scholarly journals The DNA Sensor cGAS is Decorated by Acetylation and Phosphorylation Modifications in the Context of Immune Signaling

2020 ◽  
Vol 19 (7) ◽  
pp. 1193-1208 ◽  
Author(s):  
Bokai Song ◽  
Todd M. Greco ◽  
Krystal K. Lum ◽  
Caroline E. Taber ◽  
Ileana M. Cristea
Keyword(s):  
Cyclic Gmp ◽  
Single Point ◽  
Cell Types ◽  
Post Translational Modification ◽  
Immune Signaling ◽  
Signaling Axis ◽  
Primary Fibroblasts ◽  
Elevated Surface ◽  
Simplex Virus ◽  
Hsv 1

The cyclic GMP-AMP synthase (cGAS) protein is a pattern-recognition receptor of the mammalian innate immune system that is recognized as a main cytosolic sensor of pathogenic or damaged DNA. cGAS DNA binding initiates catalytic production of the second messenger, cyclic GMP-AMP, which activates the STING-TBK1-IRF3 signaling axis to induce cytokine expression. Post-translational modification (PTM) has started to be recognized as a critical component of cGAS regulation, yet the extent of these modifications remains unclear. Here, we report the identification and functional analysis of cGAS phosphorylations and acetylations in several cell types under basal and immune-stimulated conditions. cGAS was enriched by immunoaffinity purification from human primary fibroblasts prior to and after infection with herpes simplex virus type 1 (HSV-1), as well as from immune-stimulated STING-HEK293T cells. Six phosphorylations and eight acetylations were detected, of which eight PTMs were not previously documented. PTMs were validated by parallel reaction monitoring (PRM) mass spectrometry in fibroblasts, HEK293T cells, and THP-1 macrophage-like cells. Primary sequence and structural analysis of cGAS highlighted a subset of PTM sites with elevated surface accessibility and high evolutionary sequence conservation. To assess the functional relevance of each PTM, we generated a series of single-point cGAS mutations. Stable cell lines were constructed to express cGAS with amino acid substitutions that prevented phosphorylation (Ser-to-Ala) and acetylation (Lys-to-Arg) or that mimicked the modification state (Ser-to-Asp and Lys-to-Gln). cGAS-dependent apoptotic and immune signaling activities were then assessed for each mutation. Our results show that acetyl-mimic mutations at Lys384 and Lys414 inhibit the ability of cGAS to induce apoptosis. In contrast, the Lys198 acetyl-mimic mutation increased cGAS-dependent interferon signaling when compared with the unmodified charge-mimic. Moreover, targeted PRM quantification showed that Lys198 acetylation is decreased upon infections with two herpesviruses—HSV-1 and human cytomegalovirus (HCMV), highlighting this residue as a regulatory point during virus infection.

scholarly journals Herpes Simplex Virus 1 UL24 Abrogates the DNA Sensing Signal Pathway by Inhibiting NF-κB Activation

2017 ◽  
Vol 91 (7) ◽  
Author(s):  
Haiyan Xu ◽  
Chenhe Su ◽  
Angela Pearson ◽  
Christopher H. Mody ◽  
Chunfu Zheng
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cyclic Gmp ◽  
Nuclear Translocation ◽  
Signal Pathway ◽  
Promoter Activation ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
First Time ◽  
Hsv 1

ABSTRACT Cyclic GMP-AMP synthase (cGAS) is a newly identified DNA sensor that recognizes foreign DNA, including the genome of herpes simplex virus 1 (HSV-1). Upon binding of viral DNA, cGAS produces cyclic GMP-AMP, which interacts with and activates stimulator of interferon genes (STING) to trigger the transcription of antiviral genes such as type I interferons (IFNs), and the production of inflammatory cytokines. HSV-1 UL24 is widely conserved among members of the herpesviruses family and is essential for efficient viral replication. In this study, we found that ectopically expressed UL24 could inhibit cGAS-STING-mediated promoter activation of IFN-β and interleukin-6 (IL-6), and UL24 also inhibited interferon-stimulatory DNA-mediated IFN-β and IL-6 production during HSV-1 infection. Furthermore, UL24 selectively blocked nuclear factor κB (NF-κB) but not IFN-regulatory factor 3 promoter activation. Coimmunoprecipitation analysis demonstrated that UL24 bound to the endogenous NF-κB subunits p65 and p50 in HSV-1-infected cells, and UL24 was also found to bind the Rel homology domains (RHDs) of these subunits. Furthermore, UL24 reduced the tumor necrosis factor alpha (TNF-α)-mediated nuclear translocation of p65 and p50. Finally, mutational analysis revealed that the region spanning amino acids (aa) 74 to 134 of UL24 [UL24(74–134)] is responsible for inhibiting cGAS-STING-mediated NF-κB promoter activity. For the first time, UL24 was shown to play an important role in immune evasion during HSV-1 infection. IMPORTANCE NF-κB is a critical component of the innate immune response and is strongly induced downstream of most pattern recognition receptors (PRRs), leading to the production of IFN-β as well as a number of inflammatory chemokines and interleukins. To establish persistent infection, viruses have evolved various mechanisms to counteract the host NF-κB pathway. In the present study, for the first time, HSV-1 UL24 was demonstrated to inhibit the activation of NF-κB in the DNA sensing signal pathway via binding to the RHDs of the NF-κB subunits p65 and p50 and abolishing their nuclear translocation.

scholarly journals Herpes Simplex Virus ICP0 Mutants Are Hypersensitive to Interferon

2000 ◽  
Vol 74 (4) ◽  
pp. 2052-2056 ◽  
Author(s):  
Karen L. Mossman ◽  
Holly A. Saffran ◽  
James R. Smiley
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Types ◽  
Wild Type Virus ◽  
Osteosarcoma Cell ◽  
Virion Host Shutoff ◽  
Virion Host Shutoff Protein ◽  
Severe Reduction ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Interferon (IFN) is an important immune system molecule capable of inducing an antiviral state within cells. Herpes simplex virus type 1 (HSV-1) replication is somewhat reduced in tissue culture in the presence of IFN, presumably due to decreased viral transcription. Here, we show mutations that inactivate immediate-early (IE) gene product ICP0 render HSV-1 exquisitely sensitive to IFN inhibition, resulting in greatly decreased levels of viral mRNA transcripts and the resulting polypeptides and a severe reduction in plaque formation ability. Mutations in other HSV-1 genes, including the genes coding for virion transactivator VP16 and the virion host shutoff protein vhs, IE gene ICP22, and the protein kinase UL13 gene, do not increase the IFN sensitivity of HSV-1. Interestingly, ICP0 mutants demonstrate the same level of sensitivity to IFN as wild-type virus on U2OS cells, an osteosarcoma cell line that is known to complement mutations in ICP0 and VP16. Thus, in some cell types, functional ICP0 is required for HSV-1 to efficiently bypass the inhibitory effects of IFN in order to ensure its replication. The significance of this link between ICP0 and IFN resistance is discussed.

scholarly journals A Protein Encoded by the Herpes Simplex Virus (HSV) Type 1 2-Kilobase Latency-Associated Transcript Is Phosphorylated, Localized to the Nucleus, and Overcomes the Repression of Expression from Exogenous Promoters When Inserted into the Quiescent HSV Genome

2002 ◽  
Vol 76 (8) ◽  
pp. 4056-4067 ◽  
Author(s):  
S. K. Thomas ◽  
C. E. Lilley ◽  
D. S. Latchman ◽  
R. S. Coffin
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Types ◽  
Current Evidence ◽  
Virus Growth ◽  
Exact Function ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus (HSV) is characterized by its ability to establish a latent infection in sensory neurons, from which it can periodically reactivate. The mechanisms of latency, however, remain unclear. The HSV genome is quiescent during latency except for the expression of the latency-associated transcripts (LATs). Although the exact function of the LATs remains obscure, current evidence suggests they are multifunctional and are involved in both establishment of latency and reactivation from latency. The LATs contain several open reading frames (ORFs). One or more of the functions of the LATs could therefore be protein mediated. We have previously reported that deregulated expression of the largest of the HSV type 1 (HSV-1) LAT ORFs (∼274 amino acids) greatly enhances virus growth in cell types that are normally relatively nonpermissive for HSV replication and also that it complements mutations to the immediate-early (IE) gene ICP0 (S. K. Thomas, G. Gough, D. S. Latchman, and R. S. Coffin, J. Virol. 73:6618-6625, 1999). Here we show that LAT ORF expression overcomes the repression of expression from exogenous promoters introduced into the HSV-1 genome which normally occurs in the absence of IE gene expression. To further explore LAT ORF function, we have generated an epitope-tagged LAT ORF, LATmycHis, which forms punctate structures in the infected-cell nucleus reminiscent of the structures formed by ICP0. These are associated with the appearance of a phosphorylated form of the protein and are formed adjacent to, or around the edges of, viral replication compartments. These results provide further evidence that the HSV-1 LAT ORF protein is biologically functional and that the tightly regulated expression of this protein may be important in the wild-type latency phenotype in vivo.

scholarly journals Activity and intracellular localization of the human cytomegalovirus protein pp71

2002 ◽  
Vol 83 (7) ◽  
pp. 1601-1612 ◽  
Author(s):  
Ker R. Marshall ◽  
Kate V. Rowley ◽  
Angela Rinaldi ◽  
Iain P. Nicholson ◽  
Alexander M. Ishov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Intracellular Localization ◽  
Cell Types ◽  
Independent Manner ◽  
Nuclear Domain ◽  
Protein Icp0 ◽  
Simplex Virus ◽  
Hsv 1

The human cytomegalovirus (HCMV) tegument phosphoprotein pp71 activates viral immediate early (IE) transcription and thus has a role in initiating lytic infection. Protein pp71 stimulates expression from a range of promoters in a sequence-independent manner, and in this respect behaves similarly to the herpes simplex virus type 1 (HSV-1) IE protein ICP0. The intracellular localization of pp71 was investigated after its expression from transfected plasmids or from HSV-1 mutants constructed to produce pp71 transiently. The protein colocalized with the cell promyelocytic leukaemia (PML) protein at nuclear domain 10 (ND10) structures but, unlike ICP0, pp71 did not induce disruption of ND10. The activity of pp71 in mouse sensory neurons in vivo was investigated after co-inoculation of animals with pairs of HSV-1 mutants, one expressing pp71 and the second containing the E. coli lacZ gene controlled by various promoters. In this system, pp71 stimulated β-galactosidase expression from a range of viral IE promoters when mice were analysed at 4 days postinoculation. At later times, expression of pp71 resulted in a reduction in numbers of neurons containing β-galactosidase, indicating cytotoxicity or promoter shutoff. The HSV-1 latency-active promoter was not responsive to pp71, demonstrating specificity in the activity of the protein. Pp71 was as active in mice lacking both copies of the PML gene (PML−/−) as in control animals, and in PML−/− fibroblasts pp71 stimulated gene expression as effectively as in other cell types. Therefore, neither the PML protein nor the normal ND10 structure is necessary for pp71 to stimulate gene expression.

scholarly journals Herpesvirus Entry Mediator HVEM Mediates Cell-Cell Spread in BHK(TK−) Cell Clones

1998 ◽  
Vol 72 (2) ◽  
pp. 1411-1417 ◽  
Author(s):  
Richard J. Roller ◽  
Daniel Rauch
Keyword(s):  
Cell Lines ◽  
Pseudorabies Virus ◽  
Cell Types ◽  
Single Step ◽  
Cell Clones ◽  
Step Growth ◽  
Simplex Virus ◽  
Cell Spread ◽  
Hsv 1 ◽  
Cell Cell

ABSTRACT 95-19 and US11cl19.3 are BHK(TK−)-derived cell lines that are highly resistant to postattachment entry of herpes simplex virus type 1 (HSV-1) and HSV-2 but not to later steps in single-step replication. The resistance properties of these two cell types are not identical. US11cl19.3 cells are fully susceptible to pseudorabies virus (PRV), as shown by single-step growth experiments, whereas 95-19 cells are resistant to entry of free PRV but not to entry by cell-cell spread. We have tested the ability of HVEM to overcome the block to infection in both cell lines following transient and stable transfection. HVEM was able to mediate entry of free HSV-1 into both cell lines, as shown by an increase in the number of β-galactosidase-expressing cells in cultures transiently transfected with an HVEM expression plasmid and infected withlacZ-expressing HSV-1. In stably transfected 95-19 cells, HVEM enhanced infection by free HSV-1, as shown by an increase in the number of infectious centers obtained following infection. In both cell types, HVEM strongly enhanced entry of HSV-1 and HSV-2 by cell-cell spread, suggesting that HVEM can function as an entry mediator both in entry of free virus and in entry by cell-cell spread.

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 The ESCRT-Related ATPase Vps4 Is Modulated by Interferon during Herpes Simplex Virus 1 Infection

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Jorge Ruben Cabrera ◽  
Richard Manivanh ◽  
Brian J. North ◽  
David A. Leib
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Infections ◽  
Cell Types ◽  
Primary Neurons ◽  
Herpes Simplex Virus 1 ◽  
Escrt Machinery ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTInterferons (IFNs) and autophagy are critical neuronal defenses against viral infection. IFNs alter neuronal autophagy by promoting the accumulation of IFN-dependent LC3-decorated autophagic structures, termed LC3 clusters. Here, we analyzed LC3 clusters in sensory ganglia following herpes simplex virus 1 (HSV-1) infection. In the vicinity of acutely infected neurons, antigen-negative neurons contained structures resembling accumulated autophagosomes and autolysosomes that culminated in LC3 clusters. This accumulation reflects a delayed completion of autophagy. Theendosomalsortingcomplexesrequired fortransport (ESCRT) machinery participates in autophagosome closure and is also required for HSV-1 replication. In this study, our results showed that HSV-1 infectionin vivoand in primary neurons caused a decrease in Vps4 (a key ESCRT pathway ATPase) RNA and protein with concomitant Stat1 activation and LC3 cluster induction. We also observed that IFNs were sufficient to decrease RNA and protein levels of Vps4 in primary neurons and in other cell types. The accumulation of ubiquitin was also observed at the LC3 cluster sites. Together, our results show that IFNs modulate the ESCRT machinery in neurons in response to HSV-1 infections.IMPORTANCENeurons rely on IFNs and autophagy as major defenses against viral infections, and HSV must overcome such defenses in order to replicate. In addition to controlling host immunity, HSV must also control host membranes in order to complete its life cycle. HSV uses the host ESCRT membrane scission machinery for viral production and transport. Here we present evidence of a new IFN-dependent mechanism used by the host to prevent ESCRT subversion by HSV. This activity also impacts the dynamics of autophagy, possibly explaining the presence of recently described LC3 clusters in the HSV-infected nervous system. The induced accumulations of ubiquitin observed in these LC3 clusters resembled those observed in certain neurodegenerative diseases, suggesting possible mechanistic parallels between these conditions.

scholarly journals Axin expression enhances herpes simplex virus type 1 replication by inhibiting virus-mediated cell death in L929 cells

2013 ◽  
Vol 94 (7) ◽  
pp. 1636-1646 ◽  
Author(s):  
Eun-Jin Choi ◽  
Sewoon Kim ◽  
Eek-hoon Jho ◽  
Ki-Joon Song ◽  
Sun-Ho Kee
Keyword(s):  
Cell Death ◽  
Herpes Simplex ◽  
Viral Replication ◽  
Cell Types ◽  
L929 Cells ◽  
Rgs Domain ◽  
Simplex Virus ◽  
Axin Expression ◽  
Hsv 1

Herpes simplex virus type 1 (HSV-1) replicates in various cell types and induces early cell death, which limits viral replication in certain cell types. Axin is a scaffolding protein that regulates Wnt signalling and participates in various cellular events, including cellular proliferation and cell death. The effects of axin expression on HSV-1 infection were investigated based on our initial observation that Wnt3a treatment or axin knockdown reduced HSV-1 replication. L929 cells expressed the axin protein in a doxycycline-inducible manner (L-axin) and enhanced HSV-1 replication in comparison to control cells (L-EV). HSV-1 infection induced cell death as early as 6 h after infection through the necrotic pathway and required de novo protein synthesis in L929 cells. Subsequent analysis of viral protein expression suggested that axin expression led to suppression of HSV-1-induced premature cell death, resulting in increased late gene expression. In analysis of axin deletion mutants, the regulators of the G-protein signalling (RGS) domain were involved in the axin-mediated enhancement of viral replication and reduction in cell death. These results suggest that viral replication enhancement might be mediated by the axin RGS domain.

scholarly journals Histone deacetylase inhibitors reduce the number of herpes simplex virus-1 genomes initiating expression in individual cells

2016 ◽  
Author(s):  
Shapira Lev ◽  
Ralph Maya ◽  
Tomer Enosh ◽  
Cohen Shai ◽  
Kobiler Oren
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Types ◽  
Nuclear Bodies ◽  
Host Proteins ◽  
Herpes Simplex Virus 1 ◽  
Viral Genomes ◽  
Early Protein ◽  
Simplex Virus ◽  
Hsv 1

AbstractAlthough many viral particles can enter a single cell, the number of viral genomes per cell that establish infection is limited. However, mechanisms underlying this restriction were not explored in depth. For herpesviruses, one of the possible mechanisms suggested is chromatinization and silencing of the incoming genomes. To test this hypothesis, we followed infection with three herpes simplex virus 1 (HSV-1) fluorescence-expressing recombinants in the presence or absence of histone deacetylases inhibitors (HDACi’s). Unexpectedly, a lower number of viral genomes initiated expression in the presence of these inhibitors. This phenomenon was observed using several HDACi: Trichostatin A (TSA), Suberohydroxamic Acid (SBX), Valporic Acid (VPA) and Suberoylanilide Hydoxamic Acid (SAHA). We found that HDACi presence did not change the progeny outcome from the infected cells but did alter the kinetic of the infection. Different cell types (HFF, Vero and U2OS), which vary in their capability to activate intrinsic and innate immunity, show a cell specific basal average number of viral genomes establishing infection. Importantly, in all cell types, treatment with TSA reduced the number of viral genomes. ND10 nuclear bodies are known to interact with the incoming herpes genomes and repress viral replication. The viral immediate early protein, ICP0, is known to disassemble the ND10 bodies and to induce degradation of some of the host proteins in these domains. HDACi treated cells expressed higher levels of some of the host ND10 proteins (PML and ATRX), which may down regulate the number of viral genomes initiating expression per cell. Corroborating this hypothesis, infection with three HSV-1 recombinants carrying a deletion in the gene coding for ICP0, show a reduction in the number of genomes being expressed in U2OS cells. We suggest that alterations in the levels of host proteins involved in intrinsic antiviral defense may result in differences in the number of genomes that initiate expression.

scholarly journals Anterograde Neuronal Circuit Tracers Derived from Herpes Simplex Virus 1: Development, Application, and Perspectives

2020 ◽  
Vol 21 (16) ◽  
pp. 5937
Author(s):  
Dong Li ◽  
Hong Yang ◽  
Feng Xiong ◽  
Xiangmin Xu ◽  
Wen-Bo Zeng ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Technological Development ◽  
Cell Types ◽  
Neuronal Circuit ◽  
Herpes Simplex Virus 1 ◽  
Special Value ◽  
Simplex Virus ◽  
Hsv 1

Herpes simplex virus type 1 (HSV-1) has great potential to be applied as a viral tool for gene delivery or oncolysis. The broad infection tropism of HSV-1 makes it a suitable tool for targeting many different cell types, and its 150 kb double-stranded DNA genome provides great capacity for exogenous genes. Moreover, the features of neuron infection and neuron-to-neuron spread also offer special value to neuroscience. HSV-1 strain H129, with its predominant anterograde transneuronal transmission, represents one of the most promising anterograde neuronal circuit tracers to map output neuronal pathways. Decades of development have greatly expanded the H129-derived anterograde tracing toolbox, including polysynaptic and monosynaptic tracers with various fluorescent protein labeling. These tracers have been applied to neuroanatomical studies, and have contributed to revealing multiple important neuronal circuits. However, current H129-derived tracers retain intrinsic drawbacks that limit their broad application, such as yet-to-be improved labeling intensity, potential nonspecific retrograde labeling, and high toxicity. The biological complexity of HSV-1 and its insufficiently characterized virological properties have caused difficulties in its improvement and optimization as a viral tool. In this review, we focus on the current H129-derived viral tracers and highlight strategies in which future technological development can advance its use as a tool.

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