There has been increased interest in the possible role of human cytomegalovirus (HCMV) in carcinogenesis during the last decade. HCMV seroprevalence was enhanced in patients with hepatocellular carcinoma (HCC) but a possible relationship between HCC and HCMV infection remained to be assessed. The aim of this work was to investigate the pro-tumor influence of HCMV on primary human hepatocytes (PHH) and HepG2 cells.
Following infection of PHH and HepG2 cells by two different strains of HCMV, we measured the production of IL-6 in culture supernatants by ELISA and the protein levels of STAT3, pSTAT3, JAK, cyclin D1, survivin, p53, p21, and Mdm2 by western Blotting in infected and uninfected cells. Cell proliferation and transformation were investigated using Ki67Ag expression measurement and soft-agar colony formation assay respectively.
Infection of HepG2 cells and PHH by HCMV resulted in the production of IL-6 and the subsequent activation of the IL-6R-JAK-STAT3 pathway. HCMV increased the expression of cyclin D1 and survivin. Cell proliferation was enhanced in HepG2 and PHH infected with HCMV, despite a paradoxical overexpression of p53 and p21. More importantly, we observed the formation of colonies in soft agar seeded with PHH infected with HCMV and when we challenged the HepG2 cultures to form tumorspheres, we found that the HCMV-infected cultures formed 2.5-fold more tumorspheres than uninfected cultures.
HCMV activated the IL-6-JAK-STAT3 pathway in PHH and HepG2 cells, favored cellular proliferation, induced PHH transformation and enhanced HepG2 tumorsphere formation. Our observations raise the possibility that HCMV infection might be involved in the genesis of hepatocellular carcinoma.
The interferon (IFN)-mediated antiviral response is a major defense of the host immune system. In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Herpes simplex virus 1 (HSV-1) is a large DNA virus containing more than 80 genes, many of which encode proteins that are involved in virus-host interactions and show immune modulatory capabilities. In this study, we demonstrate that the US11 protein, an RNA binding tegument protein of HSV-1, is a novel antagonist of the beta IFN (IFN-β) pathway. US11 significantly inhibited Sendai virus (SeV)-induced IFN-β production, and its double-stranded RNA (dsRNA) binding domain was indispensable for this inhibition activity. Additionally, wild-type HSV-1 coinfection showed stronger inhibition than US11 mutant HSV-1 in SeV-induced IFN-β production. Coimmunoprecipitation analysis demonstrated that the US11 protein in HSV-1-infected cells interacts with endogenous RIG-I and MDA-5 through its C-terminal RNA-binding domain, which was RNA independent. Expression of US11 in both transfected and HSV-1-infected cells interferes with the interaction between MAVS and RIG-I or MDA-5. Finally, US11 dampens SeV-mediated IRF3 activation. Taken together, the combined data indicate that HSV-1 US11 binds to RIG-I and MDA-5 and inhibits their downstream signaling pathway, preventing the production of IFN-β, which may contribute to the pathogenesis of HSV-1 infection.
Varicella-zoster virus (VZV) infection of differentiated cells within the host and establishment of latency likely requires evasion of innate immunity and limits secretion of antiviral cytokines. Here we report that its immediate-early protein ORF61 antagonizes the beta interferon (IFN-β) pathway. VZV infection down-modulated the Sendai virus (SeV)-activated IFN-β pathway, including mRNA of IFN-β and its downstream interferon-stimulated genes (ISGs), ISG54 and ISG56. Through a primary screening of VZV genes, we found that ORF61 inhibited SeV-mediated activation of IFN-β and ISRE (IFN-stimulated response element) promoter activities but only slightly affected NF-κB promoter activity, implying that the IFN-β pathway may be blocked in the IRF3 branch. An indirect immunofluorescence assay demonstrated that ectopic expression of ORF61 abrogated the detection of IRF3 in SeV-infected cells; however, it did not affect endogenous dormant IRF3 in noninfected cells. Additionally, ORF61 was shown to be partially colocalized with activated IRF3 in the nucleus upon treatment with MG132, an inhibitor of proteasomes, and the direct interaction between ORF61 and activated IRF3 was confirmed by a coimmunoprecipitation assay. Furthermore, Western blot analysis demonstrated that activated IRF3 was ubiquitinated in the presence of ORF61, suggesting that ORF61 degraded phosphorylated IRF3 via a ubiquitin-proteasome pathway. Semiquantitative reverse transcription-PCR (RT-PCR) analysis demonstrated that the level of ISG54 and ISG56 mRNAs was also downregulated by ORF61. Taken together, our results convincingly demonstrate that ORF61 down-modulates the IRF3-mediated IFN-β pathway by degradation of activated IRF3 via direct interaction, which may contribute to the pathogenesis of VZV infection.
We previously found that enveloped virus binding and penetration are necessary to initiate an interferon-independent, IRF3-mediated antiviral response. To investigate whether membrane perturbations that accompany membrane fusion-dependent enveloped-virus entry are necessary and sufficient for antiviral-state induction, we utilized a reovirus fusion-associated small transmembrane (FAST) protein. Membrane disturbances during FAST protein-mediated fusion, in the absence of additional innate immune response triggers, are sufficient to elicit interferon-stimulated gene induction and establishment of an antiviral state. Using sensors of membrane disruption to activate an IRF3-dependent, interferon-independent antiviral state may provide cells with a rapid, broad-spectrum innate immune response to enveloped-virus infections.
The cellular defense to infection depends on accurate activation of transcription factors and expression of select innate immunity genes. Interferon regulatory factor 5 (IRF5), a risk factor for systemic lupus erythematosus, is activated in response to pathogen recognition receptor engagement and downstream effector molecules. We find the nucleotide-binding oligomerization domain containing protein 2 (NOD2) receptor to be a significant activator of IRF5. Phosphorylation is key to the regulation of IRF5, but the precise phosphorylation sites in IRF5 remained to be identified. We used mass spectrometry to identify for the first time specific residues that are phosphorylated in response to TANK-binding kinase-1 (TBK-1), tumor necrosis factor receptor-associated factor 6 (TRAF6), or receptor interacting protein 2 (RIP2). RIP2, a kinase known to function downstream of NOD2, was the most effective activator of IRF5-regulated gene expression. To determine if the phosphorylated residues are required or sufficient for IRF5 activity, aspartic acid phosphomimetic substitutions or inactivating alanine substitutions were tested. Phosphorylation of carboxyl serines 451 and 462 appear the primary trigger of IRF5 function in nuclear accumulation, transcription, and apoptosis. Results indicate polyubiquitination of IRF5 does not play a major role in its transcriptional activity, and that ubiquitination and phosphorylation are independent modifications.
The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection.
Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMO-modified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection.
Viruses must evade several antiviral defences in order to establish a productive infection. These include antibody- and cell-mediated acquired immunity and interferon-regulated innate immunity. Recently, a third arm of antiviral defence has been discovered, so called intrinsic immunity. This aspect of antiviral resistance represents the first line of intracellular defence against virus infection and is mediated by pre-existing cellular factors that attempt to repress viral replication during the initial stages of infection. Like acquired and innate immunity, viruses have evolved mechanisms that overcome intrinsic defence. Here we show that in response to herpes simplex virus type-1 (HSV-1) infection an important aspect of intrinsic immunity is regulated by the small ubiquitin-like modifier (SUMO) conjugation pathway. In response to this defence, the virus induces rapid degradation of specific SUMO-conjugated proteins, followed by widespread loss of SUMO-conjugated species in general. Inactivation of the SUMO pathway inhibits the cell’s ability to efficiently repress viral replication in the absence of this viral countermeasure. Our data identifies an important regulatory pathway that mediates intrinsic resistance to HSV-1 infection and describes the biochemical mechanism that the virus utilizes in order to counteract this antiviral defence.
Components of promyelocytic leukaemia (PML) nuclear bodies (ND10) are recruited to sites associated with herpes simplex virus type 1 (HSV-1) genomes soon after they enter the nucleus. This cellular response is linked to intrinsic antiviral resistance and is counteracted by viral regulatory protein ICP0. We report that the SUMO interaction motifs of PML, Sp100 and hDaxx are required for recruitment of these repressive proteins to HSV-1 induced foci, which also contain SUMO conjugates and PIAS2β, a SUMO E3 ligase. SUMO modification of PML and elements of its tripartite motif (TRIM) are also required for recruitment in cells lacking endogenous PML. Mutants of PML isoform I and hDaxx that are not recruited to virus induced foci are unable to reproduce the repression of ICP0 null mutant HSV-1 infection mediated by their wild type counterparts. We conclude that recruitment of ND10 components to sites associated with HSV-1 genomes reflects a cellular defence against invading pathogen DNA that is regulated through the SUMO modification pathway.
Viruses encounter several different defences that impede infection, including acquired immunity mediated by the immune system and innate immunity that includes the synthesis of antiviral proteins through the interferon pathway. In recent years, a third arm of antiviral defence has been described, named intrinsic immunity or intrinsic resistance, that is conferred by constitutively expressed cellular proteins. In the case of herpesviruses, intrinsic resistance involves the action of cellular repressors that restrict viral transcription once the viral genome enters the nucleus. Several studies have presented evidence that one aspect of intrinsic resistance involves cellular proteins that form distinct nuclear structures known as ND10. Several ND10 components are known to accumulate rapidly at sites in close association with herpes simplex virus type 1 genomes. Here we report that this cellular response requires the ability of several of the proteins in question to interact with a small ubiquitin-like protein known as SUMO. In two such examples of these proteins, we show that their ability to interact with SUMO is required for their roles in repressing viral infection. We suggest that this SUMO-dependent pathway may underlie a more general mechanism by which cells protect themselves from invading foreign DNA.
Hepatitis C virus (HCV) infection causes significant morbidity, and efficient mouse models would greatly facilitate virus studies and the development of effective vaccines and new therapeutic agents. Entry factors, innate immunity, and host factors needed for viral replication represent the initial barriers that restrict HCV infection of mouse cells. Experiments in this paper consider early postentry steps of viral infection and investigate the roles of interferon regulatory factors (IRF-3 and IRF-9) and microRNA (miR-122) in promoting HCV replication in mouse embryo fibroblasts (MEFs) that contain viral subgenomic replicons. While wild-type murine fibroblasts are restricted for HCV RNA replication, deletion of IRF-3 alone can facilitate replicon activity in these cells. This effect is thought to be related to the inactivation of the type I interferon synthesis mediated by IRF-3. Additional deletion of IRF-9 to yield IRF-3−/− IRF-9−/− MEFs, which have blocked type I interferon signaling, did not increase HCV replication. Expression of liver-specific miR-122 in MEFs further stimulated the synthesis of HCV replicons in the rodent fibroblasts. The combined effects of miR-122 expression and deletion of IRF-3 produced a cooperative stimulation of HCV subgenome replication. miR-122 and IRF-3 are independent host factors that are capable of influencing HCV replication, and our findings could help to establish mouse models and other cell systems that support HCV growth and particle formation.
Herpesviral entry is a highly elaborated process requiring many proteins to act in precise conjunction. Neutralizing antibodies interfere with this process to abrogate viral infection. Based on promoter transactivation of a reporter gene we established a novel method to quantify herpesvirus entry and neutralization by antibodies. Following infection with mouse and human cytomegalovirus and Herpes simplex virus 1 we observed promoter transactivation resulting in substantial luciferase expression (>1000-fold). No induction was elicited by UV-inactivated viruses. The response was MOI-dependent and immunoblots confirmed a correlation between luciferase induction and pp72-IE1 expression. Monoclonal antibodies, immune sera and purified immunoglobulin preparations decreased virus-dependent luciferase induction dose-dependently, qualifying this approach as surrogate virus neutralization test. Besides the reduced hands-on time, this assay allows analysis of herpesvirus entry in semi-permissive and non-adherent cells, which were previously non-assessable but play significant roles in herpesvirus pathology.
Virus infection elicits a robust innate antiviral response dominated by the production of type 1 IFN. In non-professional innate immune cells such as fibroblasts, type 1 IFN is rapidly produced following the recognition of viral dsRNA and the subsequent activation of the constitutively expressed transcription factor IFN regulatory factor 3 (IRF3). While origin, localization and length are factors in mediating dsRNA recognition and binding by cellular dsRNA binding proteins, the biological significance of differential dsRNA binding is unclear, since the subsequent signaling pathways converge on IRF3. Here, we show a dsRNA length dependent activation of IRFs, IFNs and IFN stimulated genes in mouse fibroblasts. The length dependence was exacerbated in fibroblasts deficient in the mitochondria-associated adaptor IPS-1 and IRF3, suggesting that antiviral gene induction mediated by short and long dsRNA molecules is predominantly IPS-1 and IRF3-dependent and –independent, respectively. Furthermore, we provide evidence of an innate antiviral response in fibroblasts in the absence of both IRF3 and type 1 IFN induction. Even with these key modulators missing, a 60% to 90% inhibition of virus replication was observed following 24-hour treatment with short or long dsRNA molecules, respectively. These data provide evidence of a novel antiviral pathway that is dependent on dsRNA length, but independent of the type 1 IFN system.
Chronic obstructive pulmonary disease is a progressive lung disease that is punctuated by periods of exacerbations (worsening of symptoms) that are attributable to viral infections. While rhinoviruses are most commonly isolated viruses during episodes of exacerbation, influenza viruses have the potential to become even more problematic with the increased likelihood of an epidemic.
Methodology and Principal Findings
This study examined the impact of current and potential pharmacological targets namely the systemic corticosteroid dexamethasone and the peroxisome proliferator-activated receptor- gamma agonist pioglitazone on the outcome of infection in smoke-exposed mice. C57BL/6 mice were exposed to room air or cigarette smoke for 4 days and subsequently inoculated with an H1N1 influenza A virus. Interventions were delivered daily during the course of infection. We show that smoke-exposed mice have an exacerbated inflammatory response following infection. While smoke exposure did not compromise viral clearance, precision cut lung slices from smoke-exposed mice showed greater expression of CC (MCP-1, -3), and CXC (KC, MIP-2, GCP-2) chemokines compared to controls when stimulated with a viral mimic or influenza A virus. While dexamethasone treatment partially attenuated the inflammatory response in the broncho-alveolar lavage of smoke-exposed, virally-infected animals, viral-induced neutrophilia was steroid insensitive. In contrast to controls, dexamethasone-treated smoke-exposed influenza-infected mice had a worsened health status. Pioglitazone treatment of virally-infected smoke-exposed mice proved more efficacious than the steroid intervention. Further mechanistic evaluation revealed that a deficiency in CCR2 did not improve the inflammatory outcome in smoke-exposed, virally-infected animals.
Conclusions and Significance
This animal model of cigarette smoke and H1N1 influenza infection demonstrates that smoke-exposed animals are differentially primed to respond to viral insult. While providing a platform to test pharmacological interventions, this model demonstrates that treating viral exacerbations with alternative anti-inflammatory drugs, such as PPAR-gamma agonists should be further explored since they showed greater efficacy than systemic corticosteroids.
Interferon regulatory factor 3 (IRF3) is important for innate antiviral responses; accordingly, many viruses target and inactivate IRF3. The ability of the Herpes simplex virus type 1 (HSV-1) immediate early protein ICP0 to inhibit IRF3 is controversial and has not been studied solely in the context of a wild type HSV-1 infection. Discrepancies in the literature surround the mechanism by which ICP0 antagonizes the IRF3 pathway, the cellular localization of ICP0 inhibitory activity and the ability of ICP0 to interfere with interferon and interferon-stimulated gene induction. In this study, we set out to investigate the role of ICP0 localization and the requirement of the proteasome during the inhibition of IRF3 activation within the context of an HSV-1 infection. Collectively, the results presented herein demonstrate that incoming wild type HSV-1 activates IRF3 and that de novo produced ICP0 prevents sustained IRF3 activation following its translocation from the nucleus to the cytoplasm. While previous studies implicate the E3 ubiquitin ligase domain of ICP0 in mediating its biological functions, including the inhibition of IRF3, we show that cytoplasmic ICP0 does not require the proteasome for this activity. Instead, proteasome function is required to localize ICP0 to the cytoplasm where it mediates its inhibitory effect independent of E3 ubiquitin ligase activity. The importance of these findings is discussed within the context of an HSV-1 infection.
Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (ds)RNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN) responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As) are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNβ. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG) induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as ‘carriers’, facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions.
Nearly all viruses produce dsRNA during their replication cycle. This molecule is not normally found in a healthy host cell and thus functions as a flag, alerting the host to a viral infection. Cells can die by lysis during virus infections, and the intracellular dsRNA is then released into the extracellular space. This dsRNA is stable in the extracellular milieu, and is able to function as a signaling molecule, detected by neighboring cells. This has been observed experimentally, as extracellular dsRNA has been used for years to trigger host antiviral responses. It has also been suggested that extracellular dsRNA plays a role in causing pathological symptoms in virus infected patients. Our data suggests that class A scavenger receptors (SR-As) function as cell surface receptors for dsRNA. SR-As bind extracellular, viral dsRNA and mediate its entry into the cell, where it delivers the dsRNA to other known intracellular dsRNA sensors, activating intracellular antiviral responses. These findings shed new light on how the host detects and responds to virus infection.
Herpes simplex virus VP16 and ICP0 mutants replicate efficiently in U2OS osteosarcoma cells but are restricted in other cell types. We previously showed that the restrictive phenotype is dominant in a transient cell fusion assay, suggesting that U2OS cells lack an antiviral mechanism present in other cells. Recent data indicate that unscheduled membrane fusion events can activate the expression of interferon-stimulated genes (ISGs) in fibroblasts, raising the possibility that our earlier results were due to a fusion-induced antiviral state. However, we show here that the permissive phenotype is also extinguished following fusion with Vero cells in the absence of ISG induction.
Immune responses against HSV-1 and HSV-2 are complex and involve a delicate interplay between innate signaling pathways and adaptive immune responses. The innate response to HSV involves the induction of type I IFN, whose role in protection against disease is well characterized in vitro and in vivo. Cell types such as NK cells and pDCs contribute to innate anti-HSV responses in vivo. Finally, the adaptive response includes both humoral and cellular components that play important roles in antiviral control and latency. This review summarizes the innate and adaptive effectors that contribute to susceptibility, immune control and pathogenesis of HSV, and highlights the delicate interplay between these two important arms of immunity.
Herpes Simplex virus (HSV); innate immunity; antiviral signaling; type I interferon (IFN); Natural killer (NK) cells; plasmacytoid dendritic cells (pDCs); adaptive immunity
Viral infection elicits the activation of numerous cellular signal transduction pathways, leading to the induction of both innate and adaptive immune responses in the host. In particular, interferon regulatory factor 3 (IRF3) has been shown to be essential for the induction of an antiviral response. Current models suggest that virus replication causes phosphorylation of C-terminal serine and threonine residues on IRF3, leading to its dimerization and translocation to the nucleus, where it activates interferon. Upon entry of replication-deficient Newcastle disease virus (NDV) particles, however, we failed to detect IRF3 dimerization or hyperphosphorylation, despite robust interferon-stimulated gene (ISG) and antiviral state induction and confirmation by small interfering RNA knockdown that IRF3 is essential for this response. To further compare the effects of various viruses and their replication status on IRF3 activation and to determine the minimal posttranslational modification required for IRF3 activation, two-dimensional gel electrophoresis and native polyacrylamide gel electrophoresis were employed. However, we failed to identify a minimal posttranslational modification of IRF3 that correlated with downstream biological activity, and the extent of posttranslational modification observed on IRF3 did not correlate with the degree of subsequent ISG induction. Thus, current techniques used to detect IRF3 activation are insufficient to infer its role in mediating downstream biological response induction and should be utilized with caution.
Toll-like receptors (TLRs) constitute a family of innate receptors that recognize and respond to a wide spectrum of microorganisms, including fungi, bacteria, viruses, and protozoa. Previous studies have demonstrated that ligands for TLR3 and TLR9 induce potent innate antiviral responses against herpes simplex virus type 2 (HSV-2). However, the factor(s) involved in this innate protection is not well-defined. Here we report that production of beta interferon (IFN-β) but not production of IFN-α, IFN-γ, or tumor necrosis factor alpha (TNF-α) strongly correlates with innate protection against HSV-2. Local delivery of poly(I:C) and CpG oligodeoxynucleotides induced significant production of IFN-β in the genital tract and provided complete protection against intravaginal (IVAG) HSV-2 challenge. There was no detectable IFN-β in mice treated with ligands for TLR4 or TLR2, and these mice were not protected against subsequent IVAG HSV-2 challenge. There was no correlation between levels of TNF-α or IFN-γ in the genital tract and protection against IVAG HSV-2 challenge following TLR ligand delivery. Both TNF-α−/− and IFN-γ−/− mice were protected against IVAG HSV-2 challenge following local delivery of poly(I:C). To confirm that type I interferon, particularly IFN-β, mediates innate protection, mice unresponsive to type I interferons (IFN-α/βR−/− mice) and mice lacking IFN regulatory factor-3 (IRF-3−/− mice) were treated with poly(I:C) and then challenged with IVAG HSV-2. There was no protection against HSV-2 infection following poly(I:C) treatment of IFN-α/βR−/− or IRF-3−/− mice. Local delivery of murine recombinant IFN-β protected C57BL/6 and IRF-3−/− mice against IVAG HSV-2 challenge. Results from these in vivo studies clearly suggest a strong correlation between IFN-β production and innate antiviral immunity against HSV-2.
Viral infection elicits the activation of numerous cellular signal transduction pathways, leading to the induction of both innate and adaptive immunity. Previously we showed that entry of virion particles from a diverse array of enveloped virus families was capable of eliciting an interferon regulatory factor 3 (IRF-3)-mediated antiviral state in human fibroblasts in the absence of interferon production. Here we show that extracellular regulated kinase 1/2, p38 mitogen-activated protein kinase, and Jun N-terminal kinase/stress-activated protein kinase activities are not required for antiviral state induction. In contrast, treatment of cells with LY294002, an inhibitor of the phosphoinositide 3-kinase (PI3 kinase) family, prevents the induction of interferon-stimulated gene 56 (ISG56) and an antiviral response upon entry of virus particles. However, the prototypic class I p85/p110 PI3 kinase and its downstream effector Akt/PKB are dispensable for ISG and antiviral state induction. Furthermore, DNA-PK and PAK1, LY294002-sensitive members of the PI3 kinase family shown previously to be involved in IRF-3 activation, are also dispensable for ISG and antiviral state induction. The LY294002 inhibitor fails to prevent IRF-3 homodimerization or nuclear translocation upon virus particle entry. Together, these data suggest that virus entry triggers an innate antiviral response that requires the activity of a novel PI3 kinase family member.
The classical interferon (IFN)-dependent antiviral response to viral infection involves the regulation of IFN-stimulated genes (ISGs), one being the gene encoding cellular endoribonuclease RNase L, which arrests protein synthesis and induces apoptosis by nonspecifically cleaving rRNA. Recently, the herpes simplex virus type 1 (HSV-1) protein ICP0 has been shown to block the induction of ISGs by subverting the IFN pathway upstream of the 2′-5′-oligoadenylate synthetase (OAS)/RNase L pathway. We report that ICP0 also prevents rRNA degradation at late stages of HSV-1 infection, independent of its E3 ubiquitin ligase activity, and that the resultant rRNA degradation is independent of the classical RNase L antiviral pathway. Moreover, the degradation is independent of the viral RNase vhs and is independent of IFN response factor 3. These studies indicate the existence of another, previously unidentified, RNase that is part of the host antiviral response to viral infection.
Newly delivered herpes simplex virus genomes are subject to repression during the early stages of infection of human fibroblasts. This host defence strategy can limit virus replication and lead to long-term persistence of quiescent viral genomes. The viral immediate-early protein ICP0 acts to negate this negative regulation, thereby facilitating the onset of the viral replication cycle. Although few mechanistic details are available, the host repression machinery has been proposed to assemble the viral genome into a globally inaccessible configuration analogous to heterochromatin, blocking access to most or all trans-acting factors. The strongest evidence for this hypothesis is that ICP0-deficient virus is unable to reactivate quiescent viral genomes, despite its ability to undergo productive infection given a sufficiently high multiplicity of infection. However, recent studies have shown that quiescent infection induces a potent antiviral state, and that ICP0 plays a key role in disarming such host antiviral responses. These findings raise the possibility that cells containing quiescent viral genomes may be refractory to superinfection by ICP0-deficient virus, potentially providing an alternative explanation for the inability of such viruses to trigger reactivation. We therefore asked if ICP0-deficient virus is capable of replicating in cells that contain quiescent viral genomes.
We found that ICP0-deficient herpes simplex virus is able to infect quiescently infected cells, leading to expression and replication of the superinfecting viral genome. Despite this productive infection, the resident quiescent viral genome was neither expressed nor replicated, unless ICP0 was provided in trans.
These data document that quiescent HSV genomes fail to respond to the virally modified host transcriptional apparatus or viral DNA replication machinery provided in trans by productive HSV infection in the absence of ICP0. These results point to global repression as the basis for HSV genome quiescence, and indicate that ICP0 induces reactivation by overcoming this global barrier to the access of trans-acting factors.
Mammalian cells respond to virus infections by eliciting both innate and adaptive immune responses. One of the most effective innate antiviral responses is the production of alpha/beta interferon and the subsequent induction of interferon-stimulated genes (ISGs), whose products collectively limit virus replication and spread. Following viral infection, interferon is produced in a biphasic fashion that involves a number of transcription factors, including the interferon regulatory factors (IRFs) 1, 3, 7, and 9. In addition, virus infection has been shown to directly induce ISGs in the absence of prior interferon production through the activation of IRF3. This process is believed to require virus replication and results in IRF3 hyperphosphorylation, nuclear localization, and proteasome-mediated degradation. Previously, we and others demonstrated that herpes simplex virus type 1 (HSV-1) induces ISGs and an antiviral response in fibroblasts in the absence of both interferon production and virus replication. In this report, we show that the entry of enveloped virus particles from diverse virus families elicits a similar innate response. This process requires IRF3, but not IRF1, IRF7, or IRF9. Following virus replication, the large DNA viruses HSV-1 and vaccinia virus effectively inhibit ISG mRNA accumulation, whereas the small RNA viruses Newcastle disease virus, Sendai virus, and vesicular stomatitis virus do not. In addition, we found that IRF3 hyperphosphorylation and degradation do not correlate with ISG and antiviral state induction but instead serve as a hallmark of productive virus replication, particularly following a high-multiplicity infection. Collectively, these data suggest that virus entry triggers an innate antiviral response mediated by IRF3 and that subsequent virus replication results in posttranslational modification of IRF3, such as hyperphosphorylation, depending on the nature of the incoming virus.
Virus infection induces a rapid cellular response in cells characterized by the induction of interferon. While interferon itself does not induce an antiviral response, it activates a number of interferon-stimulated genes that collectively function to inhibit virus replication and spread. Previously, we and others reported that herpes simplex virus type 1 (HSV-1) induces an interferon -independent antiviral response in the absence of virus replication. Here, we report that the HSV-1 proteins ICP0 and vhs function in concert to disable the host antiviral response. In particular, we show that ICP0 blocks interferon regulatory factor IRF3- and IRF7-mediated activation of interferon-stimulated genes and that the RING finger domain of ICP0 is essential for this activity. Furthermore, we demonstrate that HSV-1 modifies the IRF3 pathway in a manner different from that of the small RNA viruses most commonly studied.
Interferon inhibits virus replication through multiple mechanisms. Here we show that herpes simplex virus proteins ICP0 and ICP34.5 overcome interferon-induced barriers to viral transcription and translation, respectively. These cytokine-induced antiviral mechanisms are differentially expressed in established cell lines: U2OS cells do not mount the IFN-induced mechanism targeted by ICP0, and Vero cells may be defective for the mechanism targeted by ICP34.5.
Virus infection induces an antiviral response that is predominantly associated with the synthesis and secretion of soluble interferon. Here, we report that herpes simplex virus type 1 virions induce an interferon-independent antiviral state in human embryonic lung cells that prevents plaquing of a variety of viruses. Microarray analysis of 19,000 human expressed sequence tags revealed induction of a limited set of host genes, the majority of which are also induced by interferon. Genes implicated in controlling the intracellular spread of virus and eliminating virally infected cells were among those induced. Induction of the cellular response occurred in the absence of de novo cellular protein synthesis and required viral penetration. In addition, this response was only seen when viral gene expression was inhibited, suggesting that a newly synthesized viral protein(s) may function as an inhibitor of this response.