Early Viral Gene Research Articles

White spot syndrome virus-encoded microRNA promotes viral replication by maintaining viral early gene expression

Disease especially caused by White spot syndrome virus (WSSV) has a huge impact on the shrimp production industry. An understanding of shrimp-WSSV interaction could lead to an effective approach to control WSSV infection. MicroRNA (miRNA) is a short small RNA which functions in post-transcriptional gene regulation and is involved in the WSSV infection. Previously, WSSV-encoded miRNAs were identified from infected P. monodon. We investigated wsv-miR-9 which showed the highest expression in WSSV-infected gill tissue. The expression of wsv-miR-9 was up-regulated upon WSSV infection and reduced in moribund shrimp. Antisense inhibition using anti-miRNA oligonucleotides (AMO) resulted in a complete inhibition of wsv-miR-9 expression. An in vitro functional study demonstrated that mimic of wsv-miR-9 could function in translational suppression by decreasing the reporter protein activity when compared to the mutant target. In shrimp, loss of wsv-miR-9 by AMO9 resulted in a delay of viral replication after 6–12 h post WSSV challenge. A reduction of WSSV early genes, ie1 and DNA polymerase mRNA expressions was observed within 6 h. These results reveal that wsv-miR-9 is important in maintaining early viral gene expression.

Varicella-zoster virus early infection but not complete replication is required for the induction of chronic hypersensitivity in rat models of postherpetic neuralgia.

Herpes zoster, the result of varicella-zoster virus (VZV) reactivation, is frequently complicated by difficult-to-treat chronic pain states termed postherpetic neuralgia (PHN). While there are no animal models of VZV-induced pain following viral reactivation, subcutaneous VZV inoculation of the rat causes long-term nocifensive behaviors indicative of mechanical and thermal hypersensitivity. Previous studies using UV-inactivated VZV in the rat model suggest viral gene expression is required for the development of pain behaviors. However, it remains unclear if complete infection processes are needed for VZV to induce hypersensitivity in this host. To further assess how gene expression and replication contribute, we developed and characterized three replication-conditional VZV using a protein degron system to achieve drug-dependent stability of essential viral proteins. Each virus was then assessed for induction of hypersensitivity in rats under replication permissive and nonpermissive conditions. VZV with a degron fused to ORF9p, a late structural protein that is required for virion assembly, induced nocifensive behaviors under both replication permissive and nonpermissive conditions, indicating that complete VZV replication is dispensable for the induction of hypersensitivity. This conclusion was confirmed by showing that a genetic deletion recombinant VZV lacking DNA packaging protein ORF54p still induced prolonged hypersensitivities in the rat. In contrast, VZV with a degron fused to the essential IE4 or IE63 proteins, which are involved in early gene regulation of expression, induced nocifensive behaviors only under replication permissive conditions, indicating importance of early gene expression events for induction of hypersensitivity. These data establish that while early viral gene expression is required for the development of nocifensive behaviors in the rat, complete replication is dispensable. We postulate this model reflects events leading to clinical PHN, in which a population of ganglionic neurons become abortively infected with VZV during reactivation and survive, but host signaling becomes altered in order to transmit ongoing pain.

Fos Facilitates Gallid Alpha-Herpesvirus 1 Infection by Transcriptional Control of Host Metabolic Genes and Viral Immediate Early Gene.

Gallid alpha-herpesvirus 1, also known as avian infectious laryngotracheitis virus (ILTV), continues to cause huge economic losses to the poultry industry worldwide. Similar to that of other herpesvirus-encoded proteins, the expression of viral genes encoded by ILTV is regulated by a cascade, and the underlying regulatory mechanism remains largely unclear. The viral immediate-early (IE) gene ICP4 plays a prominent role in the initiation of the transcription of early and late genes during ILTV replication. In this study, we identified AP-1 as the key regulator of the transcription of ILTV genes by bioinformatics analysis of genome-wide transcriptome data. Subsequent functional studies of the key members of the AP-1 family revealed that Fos, but not Jun, regulates ILTV infection through AP-1 since knockdown of Fos, but not Jun, by gene silencing significantly reduced ICP4 transcription and subsequent viral genome replication and virion production. Using several approaches, we identified ICP4 as a bona fide target gene of Fos that regulated Fos and has Fos response elements within its promoter. Neither the physical binding of Jun to the promoter of ICP4 nor the transcriptional activity of Jun was observed. In addition, knockdown of Fos reduced the transcription of MDH1 and ATP5A1, genes encoding two host rate-limiting enzymes essential for the production of the TCA intermediates OAA and ATP. The biological significance of the transcriptional regulation of MDH1 and ATP5A1 by Fos in ILTV infection was supported by the fact that anaplerosis of OAA and ATP rescued both ICP4 transcription and virion production in infected cells under when Fos was silenced. Our study identified the transcription factor Fos as a key regulator of ILTV infection through its transcription factor function on both the virus and host sides, improving the current understanding of both avian herpesvirus–host interactions and the roles of AP-1 in viral infection.

The Second-Generation XPO1 Inhibitor Eltanexor Inhibits Human Cytomegalovirus (HCMV) Replication and Promotes Type I Interferon Response.

Human cytomegalovirus (HCMV) is a ubiquitous opportunistic pathogen and can be life-threatening for immunocompromised individuals. There is currently no available vaccine for the prevention of HCMV- associated diseases and most of the available antiviral drugs that target viral DNA synthesis become ineffective in treating HCMV mutants that arise after long-term use in immunocompromised patients. Here, we examined the effects of Eltanexor, a second-generation selective inhibitor of nuclear export (SINE), on HCMV replication. Eltanexor effectively inhibits HCMV replication in human foreskin fibroblasts in a dose-dependent manner. Eltanexor does not significantly inhibit viral entry and nuclear import of viral genomic DNA, but rather suppress the transcript and protein levels of viral immediate-early (IE), early (E) and late (L) genes, and abolishes the production of infectious virions. We further found Eltanexor treatment promotes proteasome-mediated degradation of XPO1, which contributes to the nuclear retention of interferon regulatory factor 3 (IRF-3), resulting in increased expression of type I interferon as well as interferon stimulating genes ISG15 and ISG54. This study reveals a novel antiviral mechanism of Eltanexor which suggests it has potential to inhibit a broad spectrum of viral pathogens.

P048 Cytomegalovirus retinitis in a patient with rheumatoid arthritis on tofacitinib

Abstract Background/Aims Cytomegalovirus (CMV) retinitis is one of the most common opportunistic ocular infections and has traditionally been associated with immunocompromised individuals. We present a case of CMV retinitis following 3 months of treatment with tofacitinib in conjunction with methotrexate (MTX) for rheumatoid arthritis (RA) in an elderly lady with no other co-morbidities. Methods The case is presented below. Results 69-year-old lady with long standing seronegative, anti-cyclic citrullinated peptide negative, erosive RA since 2016. She was intolerant of sulphasalazine, leflunamide, methotrexate and etanercept. Hydroxychloroquine was avoided due to idiopathic cystoid macular oedema in the right eye with a baseline visual acuity of 6/18. Baseline fundus fluorescein angiogram (FFA) was negative for any vascular, inflammatory or occlusive disease. She was escalated to tofacitinib in August 2019. Approximately 3 months later, she noticed significantly reduced vision in her right eye from 6/18 down to 6/60. She had an FFA and Indocyanine Green Angiography, which showed quite extensive retinal ischaemia and vasculitis. Her bloods revealed a raised serum ACE and a T-spot test from 2018 was reactive followed by a repeat in 2019 which was equivocal. Tofacitinib was discontinued. She was commenced on rifampicin and isoniazid as 3 months prophylaxis for latent tuberculosis (LTBI). Additionally, she was started on oral prednisolone 60mg daily for presumed sarcoid related retinal vasculitis. She failed to improve despite a week’s treatment and subsequently had a vitreous aspirate which was strongly positive for CMV PCR. Her CMV viral load was 267copies/ml. The aspirate was negative for other infectious agents. She was commenced on valganciclovir for CMV retinitis. Unfortunately, her vision remained unchanged despite the viral load becoming undetectable a few weeks later. Despite having LTBI, there was no reactivation of this with her immunosuppressive therapy. Conclusion CMV retinitis is usually associated with immunocompromised individuals and is rarely encountered related to tofacitinib therapy for rheumatoid arthritis. Tofacitinib suppresses T cell function by inhibiting STAT signalling pathways, modulating T cell activation and cytokine production. Our case highlights the preferential development of CMV retinitis over active TB in the presence of LTBI, which is a recognised risk with the use of JAK kinase inhibitors. TB risk has been associated with genetic mutations in IL-12, IFN-γ and STAT1 pathways while CMV activation may be influenced by mutations in HLA ,Toll-like receptors, immunoglobulin light chain antibodies, IFN lambda region as well as alternative transcripts of the CMV viral major immediate early genes. Disclosure K. Beharry: None. S. Kaur: None. E. Papagiannuli: None. S. Moses: None. D. De Lord: None.

Interferon Gamma Inhibits Equine Herpesvirus 1 Replication in a Cell Line-Dependent Manner.

The sole equine herpesvirus 1 (EHV-1) immediate-early protein (IEP) is essential for viral replication by transactivating viral immediate-early (IE), early (E), and late (L) genes. Here, we report that treatment of mouse MH-S, equine NBL6, and human MRC-5 cells with 20 ng/mL of IFN-γ reduced EHV-1 yield by 1122-, 631-, and 10,000-fold, respectively. However, IFN-γ reduced virus yield by only 2–4-fold in mouse MLE12, mouse L-M, and human MeWo cells compared to those of untreated cells. In luciferase assays with the promoter of the EHV-1 early regulatory EICP0 gene, IFN-γ abrogated trans-activation activity of the IEP by 96% in MH-S cells, but only by 21% in L-M cells. Similar results were obtained in assays with the early regulatory UL5 and IR4 promoter reporter plasmids. IFN-γ treatment reduced IEP protein expression by greater than 99% in MH-S cells, but only by 43% in L-M cells. The expression of IEP and UL5P suppressed by IFN-γ was restored by JAK inhibitor treatment, indicating that the inhibition of EHV-1 replication is mediated by JAK/STAT1 signaling. These results suggest that IFN-γ blocks EHV-1 replication by inhibiting the production of the IEP in a cell line-dependent manner. Affymetrix microarray analyses of IFN-γ-treated MH-S and L-M cells revealed that five antiviral ISGs (MX1, SAMHD1, IFIT2, NAMPT, TREX1, and DDX60) were upregulated 3.2–18.1-fold only in MH-S cells.

Biology of Polyomavirus miRNA.

Polyomaviruses are a family of non-enveloped DNA viruses with wide host ranges. Human polyomaviruses typically cause asymptomatic infection and establish persistence but can be reactivated under certain conditions and cause severe diseases. Most well studied polyomaviruses encode a viral miRNA that regulates viral replication and pathogenesis by targeting both viral early genes and host genes. In this review, we summarize the current knowledge of polyomavirus miRNAs involved in virus infection. We review in detail the regulation of polyomavirus miRNA expression, as well as the role polyomavirus miRNAs play in viral pathogenesis by controlling both host and viral gene expression. An overview of the potential application of polyomavirus miRNA as a marker for the progression of polyomaviruses associated diseases and polyomaviruses reactivation is also included.

Profiling the Blood Compartment of Hematopoietic Stem Cell Transplant Patients During Human Cytomegalovirus Reactivation.

Human cytomegalovirus (HCMV) is a widespread pathogen establishing a latent infection in its host. HCMV reactivation is a major health burden in immunocompromised individuals, and is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Here we determined HCMV genomic levels using droplet digital PCR in different peripheral blood mononuclear cell (PBMC) populations in HCMV reactivating HSCT patients. This high sensitivity approach revealed that all PBMC populations harbored extremely low levels of viral DNA at the peak of HCMV DNAemia. Transcriptomic analysis of PBMCs from high-DNAemia samples revealed elevated expression of genes typical of HCMV specific T cells, while regulatory T cell enhancers as well as additional genes related to immune response were downregulated. Viral transcript levels in these samples were extremely low, but remarkably, the detected transcripts were mainly immediate early viral genes. Overall, our data indicate that HCMV DNAemia is associated with distinct signatures of immune response in the blood compartment, however it is not necessarily accompanied by substantial infection of PBMCs and the residual infected PBMCs are not productively infected.

Lupeol impairs herpes simplex virus type 1 replication by inhibiting the promoter activity of the viral immediate early gene α0.

Herpes simplex virus type 1 (HSV-1) is an important human pathogenic virus. It is urgent to develop novel antiviral targets because of the limited treatment options and the emergence of drug resistant strains. In this study, we tested the antiviral activity of lupeol, a triterpenoid compound, against HSV-1 and acyclovir (ACV) resistant strains. Lupeol significantly inhibited HSV-1 (F strain) and ACV-resistant strains including HSV-1/106, HSV-1/153, and HSV-1/Blue. Lupeol activity of the HSV-1α0 and α4 promoters, therefore down regulating the expression of the α0, α4, and α27 genes. Collectively, lupeol showed strong antiviral activity against HSV-1 and ACV-resistant strains, and could be a promising therapeutic candidate for HSV-1 pathogenesis. Keywords: herpes simplex virus 1; lupeol; ACV-resistant strains; promoter.

Anti-herpes virus activity of the carnivorous botanical, Sarracenia purpurea

Herpes simplex virus type-1 (HSV-1), one of the most widely spread human viruses in the Herpesviridae family, causes herpes labialis (cold sores) and keratitis (inflammation of the cornea). Conventional treatment for HSV-1 infection includes pharmaceutical drugs, such as acyclovir and docosonal, which are efficacious but maintain the potential for the development of viral drug resistance. Extracts from the carnivorous pitcher plant, Sarracenia purpurea, have previously been shown to inhibit the replication of HSV-1. In this study, we demonstrate that S. purpurea extracts can inhibit the replication of HSV-1 by two distinct mechanisms of action. These extracts directly inhibit extracellular virions or viral attachment to the human host cell as well as inhibiting the expression of viral immediate-early, early and late genes when added at various times post-infection. This botanical has previously been shown to inhibit the replication of poxviruses through the inhibition of early viral gene transcription. These results support a broader anti-viral activity of S. purpurea extracts against both pox and herpes viruses.

CqPP2A inhibits white spot syndrome virus infection by up-regulating antimicrobial substances expression in red claw crayfish Cherax quadricarinatus

Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase, a highly conserved enzyme widely expressed in eukaryotic cells, which accounts for a majority of the serine/threonine phosphatase activity in cells implicated in regulation of immune signaling pathways and antiviral response. However, most of studies about PP2A have been conducted in mammals but few in crustaceans. In this study, two subunits of PP2A (named as CqPP2Ab and CqPP2Ac) were characterized to be involved in white spot syndrome virus (WSSV) infection in the haematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. The open reading frame (ORF) of CqPP2Ab was 1341 bp encoding 446 amino acids with seven WD40 domains, and the ORF of CqPP2Ac was 930 bp encoding 309 amino acids with a PP2Ac domain. Tissue distribution analysis showed that the mRNA transcript of CqPP2Ab and CqPP2Ac were both widely expressed in all the tested tissues with the highest expression in hemocyte, followed by high expression in Hpt. The gene expressions of CqPP2Ab and CqPP2Ac were both significantly down-regulated at 6 h post WSSV infection (6 hpi) in Hpt cells. Importantly, the expression of viral immediate early gene IE1 and late viral gene envelope protein VP28 were both significantly increased post WSSV infection after gene silencing of CqPP2Ab or CqPP2Ac in Hpt cells, suggesting that CqPP2Ab and CqPP2Ac could inhibit WSSV infection in Hpt cells, probably by increasing the antimicrobial substances expression in consideration to the significantly reduced expression of anti-lipopolysaccharide factor, crustin, and lysozyme after gene silencing of CqPP2Ab or CqPP2Ac, respectively. These findings provide a new light on the mechanism of WSSV infection and the antiviral response in crustaceans.

Modeling the epithelial-mesenchymal transition process in a 3D organotypic cervical neoplasia

Here, we proposed an innovative organotypic cervical tumor model able to investigate the bi-directional crosstalk between epithelium and stroma as well as the key disease features of the epithelial-mesenchymal transition (EMT) process in vitro. By using a modular tissue assembling approach, we developed 3D cervical stromal models composed of primary human cervical fibroblasts (HCFs) or cervical cancer-associated fibroblasts (CCAFs) embedded in their own ECM to produce 3D normal cervical-instructed stroma (NCIS) or 3D cervical cancer-instructed stroma (CCIS), respectively. Then, we demonstrate the role of the tumor microenvironment (TME) in potentiating the intrinsic invasive attitude of cervical cancer derived SiHa cells and increasing their early viral gene expression by comparing the SiHa behavior when cultured on NCIS or CCIS (SiHa-NCIS or SiHa-CCIS). We proved the crucial role of the CCAFs and stromal microenvironment in the mesenchymalization of the cancer epithelial cells by analyzing several EMT markers. We further assessed the expression of the epithelial adhesion molecules, matricellular enzymes, non-collagenous proteins as well as ECM remodeling in terms of collagen fibers texture and assembly. This cervical tumor model, closely recapitulating key cervical carcinogenesis features, may provide efficient and relevant support to current approaches characterizing cancer progression and develop new anticancer therapy targeting stroma rather than cancer cells.

Global exploration of the metabolic requirements of gallid alphaherpesvirus 1.

Although therapeutics targeting viral metabolic processes have been considered as promising strategies to treat herpesvirus infection, the metabolic requirements of gallid alphaherpesvirus 1 (ILTV), which is economically important to the poultry industry worldwide, remain largely unknown. Using the ILTV-susceptible but nonpermissive chicken cell line DF-1 and the ILTV-permissive chicken cell line LMH as models, the present study explored the metabolic requirements of ILTV by global transcriptome analysis and metabolome assays of ILTV infected cell lines in combination with a set of functional validations. The extensive metabolic exploration demonstrated that ILTV infection tended to promote a metabolic shift from glycolysis to fatty acid (FA) and nucleotide biosynthesis and utilizes glutamine independently of glutaminolysis, without significant general effect on the TCA cycle. In addition, different metabolic pathways were found to be required for distinct stages of ILTV replication. Glucose and glutamine were required for the transcription of viral immediate early gene ICP4 and subsequent steps of viral replication. However, FA synthesis was essential for assembly but not required for other upstream steps of ILTV replication. Moreover, the metabolic requirements of ILTV infection revealed in chicken cell lines were further validated in chicken primary cells isolated from chicken embryo kidneys and chicken embryo livers. The present study, to the best of our knowledge, provides the first global metabolic profile of animal herpesviruses and illustrates the main characteristics of the metabolic program of ILTV.

Necroptosis-based CRISPR knockout screen reveals Neuropilin-1 as a critical host factor for early stages of murine cytomegalovirus infection

Herpesviruses are ubiquitous human pathogens that cause a wide range of health complications. Currently, there is an incomplete understanding of cellular factors that contribute to herpesvirus infection. Here, we report an antiviral necroptosis-based genetic screen to identify novel host cell factors required for infection with the β-herpesvirus murine cytomegalovirus (MCMV). Our genome-wide CRISPR-based screen harnessed the capacity of herpesvirus mutants that trigger antiviral necroptotic cell death upon early viral gene expression. Vascular endothelial growth factor (VEGF) and semaphorin-binding receptor Neuropilin-1 (Nrp-1) emerge as crucial determinants of MCMV infection. We find that elimination of Nrp-1 impairs early viral gene expression and reduces infection rates in endothelial cells, fibroblasts, and macrophages. Furthermore, preincubation of virus with soluble Nrp-1 dramatically inhibits infection by reducing virus attachment. Thus, Nrp-1 is a key determinant of the initial phase of MCMV infection.

The Differentiation of Human Cytomegalovirus Infected-Monocytes Is Required for Viral Replication

Viral dissemination is a key mechanism responsible for persistence and disease following human cytomegalovirus (HCMV) infection. Monocytes play a pivotal role in viral dissemination to organ tissue during primary infection and following reactivation from latency. For example, during primary infection, infected monocytes migrate into tissues and differentiate into macrophages, which then become a source of viral replication. In addition, because differentiated macrophages can survive for months to years, they provide a potential persistent infection source in various organ systems. We broadly note that there are three phases to infection and differentiation of HCMV-infected monocytes: (1) Virus enters and traffics to the nucleus through a virus receptor ligand engagement event that activates a unique signalsome that initiates the monocyte-to-macrophage differentiation process. (2) Following initial infection, HCMV undergoes a “quiescence-like state” in monocytes lasting for several weeks and promotes monocyte differentiation into macrophages. While, the initial event is triggered by the receptor-ligand engagement, the long-term cellular activation is maintained by chronic viral-mediated signaling events. (3) Once HCMV infected monocytes differentiate into macrophages, the expression of immediate early viral (IE) genes is detectable, followed by viral replication and long term infectious viral particles release. Herein, we review the detailed mechanisms of each phase during infection and differentiation into macrophages and discuss the biological significance of the differentiation of monocytes in the pathogenesis of HCMV.

A barrier-to-autointegration factor promotes white spot syndrome virus infection in a crustacean Cherax quadricarinatus

Barrier-to-autointegration factor (BAF) is a highly conserved DNA binding protein that participates in a variety of biological processes such as transcription, epigenetic regulation and antiviral immunity in vertebrates. However, the function of BAF is poorly understood in crustaceans. In this study, we identified a barrier-to-autointegration factor (CqBAF) from red claw crayfish Cherax quadricarinatus, which was responsive to white spot syndrome virus (WSSV) infection. The full-length cDNA sequence of CqBAF was 544 bp, including an open reading frame of 273 bp encoding 90 amino acids, a 107 bp of 5′-Untranslated Regions (5′-UTR) and a 164 bp of 3′-UTR. Gene expression analysis showed that CqBAF was distributed in all tissues examined with the highest expression in the crayfish haematopietic tissue (Hpt), which protein expression was also significantly up-regulated by WSSV infection in Hpt cells. Furthermore, the transcripts of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV were clearly reduced in Hpt cells after gene silencing of CqBAF. Importantly, the promoter activity of two immediate early genes of WSSV, including WSV051 and IE1, was strongly enhanced by the increased phosphorylation of CqBAF, which also facilitated the accumulation of CqBAF protein in the cytoplasm of Sf9 cells. Taken together, these data suggest that CqBAF is likely to increase the replication of WSSV by promoting the transcription of viral immediate early genes, probably regulated by phosphorylation of CqBAF, which sheds new light on the molecular mechanism of WSSV infection.

Hsv-1 Endocytic Entry into a Human Oligodendrocytic Cell Line is Mediated by Clathrin and Dynamin but Not Caveolin.

Endocytosis is a pathway used by viruses to enter cells that can be classified based on the proteins involved, such as dynamin, clathrin or caveolin. Although the entry of herpes simplex type 1 (HSV-1) by endocytosis has been documented in different cell types, its dependence on clathrin has not been described whereas its dependence on dynamin has been shown according to the cell line used. The present work shows how clathrin-mediated endocytosis (CME) is one way that HSV-1 infects the human oligodendroglial (HOG) cell line. Partial dynamin inhibition using dynasore revealed a relationship between decrease of infection and dynamin inhibition, measured by viral titration and immunoblot. Co-localization between dynamin and HSV-1 was verified by immunofluorescence at the moment of viral entry into the cell. Inhibition by chlorpromazine revealed that viral progeny also decreased when clathrin was partially inhibited in our cell line. RT-qPCR of immediately early viral genes, specific entry assays and electron microscopy all confirmed clathrin’s participation in HSV-1 entry into HOG cells. In contrast, caveolin entry assays showed no effect on the entry of this virus. Therefore, our results suggest the participation of dynamin and clathrin during endocytosis of HSV-1 in HOG cells.

HSV-1 Hijacks the Host DNA Damage Response in Corneal Epithelial Cells through ICP4-Mediated Activation of ATM

PurposeHerpes simplex virus type I (HSV-1) infection of corneal epithelial cells activates ataxia telangiectasia mutated (ATM), an apical kinase in the host DNA damage response pathway, whose activity is necessary for the progression of lytic HSV-1 infection. The purpose of this study is to investigate the mechanism of ATM activation by HSV-1 in the corneal epithelium, as well as its functional significance.MethodsMechanistic studies were performed in cultured human corneal epithelial cell lines (hTCEpi, HCE), as well as in esophageal (EPC2) and oral (OKF6) cell lines. Transfection-based experiments were performed in HEK293 cells. HSV-1 infection was carried out using the wild-type KOS strain, various mutant strains (tsB7, d120, 7134, i13, n208), and bacterial artificial chromosomes (fHSVΔpac, pM24). Inhibitors of ATM (KU-55933), protein synthesis (cycloheximide), and viral DNA replication (phosphonoacetic acid) were used. Outcomes of infection were assayed using Western blotting, qRT-PCR, immunofluorescence, and comet assay.ResultsThis study demonstrates that HSV-1-mediated ATM activation in corneal epithelial cells relies on the viral immediate early gene product ICP4 and requires the presence of the viral genome in the host nucleus. We show that ATM activation is independent of viral genome replication, the ICP0 protein, and the presence of DNA lesions. Interestingly, ATM activity appears to be necessary at the onset of infection, but dispensable at the later stages.ConclusionsThis study expands our understanding of HSV-1 virus-host interactions in the corneal epithelium and identifies potential areas of future investigation and therapeutic intervention in herpes keratitis.

The Human Cytomegalovirus Transmembrane Protein pUL50 Induces Loss of VCP/p97 and Is Regulated by a Small Isoform of pUL50.

The human cytomegalovirus (HCMV) UL50 gene encodes a transmembrane protein, pUL50, which acts as a core component of the nuclear egress complex (NEC) for nucleocapsids. Recently, pUL50 has been shown to have NEC-independent activities: downregulation of IRE1 to repress the unfolded protein response and degradation of UBE1L to inhibit the protein ISG15 modification pathway. Here, we demonstrate that a 26-kDa N-terminal truncated isoform of pUL50 (UL50-p26) is expressed from an internal methionine at amino acid position 199 and regulates the activity of pUL50 to induce the loss of valosin-containing protein (VCP/p97). A UL50(M199V) mutant virus expressing pUL50(M199V) but not UL50-p26 showed delayed growth at a low multiplicity of infection. There was also delayed accumulation of the viral immediate early 2 (IE2) protein in the mutant virus, and this correlated with the reduced expression of VCP/p97, which promotes IE2 expression. Infection with mutant virus did not significantly alter ISGylation levels. In transient expression assays, pUL50 induced VCP/p97 loss posttranscriptionally, and this was dependent on the presence of its transmembrane domain. In contrast, UL50-p26 did not destabilize VCP/p97 but, rather, inhibited pUL50-mediated VCP/p97 loss and the associated major IE gene suppression. Both pUL50 and UL50-p26 interacted with VCP/p97, although UL50-p26 did so more weakly than pUL50. UL50-p26 interacted with pUL50, and this interaction was much stronger than the pUL50 self-interaction. Furthermore, UL50-p26 was able to interfere with the pUL50-VCP/p97 interaction. Our study newly identifies UL50-p26 expression during HCMV infection and suggests a regulatory role for UL50-p26 in blocking pUL50-mediated VCP/p97 loss by associating with pUL50.IMPORTANCE Targeting the endoplasmic reticulum (ER) by viral proteins may affect ER-associated protein homeostasis. During human cytomegalovirus (HCMV) infection, pUL50 targets the ER through its transmembrane domain and moves to the inner nuclear membrane (INM) to form the nuclear egress complex (NEC), which facilitates capsid transport from the nucleus to the cytoplasm. Here, we demonstrate that pUL50 induces the loss of valosin-containing protein (VCP/p97), which promotes the expression of viral major immediate early gene products, in a manner dependent on its membrane targeting but that a small isoform of pUL50 is expressed to negatively regulate this pUL50 activity. This study reports a new NEC-independent function of pUL50 and highlights the fine regulation of pUL50 activity by a smaller isoform for efficient viral growth.

Inhibition of the Super Elongation Complex Suppresses Herpes Simplex Virus Immediate Early Gene Expression, Lytic Infection, and Reactivation from Latency.

Induction of herpes simplex virus (HSV) immediate early (IE) gene transcription promotes the initiation of lytic infection and reactivation from latency in sensory neurons. IE genes are transcribed by the cellular RNA polymerase II (RNAPII) and regulated by multiple transcription factors and coactivators. The HCF-1 cellular coactivator plays a central role in driving IE expression at multiple stages through interactions with transcription factors, chromatin modulation complexes, and transcription elongation components, including the active super elongation complex/P-TEFb (SEC-P-TEFb). Here, we demonstrate that the SEC occupies the promoters of HSV IE genes during the initiation of lytic infection and during reactivation from latency. Specific inhibitors of the SEC suppress viral IE expression and block the spread of HSV infection. Significantly, these inhibitors also block the initiation of viral reactivation from latency in sensory ganglia. The potent suppression of IE gene expression by SEC inhibitors indicates that transcriptional elongation represents a determining rate-limiting stage in HSV IE gene transcription and that the SEC plays a critical role in driving productive elongation during both phases of the viral life cycle. Most importantly, this supports the model that signal-mediated induction of SEC-P-TEFb levels can promote reactivation of a population of poised latent genomes.IMPORTANCE HSV infections can cause pathologies ranging from recurrent lesions to significant ocular disease. Initiation of lytic infection and reactivation from latency in sensory neurons are dependent on the induced expression of the viral immediate early genes. Transcription of these genes is controlled at multiple levels, including modulation of the chromatin state of the viral genome and appropriate recruitment of transcription factors and coactivators. Following initiation of transcription, IE genes are subject to a key regulatory stage in which transcriptional elongation rates are controlled by the activity of the super elongation complex. Inhibition of the SEC blocks both lytic infection and reactivation from latency in sensory neurons. In addition to providing insights into the mechanisms controlling viral infection and reactivation, inhibitors of critical components such as the SEC may represent novel antivirals.