scholarly journals Single-cell RNA-sequencing of herpes simplex virus 1-infected cells connects NRF2 activation to an antiviral program

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Emanuel Wyler ◽  
Vedran Franke ◽  
Jennifer Menegatti ◽  
Christine Kocks ◽  
Anastasiya Boltengagen ◽  
...  
Keyword(s):  
Host Cell ◽  
Transition Probabilities ◽  
Virus Production ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Herpes Simplex Virus 1 ◽  
Analysis Scheme ◽  
Primary Fibroblasts ◽  
Simplex Virus ◽  
Hsv 1

Abstract Herpesvirus infection initiates a range of perturbations in the host cell, which remain poorly understood at the level of individual cells. Here, we quantify the transcriptome of single human primary fibroblasts during the first hours of lytic infection with HSV-1. By applying a generalizable analysis scheme, we define a precise temporal order of early viral gene expression and propose a set-wise emergence of viral genes. We identify host cell genes and pathways relevant for infection by combining three different computational approaches: gene and pathway overdispersion analysis, prediction of cell-state transition probabilities, as well as future cell states. One transcriptional program, which correlates with increased resistance to infection, implicates the transcription factor NRF2. Consequently, Bardoxolone methyl and Sulforaphane, two known NRF2 agonists, impair virus production, suggesting that NRF2 activation restricts viral infection. Our study provides insights into early stages of HSV-1 infection and serves as a general blueprint for the investigation of heterogeneous cell states in virus infection.

scholarly journals Single-cell RNA-sequencing of Herpes simplex virus 1-infected cells identifies NRF2 activation as an antiviral program

2019 ◽  
Author(s):  
Emanuel Wyler ◽  
Vedran Franke ◽  
Jennifer Menegatti ◽  
Kocks Christine ◽  
Anastasiya Boltengagen ◽  
...  
Keyword(s):  
Host Cell ◽  
Transition Probabilities ◽  
Virus Production ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Herpes Simplex Virus 1 ◽  
Nrf2 Activation ◽  
Analysis Scheme ◽  
Infected Cells ◽  
Hsv 1

AbstractHerpesvirus infection initiates a range of perturbations in the host cell, which remain poorly understood at the level of individual cells. Here, we quantified the transcrips of single human primary fibroblasts during the first hours of lytic infection with HSV-1. By applying a generalizable analysis scheme, we defined a precise temporal order of early viral gene expression and found unexpected bifurcations and bottlenecks. We identified individual host cell genes and pathways relevant in early infection by combining three different computational approaches: gene and pathway overdispersion analysis, prediction of cell-state transition probabilities as well as future cell states. One transcriptional program, which was turned on in infected cells and correlated with increased resistance to infection, implicated the transcription factor NRF2. Consequently, Bardoxolone methyl, a known NRF2 agonist, impaired virus production, suggesting that NRF2 activation restricts the progression of viral infection. Our study provides novel insights into early stages of HSV-1 infection and serves as a general blueprint for the investigation of heterogenous cell states in virus infection.

scholarly journals Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Benjamin A. Diner ◽  
Krystal K. Lum ◽  
Jared E. Toettcher ◽  
Ileana M. Cristea
Keyword(s):  
Gene Expression ◽  
Live Cell Imaging ◽  
Nuclear Periphery ◽  
Viral Gene Expression ◽  
Live Cell ◽  
Viral Gene ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTThe human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses.IMPORTANCEHow mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16 rapidly oligomerizes at incoming herpesvirus genomes at the nuclear periphery to transcriptionally repress viral gene expression and limit viral replicative capacity. We further demonstrate that IFI16 does not initiate upstream activation of the canonical STING/TBK-1/IRF3 signaling pathway but is required for downstream antiviral cytokine expression. In contrast, we find that, upon DNA sensing during herpesvirus infection, cGAS triggers apoptosis in a STING-dependent manner. Our live-cell imaging, mass spectrometry-based proteomics, CRISPR-based cellular assays, and optogenetics underscore the value of integrative approaches to uncover complex cellular responses against pathogens.

scholarly journals Differential Expression of Immune Checkpoint Molecules on CD8+ T Cells Specific for Immunodominant and Subdominant Herpes Simplex Virus 1 Epitopes

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Kate L. Carroll ◽  
Lyndsay Avery ◽  
Benjamin R. Treat ◽  
Lawrence P. Kane ◽  
Paul R. Kinchington ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Viral Gene Expression ◽  
Viral Reactivation ◽  
Viral Gene ◽  
Herpes Simplex Virus 1 ◽  
Latently Infected ◽  
Checkpoint Molecule ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) causes a lifelong infection of neurons that innervate barrier sites like the skin and mucosal surfaces like the eye. After primary infection of the cornea, the virus enters latency within the trigeminal ganglion (TG), from which it can reactivate throughout the life of the host. Viral latency is maintained, in part, by virus-specific CD8+ T cells that nonlethally interact with infected neurons. When CD8+ T cell responses are inhibited, HSV-1 can reactivate, and these recurrent reactivation events can lead to blinding scarring of the cornea. In the C57BL/6 mouse, CD8+ T cells specific for the immunodominant epitope from glycoprotein B maintain functionality throughout latency, while CD8+ T cells specific for subdominant epitopes undergo functional impairment that is associated with the expression of the inhibitory checkpoint molecule programmed death 1 (PD-1). Here, we investigate the checkpoint molecule T cell immunoglobulin and mucin domain-containing 3 (Tim-3), which has traditionally been associated with CD8+ T cell exhaustion. Unexpectedly, we found that Tim-3 was preferentially expressed on highly functional ganglionic CD8+ T cells during acute and latent HSV-1 infection. This, paired with data that show that Tim-3 expression on CD8+ T cells in the latently infected TG is influenced by viral gene expression, suggests that Tim-3 is an indicator of recent T cell stimulation, rather than functional compromise, in this model. We conclude that Tim-3 expression is not sufficient to define functional compromise during latency; however, it may be useful in identifying activated cells within the TG during HSV-1 infection. IMPORTANCE Without an effective means of eliminating HSV-1 from latently infected neurons, efforts to control the virus have centered on preventing viral reactivation from latency. Virus-specific CD8+ T cells within the infected TG have been shown to play a crucial role in inhibiting viral reactivation, and with a portion of these cells exhibiting functional impairment, checkpoint molecule immunotherapies have presented a potential solution to enhancing the antiviral response of these cells. In pursuing this potential treatment strategy, we found that Tim-3 (often associated with CD8+ T cell functional exhaustion) is not upregulated on impaired cells but instead is upregulated on highly functional cells that have recently received antigenic stimulation. These findings support a role for Tim-3 as a marker of activation rather than exhaustion in this model, and we provide additional evidence for the hypothesis that there is persistent viral gene expression in the HSV-1 latently infected TG.

scholarly journals Regulation of viral gene expression by the herpes simplex virus 1 UL24 protein (HSV-1 UL24 inhibits accumulation of viral transcripts)

Virology ◽  
2016 ◽  
Vol 495 ◽  
pp. 148-160 ◽  
Author(s):  
Carolina Sanabria-Solano ◽  
Carmen Elena Gonzalez ◽  
Nicolas Richerioux ◽  
Luc Bertrand ◽  
Slimane Dridi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Hsv 1

scholarly journals Dynamic Response of IFI16 and Promyelocytic Leukemia Nuclear Body Components to Herpes Simplex Virus 1 Infection

2015 ◽  
Vol 90 (1) ◽  
pp. 167-179 ◽  
Author(s):  
Roger D. Everett
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Genome ◽  
Viral Gene Expression ◽  
Nuclear Body ◽  
Viral Gene ◽  
Intrinsic Resistance ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTIntrinsic immunity is an aspect of antiviral defense that operates through diverse mechanisms at the intracellular level through a wide range of constitutively expressed cellular proteins. In the case of herpesviruses, intrinsic resistance involves the repression of viral gene expression during the very early stages of infection, a process that is normally overcome by viral tegument and/or immediate-early proteins. Thus, the balance between cellular repressors and virus-counteracting proteins determines whether or not a cell becomes productively infected. One aspect of intrinsic resistance to herpes simplex virus 1 (HSV-1) is conferred by components of promyelocytic leukemia nuclear bodies (PML NBs), which respond to infection by accumulating at sites that are closely associated with the incoming parental HSV-1 genomes. Other cellular proteins, including IFI16, which has been implicated in sensing pathogen DNA and initiating signaling pathways that lead to an interferon response, also respond to viral genomes in this manner. Here, studies of the dynamics of the response of PML NB components and IFI16 to invading HSV-1 genomes demonstrated that this response is extremely rapid, occurring within the first hour after addition of the virus, and that human Daxx (hDaxx) and IFI16 respond more rapidly than PML. In the absence of HSV-1 regulatory protein ICP0, which counteracts the recruitment process, the newly formed, viral-genome-induced PML NB-like foci can fuse with existing PML NBs. These data are consistent with a model involving viral genome sequestration into such structures, thereby contributing to the low probability of initiation of lytic infection in the absence of ICP0.IMPORTANCEHerpesviruses have intimate interactions with their hosts, with infection leading either to the productive lytic cycle or to a quiescent infection in which viral gene expression is suppressed while the viral genome is maintained in the host cell nucleus. Whether a cell becomes lytically or quiescently infected can be determined through the competing activities of cellular repressors and viral activators, some of which counteract cell-mediated repression. Therefore, the events that occur within the earliest stages of infection can be of crucial importance. This paper describes the extremely rapid response to herpes simplex virus 1 infection of cellular protein IFI16, a sensor of pathogen DNA, and also of the PML nuclear body proteins PML and hDaxx, as revealed by live-cell microscopy. The data imply that these proteins can accumulate on or close to the viral genomes in a sequential manner which may lead to their sequestration and repression.

scholarly journals RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression

2020 ◽  
Vol 94 (22) ◽  
Author(s):  
Daniel J. Kim ◽  
William Khoury-Hanold ◽  
Priyanka Caroline Jain ◽  
Jonathan Klein ◽  
Yong Kong ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Sites ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Drg Neurons ◽  
Herpes Simplex Virus 1 ◽  
Viral Genes ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) and HSV-2 can efficiently establish lifelong, transcriptionally silent latency states in sensory neurons to escape host detection. While host factors have previously been associated with long-range insulators in the viral genome, it is still unknown whether host transcription factors can repress viral genes more proximately to promote latency in dorsal root ganglion (DRG) neurons. Here, we assessed whether RUNX (runt-related transcription factor) transcription factors, which are critical in the development of sensory neurons, could be binding HSV-1 genome directly to suppress viral gene expression and lytic infection. Using previously published transcriptome sequencing data, we confirmed that mouse DRG neurons highly express Runx1 mRNA. Through computational analysis of HSV-1 and HSV-2 genomes, we observed that putative RUNX consensus binding sites (CBSs) were more enriched and more closely located to viral gene transcription start sites than would be expected by chance. We further found that RUNX CBSs were significantly more enriched among genomes of herpesviruses compared to those of nonherpesviruses. Utilizing an in vitro model of HSV-1 infection, we found that overexpressed RUNX1 could bind putative binding sites in the HSV-1 genome, repress numerous viral genes spanning all three kinetic classes, and suppress productive infection. In contrast, knockdown of RUNX1 in neuroblastoma cells induced viral gene expression and increased HSV-1 infection in vitro. In sum, these data support a novel role for RUNX1 in directly binding herpesvirus genome, silencing the transcription of numerous viral genes, and ultimately limiting overall infection. IMPORTANCE Infecting 90% of the global population, HSV-1 and HSV-2 represent some of the most prevalent viruses in the world. Much of their success can be attributed to their ability to establish lifelong latent infections in the dorsal root ganglia (DRG). It is still largely unknown, however, how host transcription factors are involved in establishing this latency. Here, we report that RUNX1, expressed highly in DRG, binds HSV-1 genome, represses transcription of numerous viral genes, and suppresses productive in vitro infection. Our computational work further suggests this strategy may be used by other herpesviruses to reinforce latency in a cell-specific manner.

scholarly journals Infection by Herpes Simplex Virus 1 Causes Near-Complete Loss of RNA Polymerase II Occupancy on the Host Cell Genome

2015 ◽  
Vol 90 (5) ◽  
pp. 2503-2513 ◽  
Author(s):  
Robert G. Abrisch ◽  
Tess M. Eidem ◽  
Petro Yakovchuk ◽  
Jennifer F. Kugel ◽  
James A. Goodrich
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Polymerase ◽  
Host Cell ◽  
Rna Polymerase Ii ◽  
Viral Gene ◽  
Herpes Simplex Virus 1 ◽  
Pol Ii ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTLytic infection by herpes simplex virus 1 (HSV-1) triggers a change in many host cell programs as the virus strives to express its own genes and replicate. Part of this process is repression of host cell transcription by RNA polymerase II (Pol II), which also transcribes the viral genome. Here, we describe a global characterization of Pol II occupancy on the viral and host genomes in response to HSV-1 infection using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). The data reveal near-complete loss of Pol II occupancy throughout host cell mRNA genes, in both their bodies and promoter-proximal regions. Increases in Pol II occupancy of host cell genes, which would be consistent with robust transcriptional activation, were not observed. HSV-1 infection induced a more potent and widespread repression of Pol II occupancy than did heat shock, another cellular stress that widely represses transcription. Concomitant with the loss of host genome Pol II occupancy, we observed Pol II covering the HSV-1 genome, reflecting a high level of viral gene transcription. Interestingly, the positions of the peaks of Pol II occupancy at HSV-1 and host cell promoters were different. The primary peak of Pol II occupancy at HSV-1 genes is ∼170 bp upstream of where it is positioned at host cell genes, suggesting that specific steps in transcription are regulated differently at HSV-1 genes than at host cell mRNA genes.IMPORTANCEWe investigated the effect of herpes simplex virus 1 (HSV-1) infection on transcription of host cell and viral genes by RNA polymerase II (Pol II). The approach we used was to determine how levels of genome-bound Pol II changed after HSV-1 infection. We found that HSV-1 caused a profound loss of Pol II occupancy across the host cell genome. Increases in Pol II occupancy were not observed, showing that no host genes were activated after infection. In contrast, Pol II occupied the entire HSV-1 genome. Moreover, the pattern of Pol II at HSV-1 genes differed from that on host cell genes, suggesting a unique mode of viral gene transcription. These studies provide new insight into how HSV-1 causes changes in the cellular program of gene expression and how the virus coopts host Pol II for its own use.

scholarly journals Cellular Transcriptional Coactivator RanBP10 and Herpes Simplex Virus 1 ICP0 Interact and Synergistically Promote Viral Gene Expression and Replication

2016 ◽  
Vol 90 (6) ◽  
pp. 3173-3186 ◽  
Author(s):  
Yuka Sato ◽  
Akihisa Kato ◽  
Yuhei Maruzuru ◽  
Masaaki Oyama ◽  
Hiroko Kozuka-Hata ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromatin Remodeling ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Null Mutation ◽  
Null Mutant ◽  
Transcriptional Coactivator ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTTo investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) regulatory protein ICP0 promotes viral gene expression and replication, we screened cells overexpressing ICP0 for ICP0-binding host cell proteins. Tandem affinity purification of transiently expressed ICP0 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that ICP0 interacted with cell protein RanBP10, a known transcriptional coactivator, in HSV-1-infected cells. Knockdown of RanBP10 in infected HEp-2 cells resulted in a phenotype similar to that observed with the ICP0-null mutation, including reduction in viral replication and in the accumulation of viral immediate early (ICP27), early (ICP8), and late (VP16) mRNAs and proteins. In addition, RanBP10 knockdown or the ICP0-null mutation increased the level of histone H3 association with the promoters of these viral genes, which is known to repress transcription. These effects observed in wild-type HSV-1-infected HEp-2 RanBP10 knockdown cells or those observed in ICP0-null mutant virus-infected control HEp-2 cells were remarkably increased in ICP0-null mutant virus-infected HEp-2 RanBP10 knockdown cells. Our results suggested that ICP0 and RanBP10 redundantly and synergistically promoted viral gene expression by regulating chromatin remodeling of the HSV-1 genome for efficient viral replication.IMPORTANCEUpon entry of herpesviruses into a cell, viral gene expression is restricted by heterochromatinization of the viral genome. Therefore, HSV-1 has evolved multiple mechanisms to counteract this epigenetic silencing for efficient viral gene expression and replication. HSV-1 ICP0 is one of the viral proteins involved in counteracting epigenetic silencing. Here, we identified RanBP10 as a novel cellular ICP0-binding protein and showed that RanBP10 and ICP0 appeared to act synergistically to promote viral gene expression and replication by modulating viral chromatin remodeling. Our results provide insight into the mechanisms by which HSV-1 regulates viral chromatin remodeling for efficient viral gene expression and replication.

scholarly journals A Functional Interaction between Herpes Simplex Virus 1 Glycoprotein gH/gL Domains I and II and gD Is Defined by Using Alphaherpesvirus gH and gL Chimeras

2015 ◽  
Vol 89 (14) ◽  
pp. 7159-7169 ◽  
Author(s):  
Qing Fan ◽  
Richard Longnecker ◽  
Sarah A. Connolly
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Host Cell ◽  
Current Model ◽  
Viral Envelope ◽  
Functional Interaction ◽  
Herpes Simplex Virus 1 ◽  
Homotypic Interaction ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTWhereas most viruses require only a single protein to bind to and fuse with cells, herpesviruses use multiple glycoproteins to mediate virus entry, and thus communication among these proteins is required. For most alphaherpesviruses, the minimal set of viral proteins required for fusion with the host cell includes glycoproteins gD, gB, and a gH/gL heterodimer. In the current model of entry, gD binds to a cellular receptor and transmits a signal to gH/gL. This signal then triggers gB, the conserved fusion protein, to insert into the target membrane and refold to merge the viral and cellular membranes. We previously demonstrated that gB homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1), were interchangeable. In contrast, neither gD nor gH/gL functioned with heterotypic entry glycoproteins, indicating that gD and gH/gL exhibit an essential type-specific functional interaction. To map this homotypic interaction site on gH/gL, we generated HSV-1/SaHV-1 gH and gL chimeras. The functional interaction with HSV-1 gD mapped to the N-terminal domains I and II of the HSV-1 gH ectodomain. The core of HSV-1 gL that interacts with gH also was required for functional homotypic interaction. The N-terminal gH/gL domains I and II are the least conserved and may have evolved to support species-specific glycoprotein interactions.IMPORTANCEThe first step of the herpesvirus life cycle is entry into a host cell. A coordinated interaction among multiple viral glycoproteins is required to mediate fusion of the viral envelope with the cell membrane. The details of how these glycoproteins interact to trigger fusion are unclear. By swapping the entry glycoproteins of two alphaherpesviruses (HSV-1 and SaHV-1), we previously demonstrated a functional homotypic interaction between gD and gH/gL. To define the gH and gL requirements for homotypic interaction, we evaluated the function of a panel of HSV-1/SaHV-1 gH and gL chimeras. We demonstrate that domains I and II of HSV-1 gH are sufficient to promote a functional, albeit reduced, interaction with HSV-1 gD. These findings contribute to our model of how the entry glycoproteins cooperate to mediate herpesvirus entry into the cell.

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