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1.
Nat Commun ; 15(1): 4018, 2024 May 13.
Article in English | MEDLINE | ID: mdl-38740820

ABSTRACT

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.


Subject(s)
Dependovirus , Gene Editing , Herpes Simplex , Herpesvirus 1, Human , Viral Load , Virus Shedding , Animals , Gene Editing/methods , Female , Dependovirus/genetics , Mice , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/physiology , Herpes Simplex/genetics , Herpes Simplex/virology , Herpes Simplex/therapy , Disease Models, Animal , Virus Latency/genetics , Humans , Genetic Vectors/genetics , Vero Cells , Genetic Therapy/methods , Herpes Genitalis/therapy , Herpes Genitalis/virology , DNA, Viral/genetics
2.
Nature ; 628(8009): 844-853, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38570685

ABSTRACT

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.


Subject(s)
Alleles , DNA Polymerase gamma , Encephalitis Viruses, Tick-Borne , Herpesvirus 1, Human , Immune Tolerance , SARS-CoV-2 , Animals , Female , Humans , Male , Mice , Age of Onset , COVID-19/immunology , COVID-19/virology , COVID-19/genetics , DNA Polymerase gamma/genetics , DNA Polymerase gamma/immunology , DNA Polymerase gamma/metabolism , DNA, Mitochondrial/immunology , DNA, Mitochondrial/metabolism , Encephalitis Viruses, Tick-Borne/immunology , Encephalitis, Tick-Borne/genetics , Encephalitis, Tick-Borne/immunology , Encephalitis, Tick-Borne/virology , Founder Effect , Gene Knock-In Techniques , Herpes Simplex/genetics , Herpes Simplex/immunology , Herpes Simplex/virology , Herpesvirus 1, Human/immunology , Immune Tolerance/genetics , Immune Tolerance/immunology , Immunity, Innate/genetics , Immunity, Innate/immunology , Interferon Type I/immunology , Mitochondrial Diseases/enzymology , Mitochondrial Diseases/genetics , Mitochondrial Diseases/immunology , Mutation , RNA, Mitochondrial/immunology , RNA, Mitochondrial/metabolism , SARS-CoV-2/immunology
3.
Shock ; 61(6): 894-904, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38662585

ABSTRACT

ABSTRACT: Objective: We conducted a two-sample bidirectional Mendelian randomization (MR) study to investigate the causal relationships between herpes viruses and sepsis. Methods: Publicly available genome-wide association study data were used. Four viruses, HSV-1, HSV-2, EBV, and CMV, were selected, with serum positivity and levels of antibody in serum as the herpes virus data. Results: In forward MR, susceptibility to HSV-1 was a risk factor for sepsis. The susceptibility to CMV showed a severity-dependent effect on sepsis and was a risk factor for the 28-day mortality from sepsis, and was also a risk factor for 28-day sepsis mortality in critical care admission. The EBV EA-D antibody level after EBV infection was a protective factor for 28-day sepsis mortality in critical care admission, and CMV pp28 antibody level was a risk factor for 28-day sepsis mortality in critical care admission. No statistically significant causal relationships between HSV-2 and sepsis were found. No exposures having statistically significant association with sepsis critical care admission as an outcome were found. In reverse MR, the sepsis critical care admission group manifested a decrease in CMV pp52 antibody levels. No causal relationships with statistical significance between sepsis exposure and other herpes virus outcomes were found. Conclusion: Our study identifies HSV-1 susceptibility as a sepsis risk, with CMV susceptibility elevating severity. Varied effects of EBV and CMV antibodies on sepsis severity are noted. Severe sepsis results in a decline in CMV antibody levels. Our results help prognostic and predictive enrichment and offer valuable information for precision sepsis treatment.


Subject(s)
Herpesvirus 1, Human , Mendelian Randomization Analysis , Sepsis , Humans , Sepsis/genetics , Herpesvirus 1, Human/immunology , Risk Factors , Cytomegalovirus Infections/genetics , Cytomegalovirus/genetics , Herpes Simplex/genetics , Genome-Wide Association Study , Male , Genetic Predisposition to Disease , Severity of Illness Index , Female
4.
Nat Commun ; 15(1): 1991, 2024 Mar 05.
Article in English | MEDLINE | ID: mdl-38443365

ABSTRACT

Herpes simplex virus 1 (HSV-1) latent infection entails repression of viral lytic genes in neurons. By functional screening using luciferase-expressing HSV-1, we identify ten neuron-specific microRNAs potentially repressing HSV-1 neuronal replication. Transfection of miR-9, the most active candidate from the screen, decreases HSV-1 replication and gene expression in Neuro-2a cells. Ectopic expression of miR-9 from lentivirus or recombinant HSV-1 suppresses HSV-1 replication in male primary mouse neurons in culture and mouse trigeminal ganglia in vivo, and reactivation from latency in the primary neurons. Target prediction and validation identify transcription factors Oct-1, a known co-activator of HSV transcription, and all three Onecut family members as miR-9 targets. Knockdown of ONECUT2 decreases HSV-1 yields in Neuro-2a cells. Overexpression of each ONECUT protein increases HSV-1 replication in Neuro-2a cells, human induced pluripotent stem cell-derived neurons, and primary mouse neurons, and accelerates reactivation from latency in the mouse neurons. Mutagenesis, ChIP-seq, RNA-seq, ChIP-qPCR and ATAC-seq results suggest that ONECUT2 can nonspecifically bind to viral genes via its CUT domain, globally stimulate viral gene transcription, reduce viral heterochromatin and enhance the accessibility of viral chromatin. Thus, neuronal miR-9 promotes viral epigenetic silencing and latency by targeting multiple host transcription factors important for lytic gene activation.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Induced Pluripotent Stem Cells , MicroRNAs , Humans , Male , Animals , Mice , Herpesvirus 1, Human/genetics , MicroRNAs/genetics , Neurons , Herpes Simplex/genetics , Transcription Factors , Epigenesis, Genetic , Homeodomain Proteins
5.
Curr Pharm Des ; 30(9): 649-665, 2024.
Article in English | MEDLINE | ID: mdl-38347772

ABSTRACT

Simplexvirus humanalpha1 (Herpes simplex virus type 1 [HSV-1]) infects millions of people globally, manifesting as vesiculo-ulcerative lesions of the oral or genital mucosa. After primary infection, the virus establishes latency in the peripheral neurons and reactivates sporadically in response to various environmental and genetic factors. A unique feature of herpesviruses is their ability to encode tiny noncoding RNAs called microRNA (miRNAs). Simplexvirus humanalpha1 encodes eighteen miRNA precursors that generate twentyseven different mature miRNA sequences. Unique Simplexvirus humanalpha1 miRNAs repertoire is expressed in lytic and latent stages and exhibits expressional disparity in various cell types and model systems, suggesting their key pathological functions. This review will focus on elucidating the mechanisms underlying the regulation of host-virus interaction by HSV-1 encoded viral miRNAs. Numerous studies have demonstrated sequence- specific targeting of both viral and host transcripts by Simplexvirus humanalpha1 miRNAs. While these noncoding RNAs predominantly target viral genes involved in viral life cycle switch, they regulate host genes involved in antiviral immunity, thereby facilitating viral evasion and lifelong viral persistence inside the host. Expression of Simplexvirus humanalpha1 miRNAs has been associated with disease progression and resolution. Systemic circulation and stability of viral miRNAs compared to viral mRNAs can be harnessed to utilize their potential as diagnostic and prognostic markers. Moreover, functional inhibition of these enigmatic molecules may allow us to devise strategies that have therapeutic significance to contain Simplexvirus humanalpha1 infection.


Subject(s)
Herpesvirus 1, Human , MicroRNAs , MicroRNAs/genetics , MicroRNAs/metabolism , Humans , Herpesvirus 1, Human/genetics , RNA, Viral/genetics , Herpes Simplex/virology , Herpes Simplex/genetics , Animals
6.
J Virol ; 97(12): e0143823, 2023 Dec 21.
Article in English | MEDLINE | ID: mdl-37991364

ABSTRACT

IMPORTANCE: Herpes simplex virus 1 (HSV-1) establishes lifelong latency in neuronal cells. Following a stressor, the virus reactivates from latency, virus is shed at the periphery and recurrent disease can occur. During latency, the viral lncRNA termed the latency-associated transcript (LAT) is known to accumulate to high abundance. The LAT is known to impact many aspects of latency though the molecular events involved are not well understood. Here, we utilized a human neuronal cell line model of HSV latency and reactivation (LUHMES) to identify the molecular-binding partners of the LAT during latency. We found that the LAT binds to both the cellular protein, TMEM43, and HSV-1 genomes in LUHMES cells. Additionally, we find that knockdown of TMEM43 prior to infection results in a decreased ability of HSV-1 to establish latency. This work highlights a potential mechanism for how the LAT facilitates the establishment of HSV-1 latency in human neurons.


Subject(s)
Cell Nucleus , Genome, Viral , Herpes Simplex , Herpesvirus 1, Human , RNA, Long Noncoding , Virus Latency , Humans , Cell Line , Herpes Simplex/genetics , Herpes Simplex/metabolism , Herpes Simplex/virology , Herpesvirus 1, Human/genetics , RNA, Long Noncoding/genetics , Virus Activation/genetics , Virus Latency/genetics , Cell Nucleus/metabolism , Cell Nucleus/virology , Neurons/metabolism , Neurons/virology , Membrane Proteins/deficiency , Membrane Proteins/metabolism , Genome, Viral/genetics
7.
Nat Commun ; 14(1): 7000, 2023 11 02.
Article in English | MEDLINE | ID: mdl-37919266

ABSTRACT

Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generate a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures have minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affects glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Humans , Herpesvirus 1, Human/genetics , Herpes Simplex/genetics , Glycoproteins/metabolism , Keratinocytes/metabolism , Polysaccharides/metabolism , Viral Envelope Proteins/metabolism
8.
J Virol ; 97(10): e0130523, 2023 10 31.
Article in English | MEDLINE | ID: mdl-37823644

ABSTRACT

IMPORTANCE: A correlation exists between stress and increased episodes of human alpha-herpes virus 1 reactivation from latency. Stress increases corticosteroid levels; consequently, the glucocorticoid receptor (GR) is activated. Recent studies concluded that a GR agonist, but not an antagonist, accelerates productive infection and reactivation from latency. Furthermore, GR and certain stress-induced transcription factors cooperatively transactivate promoters that drive the expression of infected cell protein 0 (ICP0), ICP4, and VP16. This study revealed female mice expressing a GR containing a serine to alanine mutation at position 229 (GRS229A) shed significantly lower levels of infectious virus during explant-induced reactivation compared to male GRS229A or wild-type parental C57BL/6 mice. Furthermore, female GRS229A mice contained fewer VP16 + TG neurons compared to male GRS229A mice or wild-type mice during the early stages of explant-induced reactivation from latency. Collectively, these studies revealed that GR transcriptional activity has female-specific effects, whereas male mice can compensate for the loss of GR transcriptional activation.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Receptors, Glucocorticoid , Virus Activation , Animals , Female , Male , Mice , Herpes Simplex/genetics , Herpes Simplex/virology , Herpesvirus 1, Human/physiology , Immediate-Early Proteins/metabolism , Mice, Inbred C57BL , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Trigeminal Ganglion , Ubiquitin-Protein Ligases/metabolism , Virus Activation/genetics , Virus Latency/genetics
9.
Mol Immunol ; 163: 63-74, 2023 11.
Article in English | MEDLINE | ID: mdl-37748280

ABSTRACT

Microglia are the first-line defenders against invading pathogens in the brain whose activation mediates virus clearance and leads to neurotoxicity as well. This work studies the role of Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1)/vacuole membrane protein 1 (VMP1) interaction in the activation of microglia and neuroinflammation following herpes simplex virus 1 (HSV-1) infection. Aberrantly expressed genes after HSV-1 infection were screened by analyzing the GSE35943 dataset. C57BL/6J mice and mouse microglia BV2 were infected with HSV-1 for in vivo and in vitro assays. VMP1 was downregulated but WHSC1L1 was upregulated in HSV-1-infected mouse brain tissues as well as in BV2 cells. The VMP1 overexpression enhanced mitophagy activity and suppressed oxidative stress and inflammatory activation of BV2 cells, but these effects were blocked by the autophagy antagonist 3-methyladenine. WHSC1H1 suppressed VMP1 transcription through H3K36me2-recruited DNMT3A. Downregulation of WHSC1H1 similarly enhanced mitophagy in BV2 cells, and it alleviated microglia activation, nerve cell inflammation, and brain tissue damage in HSV-1-infected mice. However, the alleviating roles of WHSC1H1 silencing were negated by further VMP1 silencing. Taken together. this study demonstrates that WHSC1L1 upregulation following HSV-1 infection leads to mitophagy impairment and neuroinflammation through epigenetic suppression of VMP1.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Animals , Mice , Down-Regulation , Epigenesis, Genetic , Herpes Simplex/genetics , Mice, Inbred C57BL , Microglia/metabolism , Mitophagy/genetics , Neuroinflammatory Diseases
10.
J Med Virol ; 95(8): e29013, 2023 08.
Article in English | MEDLINE | ID: mdl-37537877

ABSTRACT

TANK-binding kinase 1 (TBK1) is crucial in producing type Ⅰ interferons (IFN-Ⅰ) that play critical functions in antiviral innate immunity. The tight regulation of TBK1, especially its activation, is very important. Here we identify NLRC4 as a positive regulator of TBK1. Ectopic expression of NLRC4 facilitates the activation of the IFN-ß promoter, the mRNA levels of IFN-ß, ISG54, and ISG56, and the nuclear translocation of interferon regulatory factor 3 induced by cGAS and STING. Consistently, under herpes simplex virus-1 (HSV-1) infection, knockdown or knockout of NLRC4 in BJ cells and primary peritoneal macrophages from Nlrc4-deficient (Nlrc4-/- ) mice show attenuated Ifn-ß, Isg54, and Isg56 mRNA transcription, TBK1 phosphorylation, and augmented viral replications. Moreover, Nlrc4-/- mice show higher mortality upon HSV-1 infection. Mechanistically, NLRC4 facilitates the interaction between TBK1 and the E3 ubiquitin ligase CBL to enhance the K63-linked polyubiquitination of TBK1. Our study elucidates a previously uncharacterized function for NLRC4 in upregulating the cGAS-STING signaling pathway and antiviral innate immunity.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Signal Transduction , Animals , Mice , Antiviral Agents/metabolism , Herpes Simplex/genetics , Herpesvirus 1, Human/genetics , Immunity, Innate , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Phosphorylation , Signal Transduction/genetics , Ubiquitination
11.
Nat Commun ; 14(1): 4591, 2023 07 31.
Article in English | MEDLINE | ID: mdl-37524699

ABSTRACT

Herpes simplex virus 1 (HSV-1) infection and stress responses disrupt transcription termination by RNA Polymerase II (Pol II). In HSV-1 infection, but not upon salt or heat stress, this is accompanied by a dramatic increase in chromatin accessibility downstream of genes. Here, we show that the HSV-1 immediate-early protein ICP22 is both necessary and sufficient to induce downstream open chromatin regions (dOCRs) when transcription termination is disrupted by the viral ICP27 protein. This is accompanied by a marked ICP22-dependent loss of histones downstream of affected genes consistent with impaired histone repositioning in the wake of Pol II. Efficient knock-down of the ICP22-interacting histone chaperone FACT is not sufficient to induce dOCRs in ΔICP22 infection but increases dOCR induction in wild-type HSV-1 infection. Interestingly, this is accompanied by a marked increase in chromatin accessibility within gene bodies. We propose a model in which allosteric changes in Pol II composition downstream of genes and ICP22-mediated interference with FACT activity explain the differential impairment of histone repositioning downstream of genes in the wake of Pol II in HSV-1 infection.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Immediate-Early Proteins , Humans , Histones/metabolism , Herpesvirus 1, Human/genetics , Transcription, Genetic , Viral Proteins/genetics , Viral Proteins/metabolism , Herpes Simplex/genetics , Chromatin/genetics , Chromatin/metabolism , Immediate-Early Proteins/genetics , Immediate-Early Proteins/metabolism
12.
Virology ; 585: 34-41, 2023 08.
Article in English | MEDLINE | ID: mdl-37271042

ABSTRACT

DNA virus infection triggers an antiviral type I interferon (IFN) response in cells that suppresses infection of surrounding cells. Consequently, viruses have evolved mechanisms to inhibit the IFN response for efficient replication. The cellular cGAS protein binds to double-stranded DNA and synthesizes the small molecule cGAMP to initiate DNA-dependent type I IFN production. We showed previously that cGAMP production is relatively low during HSV-1 infection compared to plasmid DNA transfection. Therefore, we hypothesized that HSV-1 produces antagonists of the cGAS DNA sensing pathway. In this study, we found that the HSV-1 ICP8 protein is required for viral inhibition of the cGAS pathway by reducing cGAMP levels stimulated by double-stranded DNA transfection. ICP8 alone inhibited the cGAMP response and may inhibit cGAS action by direct interaction with DNA, cGAS, or other infected cell proteins. Our results reveal another cGAS antiviral pathway inhibitor and highlight the importance of countering IFN for efficient viral replication.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Humans , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Herpesvirus 1, Human/physiology , Virus Replication , DNA/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/metabolism , Herpes Simplex/genetics
13.
PLoS Pathog ; 19(6): e1010966, 2023 06.
Article in English | MEDLINE | ID: mdl-37343008

ABSTRACT

Herpes simplex virus 1 (HSV1) expresses its genes in a classical cascade culminating in the production of large amounts of structural proteins to facilitate virus assembly. HSV1 lacking the virus protein VP22 (Δ22) exhibits late translational shutoff, a phenotype that has been attributed to the unrestrained activity of the virion host shutoff (vhs) protein, a virus-encoded endoribonuclease which induces mRNA degradation during infection. We have previously shown that vhs is also involved in regulating the nuclear-cytoplasmic compartmentalisation of the virus transcriptome, and in the absence of VP22 a number of virus transcripts are sequestered in the nucleus late in infection. Here we show that despite expressing minimal amounts of structural proteins and failing to plaque on human fibroblasts, the strain 17 Δ22 virus replicates and spreads as efficiently as Wt virus, but without causing cytopathic effect (CPE). Nonetheless, CPE-causing virus spontaneously appeared on Δ22-infected human fibroblasts, and four viruses isolated in this way had all acquired point mutations in vhs which rescued late protein translation. However, unlike a virus deleted for vhs, these viruses still induced the degradation of both cellular and viral mRNA suggesting that vhs mutation in the absence of VP22 is necessary to overcome a more complex disturbance in mRNA metabolism than mRNA degradation alone. The ultimate outcome of secondary mutations in vhs is therefore the rescue of virus-induced CPE caused by late protein synthesis, and while there is a clear selective pressure on HSV1 to mutate vhs for optimal production of late structural proteins, the purpose of this is over and above that of virus production.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Humans , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/metabolism , Transcriptome , Ribonucleases/metabolism , Virion/metabolism , RNA, Messenger/genetics , Herpes Simplex/genetics , Herpes Simplex/metabolism
14.
J Clin Invest ; 133(11)2023 06 01.
Article in English | MEDLINE | ID: mdl-37097753

ABSTRACT

Neonatal herpes simplex virus (HSV) infection is a devastating disease with substantial morbidity and mortality. The genetic basis of susceptibility to HSV in neonates remains undefined. We evaluated a male infant with neonatal skin/eye/mouth (SEM) HSV-1 disease, who had complete recovery after acyclovir but developed HSV-1 encephalitis at 1 year of age. An immune workup showed an anergic PBMC cytokine response to TLR3 stimulation but no other TLRs. Exome sequencing identified rare missense variants in IFN-regulatory factor 7 (IRF7) and UNC-93 homolog B1 (UNC93B1). PBMC single-cell RNA-Seq done during childhood revealed decreased expression of several innate immune genes and a repressed TLR3 pathway signature at baseline in several immune cell populations, including CD14 monocytes. Functional studies in fibroblasts and human leukemia monocytic THP1 cells showed that both variants individually suppressed TLR3-driven IRF3 transcriptional activity and the type I IFN response in vitro. Furthermore, fibroblasts expressing the IRF7 and UNC93B1 variants had higher intracellular viral titers with blunting of the type I IFN response upon HSV-1 challenge. This study reports an infant with recurrent HSV-1 disease complicated by encephalitis associated with deleterious variants in the IRF7 and UNC93B1 genes. Our results suggest that TLR3 pathway mutations may predispose neonates to recurrent, severe HSV.


Subject(s)
Encephalitis, Herpes Simplex , Herpes Simplex , Herpesvirus 1, Human , Interferon Type I , Humans , Infant , Infant, Newborn , Male , Encephalitis, Herpes Simplex/genetics , Herpes Simplex/genetics , Leukocytes, Mononuclear/metabolism , Membrane Transport Proteins , Toll-Like Receptor 3/genetics
15.
Virus Res ; 328: 199087, 2023 04 15.
Article in English | MEDLINE | ID: mdl-36894069

ABSTRACT

Piwi-interacting RNAs (piRNAs) are a class of non-coding RNAs that play a key role in spermatogenesis. However, little is known about their expression characterization and role in somatic cells infected with herpes simplex virus type 1 (HSV-1). In this study, we systematically investigated the cellular piRNA expression profiles of HSV-1-infected human lung fibroblasts. Compared with the control group, 69 differentially expressed piRNAs were identified in the infection group, among which 52 were up-regulated and 17 were down-regulated. The changes in the expression of 8 piRNAs were further verified by RT-qPCR with a similar expression trend. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the target genes of piRNAs were mainly involved in antiviral immunity and various human disease-related signaling pathways. Furthermore, we tested the effects of four up-regulated piRNAs on viral replication by transfecting piRNA mimics. The results showed that the virus titers of the group transfected with piRNA-hsa-28,382 (alias piR-36,233) mimic decreased significantly, and that of the group transfected piRNA-hsa-28,190 (alias piR-36,041) mimic significantly increased. Overall, our results revealed the expression characteristics of piRNAs in HSV-1-infected cells. We also screened two piRNAs that potentially regulate HSV-1 replication. These results may promote a better understanding of the regulatory mechanism of pathophysiological changes induced by HSV-1 infection.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Piwi-Interacting RNA , Humans , Male , Fibroblasts/metabolism , Herpesvirus 1, Human/metabolism , Herpesvirus 1, Human/pathogenicity , Piwi-Interacting RNA/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Herpes Simplex/genetics , Herpes Simplex/metabolism , Gene Expression Profiling
16.
Microbiol Spectr ; 11(1): e0194322, 2023 02 14.
Article in English | MEDLINE | ID: mdl-36537798

ABSTRACT

We utilized a high-throughput cell-based assay to screen several chemical libraries for inhibitors of herpes simplex virus 1 (HSV-1) gene expression. From this screen, four aurora kinase inhibitors were identified that potently reduced gene expression during HSV-1 lytic infection. HSV-1 is known to interact with cellular kinases to regulate gene expression by modulating the phosphorylation and/or activities of viral and cellular proteins. To date, the role of aurora kinases in HSV-1 lytic infection has not been reported. We demonstrated that three aurora kinase inhibitors strongly reduced the transcript levels of immediate-early (IE) genes ICP0, ICP4, and ICP27 and impaired HSV-1 protein expression from all classes of HSV-1, including ICP0, ICP4, ICP8, and gC. These restrictions caused by the aurora kinase inhibitors led to potent reductions in HSV-1 viral replication. The compounds TAK 901, JNJ 7706621, and PF 03814735 decreased HSV-1 titers by 4,500-, 13,200-, and 8,400-fold, respectively, when present in a low micromolar range. The antiviral activity of these compounds correlated with an apparent decrease in histone H3 phosphorylation at serine 10 (H3S10ph) during viral infection, suggesting that the phosphorylation status of H3 influences HSV-1 gene expression. Furthermore, we demonstrated that the aurora kinase inhibitors also impaired the replication of other RNA and DNA viruses. These inhibitors significantly reduced yields of vaccinia virus (a poxvirus, double-stranded DNA, cytoplasmic replication) and mouse hepatitis virus (a coronavirus, positive-sense single-strand RNA [ssRNA]), whereas vesicular stomatitis virus (rhabdovirus, negative-sense ssRNA) yields were unaffected. These results indicated that the activities of aurora kinases play pivotal roles in the life cycles of diverse viruses. IMPORTANCE We have demonstrated that aurora kinases play a role during HSV-1 lytic infection. Three aurora kinase inhibitors significantly impaired HSV-1 immediate-early gene expression. This led to a potent reduction in HSV-1 protein expression and viral replication. Together, our results illustrate a novel role for aurora kinases in the HSV-1 lytic cycle and demonstrate that aurora kinase inhibitors can restrict HSV-1 replication. Furthermore, these aurora kinase inhibitors also reduced the replication of murine coronavirus and vaccinia virus, suggesting that multiple viral families utilize the aurora kinases for their own replication.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Immediate-Early Proteins , RNA Viruses , Animals , Mice , Herpesvirus 1, Human/genetics , Immediate-Early Proteins/genetics , Viral Proteins/genetics , Viral Proteins/metabolism , Cell Line , Herpes Simplex/genetics , DNA/metabolism , RNA/metabolism , Life Cycle Stages
17.
Clin Infect Dis ; 76(3): 521-527, 2023 02 08.
Article in English | MEDLINE | ID: mdl-36573283

ABSTRACT

BACKGROUND: IFNL4 genetic variants that are strongly associated with clearance of hepatitis C virus have been linked to risk of certain opportunistic infections (OIs) and cancers, including Kaposi sarcoma, cytomegalovirus infection, and herpes simplex virus infection. As the interferon (IFN) λ family plays a role in response to viral, bacterial, and fungal infections, IFNL4 genotype might affect risk for a wide range of OIs/cancers. METHODS: We examined associations between genotype for the functional IFNL4 rs368234815 polymorphism and incidence of 16 OIs/cancers among 2310 men with human immunodeficiency virus (2038 white; 272 black) enrolled in the Multicenter AIDS Cohort Study during 1984-1990. Our primary analyses used Cox proportional hazards models adjusted for self-reported racial ancestry to estimate hazard ratios with 95% confidence intervals, comparing participants with the genotypes that generate IFN-λ4 and those with the genotype that abrogates IFN-λ4. We censored follow-up at the introduction of highly effective antiretroviral therapies. RESULTS: We found no statistically significant association between IFNL4 genotype and the incidence of Kaposi sarcoma (hazard ratio, 0.92 [95% confidence interval, .76-1.11]), cytomegalovirus infection (0.94 [.71-1.24]), herpes simplex virus infection (1.37 [.68-2.93]), or any other OI/cancer. We observed consistent results using additive genetic models and after controlling for CD4 cell count through time-dependent adjustment or restriction to participants with a low CD4 cell count. CONCLUSIONS: The absence of associations between IFNL4 genotype and these OIs/cancers provides evidence that this gene does not affect the risk of disease from opportunistic pathogens.


Subject(s)
Cytomegalovirus Infections , HIV Infections , HIV-1 , Herpes Simplex , Opportunistic Infections , Sarcoma, Kaposi , Male , Humans , Cohort Studies , Genotype , HIV Infections/complications , HIV Infections/genetics , Herpes Simplex/complications , Herpes Simplex/epidemiology , Herpes Simplex/genetics , Cytomegalovirus Infections/complications , Cytomegalovirus Infections/epidemiology , Cytomegalovirus Infections/genetics , Interleukins/genetics , Polymorphism, Single Nucleotide
18.
Int J Mol Sci ; 23(23)2022 Nov 27.
Article in English | MEDLINE | ID: mdl-36499174

ABSTRACT

Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.


Subject(s)
Herpes Simplex , Herpesviridae Infections , Herpesviridae , Herpesvirus 1, Human , Humans , CRISPR-Cas Systems/genetics , Herpesvirus 1, Human/genetics , Herpes Simplex/genetics , Herpesviridae Infections/genetics , CRISPR-Associated Protein 9/genetics
19.
J Virol ; 96(24): e0092022, 2022 12 21.
Article in English | MEDLINE | ID: mdl-36453882

ABSTRACT

Real-time imaging tools for single-virus tracking provide spatially resolved, quantitative measurements of viral replication and virus-host interactions. However, efficiently labeling both parental and progeny viruses in living host cells remains challenging. Here, we developed a novel strategy using the CRISPR-Tag system to detect herpes simplex virus 1 (HSV-1) DNA in host cells. We created recombinant HSV-1 harboring an ~600-bp CRISPR-Tag sequence which can be sufficiently recognized by dCas9-fluorescent protein (FP) fusion proteins. CRISPR-assisted single viral genome tracking (CASVIT) allows us to assess the temporal and spatial information of viral replication at the single-cell level. Combining the advantages of SunTag and tandem split green fluorescent protein (GFP) in amplifying fluorescent signals, dSaCas9-tdTomato10x and dSpCas9-GFP14x were constructed to enable efficient two-color CASVIT detection. Real-time two-color imaging indicates that replication compartments (RCs) frequently come into contact with each other but do not mix, suggesting that RC territory is highly stable. Last, two-color CASVIT enables simultaneous tracking of viral DNA and host chromatin, which reveals that a dramatic loss of telomeric and centromeric DNA occurs in host cells at the early stage of viral replication. Overall, our work has established a framework for developing CRISPR-Cas9-based imaging tools to study DNA viruses in living cells. IMPORTANCE Herpes simplex virus 1 (HSV-1), a representative of the family Herpesviridae, is a ubiquitous pathogen that can establish lifelong infections and widely affects human health. Viral infection is a dynamic process that involves many steps and interactions with various cellular structures, including host chromatin. A common viral replication strategy is to form RCs that concentrate factors required for viral replication. Efficient strategies for imaging the dynamics of viral genomes, RC formation, and the interaction between the virus and host offer the opportunity to dissect the steps of the infection process and determine the mechanism underlying each step. We have developed an efficient two-color imaging system based on CRISPR-Cas9 technology to detect HSV-1 genomes quantitatively in living cells. Our results shed light on novel aspects of RC dynamics and virus-host interactions.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Host Microbial Interactions , Virus Replication , Humans , Cell Line , Chromatin , Herpes Simplex/genetics , Herpesvirus 1, Human/genetics , Host Microbial Interactions/genetics , Virus Replication/genetics , DNA, Viral/analysis , DNA, Viral/genetics
20.
J Virol ; 96(24): e0142922, 2022 12 21.
Article in English | MEDLINE | ID: mdl-36448808

ABSTRACT

We investigated whether A-type lamins (lamin A/C) and lamin B receptor (LBR) are redundant during herpes simplex virus 1 (HSV-1) infection in HeLa cells expressing lamin A/C and LBR. Lamin A/C and LBR double knockout (KO) in HSV-1-infected HeLa cells significantly impaired expressions of HSV-1 early and late genes, maturation of replication compartments, marginalization of host chromatin to the nuclear periphery, enlargement of host cell nuclei, and viral DNA replication. Phenotypes of HSV-1-infected HeLa cells were restored by the ectopic expression of lamin A/C or LBR in lamin A/C and LBR double KO cells. Of note, lamin A/C single KO, but not LBR single KO, promoted the aberrant accumulation of virus particles outside the inner nuclear membrane (INM) and viral replication, as well as decreasing the frequency of virus particles inside the INM without affecting viral gene expression and DNA replication, time-spatial organization of replication compartments and host chromatin, and nuclear enlargement. These results indicated that lamin A/C and LBR had redundant and specific roles during HSV-1 infection. Thus, lamin A/C and LBR redundantly regulated the dynamics of the nuclear architecture, including the time-spatial organization of replication compartments and host chromatin, as well as promoting nuclear enlargement for efficient HSV-1 gene expression and DNA replication. In contrast, lamin A/C inhibited HSV-1 nuclear export through the INM during viral nuclear egress, which is a unique property of lamin A/C. IMPORTANCE This study demonstrated that lamin A/C and LBR had redundant functions associated with HSV-1 gene expression and DNA replication by regulating the dynamics of the nuclear architecture during HSV-1 infection. This is the first report to demonstrate the redundant roles of lamin A/C and LBR as well as the involvement of LBR in the regulation of these viral and cellular features in HSV-1-infected cells. These findings provide evidence for the specific property of lamin A/C to inhibit HSV-1 nuclear egress, which has long been considered but without direct proof.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Lamins , Humans , Chromatin/metabolism , DNA Replication , DNA, Viral/genetics , DNA, Viral/metabolism , HeLa Cells , Herpes Simplex/genetics , Herpes Simplex/metabolism , Herpesvirus 1, Human/physiology , Lamin Type A/genetics , Lamin Type A/metabolism , Lamins/genetics , Lamins/metabolism , Virus Replication , Lamin B Receptor
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