IE and L gene expression by fluorescent protein detection. The dual-reporter HSV-1 visualizes IE gene expression by a CMV promotor-driven YFP, and L gene expression by an endogenous mCherry-VP26 fusion. We confirmed that viral gene expression, replication and spread of infection in epithelial cells is not altered by the incorporation of the fluorescent reporters. Interference with viral DNA polymerase activity abolishes VP26-mCherry detection late in HSV-1 infection, visually reporting the failure to complete viral replication. Viral replication in primary neurons is not altered, but retrograde-directed inoculation of the dual-reporter HSV-1 exhibits a modest reduction in titer, compared to unlabeled HSV-1. Low-dose axonal inoculation in the presence of small molecule modulation of neuronal signaling results in divergent outcomes of YFP and mCherry detection, suggesting different states of latent and lytic replication. Rigorous characterization of this dual-reporter HSV-1 recombinant has demonstrated the utility of temporal observation of HSV-1 replication in live cells and the potential for further insight into the dynamics of infection.

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Section: Discussionmentioning

confidence: 99%

A dual-fluorescent recombinant for live observation of Herpes simplex-type 1 infection outcomes

Domanico,

Dunn,

Kobiler

et al. 2023

Preprint

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show abstract

“…Phase I gene expression precedes "full reactivation" (also referred to as "Phase II") as a transcriptional burst of all classes of lytic viral genes, with late gene expression being uncoupled from viral DNA replication. This Phase I gene expression phenomenon has been observed in both in vitro and ex vivo models of HSV reactivation (21,25,28,31,41,42). The use of in vitro model systems has enabled the molecular mechanisms of Phase I and Phase II reactivation to be further teased apart.…”

Section: Introductionmentioning

confidence: 94%

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

Dochnal,

Whitford,

Francois

et al. 2023

Preprint

Self Cite

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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 thatde novoHSV-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.

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Impact of Cultured Neuron Models on α-Herpesvirus Latency Research

Wilson

2022

Viruses

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A signature trait of neurotropic α-herpesviruses (α-HV) is their ability to establish stable non-productive infections of peripheral neurons termed latency. This specialized gene expression program is the foundation of an evolutionarily successful strategy to ensure lifelong persistence in the host. Various physiological stresses can induce reactivation in a subset of latently-infected neurons allowing a new cycle of viral productive cycle gene expression and synthesis of infectious virus. Recurring reactivation events ensure transmission of the virus to new hosts and contributes to pathogenesis. Efforts to define the molecular basis of α-HV latency and reactivation have been notoriously difficult because the neurons harboring latent virus in humans and in experimentally infected live-animal models, are rare and largely inaccessible to study. Increasingly, researchers are turning to cultured neuron infection models as simpler experimental platforms from which to explore latency and reactivation at the molecular level. In this review, I reflect on the strengths and weaknesses of existing neuronal models and briefly summarize the important mechanistic insights these models have provided. I also discuss areas where prioritization will help to ensure continued progress and integration.

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Ex Vivo Herpes Simplex Virus Reactivation Involves a DLK-Dependent Wave of Lytic Gene Expression that is Independent of Histone Demethylase Activity and Viral Genome Synthesis

Cited by 3 publications

References 54 publications

A dual-fluorescent recombinant for live observation of Herpes simplex-type 1 infection outcomes

A dual-fluorescent recombinant for live observation of Herpes simplex-type 1 infection outcomes

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

Impact of Cultured Neuron Models on α-Herpesvirus Latency Research

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