The herpes simplex virus ICP0 RING finger domain inhibits IRF3- and IRF7-mediated activation of interferon-stimulated genes

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.     

Rhesus cytomegalovirus particles prevent activation of interferon regulatory factor 3

One of the most important innate host defense mechanisms against viral infection is the induction of interferon (IFN)-stimulated genes (ISGs). Immediately upon entry, viruses activate interferon-regulatory factor 3 (IRF3), as well as nuclear factor kappaB (NF-kappaB), which transactivate a subset of ISGs, proinflammatory genes, as well as IFN genes. Most large DNA viruses exhibit countermeasures against induction of this response. However, whereas human cytomegalovirus (HCMV) inhibits IFN-dependent induction of ISGs, IFN-independent induction of ISGs is observed both in the presence and, even moreso, in the absence of viral gene expression. Rhesus CMV (RhCMV) is an emerging animal model for HCMV sharing important similarities in primary structure, epidemiology, and pathogenesis. To determine whether RhCMV would similarly induce ISGs, we performed DNA microarray and quantitative PCR analysis of ISG expression in rhesus fibroblasts infected with RhCMV or HCMV. In contrast to HCMV, however, RhCMV did not induce expression of ISGs or proinflammatory genes at any time after infection. Moreover, dimerization and nuclear accumulation of IRF3, readily observed in HCMV-infected cells, was absent from RhCMV-infected cells, whereas neither virus seemed to activate NFkappaB. RhCMV also blocked IRF3 activation by live or UV-inactivated HCMV, suggesting that RhCMV inhibits viral IRF3 activation and the resultant ISG induction with extraordinary efficiency. Since infection during inhibition of protein expression by cycloheximide or inactivation of viral gene expression by UV treatment did not trigger IRF3 activation or ISG expression by RhCMV, we conclude that RhCMV virions contain a novel inhibitor of IFN-independent viral induction of ISG expression by IRF3.     

Type I interferon-mediated immune response against influenza A virus is attenuated in the absence of p53

Influenza A virus (IAV) infection induces secretion of type I interferon (IFN) and activation of p53, which play essential roles in the host defense against tumor development and viral infection. In this study, we knocked down p53 expression by RNA interference. The expression levels of IFN-stimulated genes (ISGs) including IFN regulatory factor (IRF) 5, IRF9, ISG15, ISG20, guanylate-binding protein 1, retinoic acid-inducible gene-I and 2′-5′-oligoadenylate synthetase 1 were significantly attenuated in response to IAV infection and IFN-α stimulation in p53-knockdown cells. This attenuated expression of ISGs was associated with enhanced replication of IAV. Pretreatment of p53-knockdown cells with IFN-α failed to inhibit IAV replication, indicating impaired antiviral activity. These findings indicate that p53 plays an essential role in the enhancement of the type I IFN-mediated immune response against IAV infection.     

MiR-23a Facilitates the Replication of HSV-1 through the Suppression of Interferon Regulatory Factor 1

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression. It has been reported that miRNAs are involved in host-virus interaction, but evidence that cellular miRNAs promote virus replication has been limited. Here, we found that miR-23a promoted the replication of human herpes simplex virus type 1 (HSV-1) in HeLa cells, as demonstrated by a plaque-formation assay and quantitative real-time PCR. Furthermore, interferon regulatory factor 1 (IRF1), an innate antiviral molecule, is targeted by miR-23a to facilitate viral replication. MiR-23a binds to the 3′UTR of IRF1 and down-regulates its expression. Suppression of IRF1 expression reduced RSAD2 gene expression, augmenting HSV-1 replication. Ectopic expression of IRF1 abrogated the promotion of HSV-1 replication induced by miR-23a. Notably, IRF1 contributes to innate antiviral immunity by binding to IRF-response elements to regulate the expression of interferon-stimulated genes (ISGs) and apoptosis, revealing a complex interaction between miR-23a and HSV-1. MiR-23a thus contributes to HSV-1 replication through the regulation of the IRF1-mediated antiviral signal pathway, which suggests that miR-23a may represent a promising target for antiviral treatments.     

The IFITM proteins inhibit HIV-1 infection

Type I interferon protects cells from virus infection through the induction of a group of genes collectively named interferon-stimulated genes (ISGs). In this study, we utilized short hairpin RNA (shRNA) to deplete ISGs in SupT1 cells in order to identify ISGs that suppress the production of human immunodeficiency virus type 1 (HIV-1). Among the ISG candidates thus identified were interferon-induced transmembrane (IFITM) proteins, including IFITM1, IFITM2, and IFITM3, that potently inhibit HIV-1 replication at least partially through interfering with virus entry. Further mutagenesis analysis shows that the intracellular region, rather than the N- and C-terminal extracellular domains, is essential for the antiviral activity of IFITM1. Altogether, these data suggest that the IFITM proteins serve as important components of the innate immune system to restrict HIV-1 infection.     

Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo

The innate immune response, and in particular the alpha/beta interferon (IFN-alpha/beta) system, plays a critical role in the control of viral infections. Interferons alpha and beta exert their antiviral effects through the induction of hundreds of interferon-induced (or -stimulated) genes (ISGs). While several of these ISGs have characterized antiviral functions, their actions alone do not explain all of the effects mediated by IFN-alpha/beta. To identify additional IFN-induced antiviral molecules, we utilized a recombinant chimeric Sindbis virus to express selected ISGs in IFN-alpha/beta receptor (IFN-alpha/betaR)(-/-) mice and looked for attenuation of Sindbis virus infection. Using this approach, we identified a ubiquitin homolog, interferon-stimulated gene 15 (ISG15), as having antiviral activity. ISG15 expression protected against Sindbis virus-induced lethality and decreased Sindbis virus replication in multiple organs without inhibiting the spread of virus throughout the host. We establish that, much like ubiquitin, ISG15 requires its C-terminal LRLRGG motif to form intracellular conjugates. Finally, we demonstrate that ISG15's LRLRGG motif is also required for its antiviral activity. We conclude that ISG15 can be directly antiviral.     

TRAF6 and IRF7 control HIV replication in macrophages

The innate immune system recognizes virus infection and evokes antiviral responses which include producing type I interferons (IFNs). The induction of IFN provides a crucial mechanism of antiviral defense by upregulating interferon-stimulated genes (ISGs) that restrict viral replication. ISGs inhibit the replication of many viruses by acting at different steps of their viral cycle. Specifically, IFN treatment prior to in vitro human immunodeficiency virus (HIV) infection stops or significantly delays HIV-1 production indicating that potent inhibitory factors are generated. We report that HIV-1 infection of primary human macrophages decreases tumor necrosis factor receptor-associated factor 6 (TRAF6) and virus-induced signaling adaptor (VISA) expression, which are both components of the IFN signaling pathway controlling viral replication. Knocking down the expression of TRAF6 in macrophages increased HIV-1 replication and augmented the expression of IRF7 but not IRF3. Suppressing VISA had no impact on viral replication. Overexpression of IRF7 resulted in enhanced viral replication while knocking down IRF7 expression in macrophages significantly reduced viral output. These findings are the first demonstration that TRAF6 can regulate HIV-1 production and furthermore that expression of IRF7 promotes HIV-1 replication.     

Vpu mediates depletion of interferon regulatory factor 3 during HIV infection by a lysosome-dependent mechanism

HIV has evolved sophisticated mechanisms to avoid restriction by intracellular innate immune defenses that otherwise serve to control acute viral infection and virus dissemination. Innate defenses are triggered when pattern recognition receptor (PRR) proteins of the host cell engage pathogen-associated molecule patterns (PAMPs) present in viral products. Interferon regulatory factor 3 (IRF3) plays a central role in PRR signaling of innate immunity to drive the expression of type I interferon (IFN) and interferon-stimulated genes (ISGs), including a variety of HIV restriction factors, that serve to limit viral replication directly and/or program adaptive immunity. Productive infection of T cells by HIV is dependent upon the targeted proteolysis of IRF3 that occurs through a virus-directed mechanism that results in suppression of innate immune defenses. However, the mechanisms by which HIV controls innate immune signaling and IRF3 function are not defined. Here, we examined the innate immune response induced by HIV strains identified through their differential control of PRR signaling. We identified viruses that, unlike typical circulating HIV strains, lack the ability to degrade IRF3. Our studies show that IRF3 regulation maps specifically to the HIV accessory protein Vpu. We define a molecular interaction between Vpu and IRF3 that redirects IRF3 to the endolysosome for proteolytic degradation, thus allowing HIV to avoid the innate antiviral immune response. Our studies reveal that Vpu is an important IRF3 regulator that supports acute HIV infection through innate immune suppression. These observations define the Vpu-IRF3 interface as a novel target for therapeutic strategies aimed at enhancing the immune response to HIV.     

Identification of a novel pathway essential for the immediate-early, interferon-independent antiviral response to enveloped virions

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.     

Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-α/β) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-α/β production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.     

The interferon-inducible gene viperin restricts West Nile virus pathogenesis

Type I interferon (IFN) signaling coordinates an early antiviral program in infected and uninfected cells by inducing IFN-stimulated genes (ISGs) that modulate viral entry, replication, and assembly. However, the specific antiviral functions in vivo of most ISGs remain unknown. Here, we examined the contribution of the ISG viperin to the control of West Nile virus (WNV) in genetically deficient cells and mice. While modest increases in levels of WNV replication were observed for primary viperin(-/-) macrophages and dendritic cells, no appreciable differences were detected in deficient embryonic cortical neurons or fibroblasts. In comparison, viperin(-/-) adult mice infected with WNV via the subcutaneous or intracranial route showed increased lethality and/or enhanced viral replication in central nervous system (CNS) tissues. In the CNS, viperin expression was induced in both WNV-infected and adjacent uninfected cells, including activated leukocytes at the site of infection. Our experiments suggest that viperin restricts the infection of WNV in a tissue- and cell-type-specific manner and may be an important ISG for controlling viral infections that cause CNS disease.     

Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells

The unparalleled spread of highly pathogenic avian influenza A (HPAI) H5N1 viruses has resulted in devastating outbreaks in domestic poultry and sporadic human infections with a high fatality rate. To better understand the mechanism(s) of H5N1 virus pathogenesis and host responses in humans, we utilized a polarized human bronchial epithelial cell model that expresses both avian alpha-2,3- and human alpha-2,6-linked sialic acid receptors on the apical surface and supports productive replication of both H5N1 and H3N2 viruses. Using this model, we compared the abilities of selected 2004 HPAI H5N1 viruses isolated from humans and a recent human H3N2 virus to trigger the type I interferon (IFN) response. H5N1 viruses elicited significantly less IFN regulatory factor 3 (IRF3) nuclear translocation, as well as delayed and reduced production of IFN-beta compared with the H3N2 virus. Furthermore, phosphorylation of Stat2 and induction of IFN-stimulated genes (ISGs), such as MX1, ISG15, IRF7, and retinoic acid-inducible gene I, were substantially delayed and reduced in cells infected with H5N1 viruses. We also observed that the highly virulent H5N1 virus replicated more efficiently and induced a weaker IFN response than the H5N1 virus that exhibited low virulence in mammals in an earlier study. Our data suggest that the H5N1 viruses tested, especially the virus with the high-pathogenicity phenotype, possess greater capability to attenuate the type I IFN response than the human H3N2 virus. The attenuation of this critical host innate immune defense may contribute to the virulence of H5N1 viruses observed in humans.