BACKGROUND

Innate immune responses have recently been appreciated to play an important role in the pathogenesis of HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control and eradicate infection, persistent inflammatory responses later during infection contribute in driving chronic immune activation and development of immunodeficiency. However, knowledge on specific HIV PAMPs and cellular PRRs responsible for inducing innate immune responses remains sparse.

RESULTS

Here we demonstrate a major role for RIG-I and the adaptor protein MAVS in induction of innate immune responses to HIV genomic RNA. We found that secondary structured HIV-derived RNAs induced a response similar to genomic RNA. In primary human peripheral blood mononuclear cells and primary human macrophages, HIV RNA induced expression of IFN-stimulated genes, whereas only low levels of type I IFN and tumor necrosis factor α were produced. Furthermore, secondary structured HIV-derived RNA activated pathways to NF-κB, MAP kinases, and IRF3 and co-localized with peroxisomes, suggesting a role for this organelle in RIG-I-mediated innate immune sensing of HIV RNA.

CONCLUSIONS

These results establish RIG-I as an innate immune sensor of cytosolic HIV genomic RNA with secondary structure, thereby expanding current knowledge on HIV molecules capable of stimulating the innate immune system.

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.

Crimean Congo hemorrhagic fever virus (CCHFV) is a deadly human pathogen that evades innate immune responses by efficiently interfering with antiviral signaling pathways mediated by NF-κB, IRF3, and IFNα/β. These pathways rely on protein ubiquitination for their activation, and one outcome is the modification of proteins with the ubiquitin (Ub)-like modifier interferon-stimulated gene (ISG)15. CCHFV and related viruses encode a deubiquitinase (DUB) of the ovarian tumor (OTU) family, which unlike eukaryotic OTU DUBs also targets ISG15 modifications. Here we characterized the viral OTU domain of CCHFV (vOTU) biochemically and structurally, revealing that it hydrolyzes four out of six tested Ub linkages, but lacks activity against linear and K29-linked Ub chains. vOTU cleaved Ub and ISG15 with similar kinetics, and we were able to understand vOTU cross-reactivity at the molecular level from crystal structures of vOTU in complex with Ub and ISG15. An N-terminal extension in vOTU not present in eukaryotic OTU binds to the hydrophobic Ile44 patch of Ub, which results in a dramatically different Ub orientation compared to a eukaryotic OTU-Ub complex. The C-terminal Ub-like fold of ISG15 (ISG15-C) adopts an equivalent binding orientation. Interestingly, ISG15-C contains an additional second hydrophobic surface that is specifically contacted by vOTU. These subtle differences in Ub/ISG15 binding allowed the design of vOTU variants specific for either Ub or ISG15, which will be useful tools to understand the relative contribution of ubiquitination vs. ISGylation in viral infection. Furthermore, the crystal structures will allow structure-based design of antiviral agents targeting this enzyme.

The cellular innate immune system recognizing pathogen infection is essential for host defense against viruses. In parallel, viruses have developed a variety of strategies to evade the innate immunity. The hepatitis B virus (HBV), a DNA virus that causes chronic hepatitis, has been shown to inhibit RNA helicase RIG-I-mediated interferon (IFN) induction. However, it is still unknown whether HBV could affect the host DNA-sensing pathways. Here we report that in transiently HBV-transfected Huh7 cells, the stably HBV-producing cell line HepAD38, and HBV-infected HepaRG cells and primary human hepatocytes, HBV markedly interfered with IFN-β induction and antiviral immunity mediated by the stimulator of interferon genes (STING), which has been identified as a central factor in foreign DNA recognition and antiviral innate immunity. Screening analysis demonstrated that the viral polymerase (Pol), but not other HBV-encoded proteins, was able to inhibit STING-stimulated interferon regulatory factor 3 (IRF3) activation and IFN-β induction. Moreover, the reverse transcriptase (RT) and the RNase H (RH) domains of Pol were identified to be responsible for the inhibitory effects. Furthermore, Pol was shown to physically associate with STING and dramatically decrease the K63-linked polyubiquitination of STING via its RT domain without altering the expression level of STING. Taken together, these observations suggest that besides its inherent catalytic function, Pol has a role in suppression of IFN-β production by direct interaction with STING and subsequent disruption of its K63-linked ubiquitination, providing a new mechanism for HBV to counteract the innate DNA-sensing pathways.

OBJECTIVE

Although whether and how HBV infection induces the innate immune responses are still controversial, it has become increasingly clear that HBV has developed strategies to counteract the pattern recognition receptor-mediated signaling pathways. Previous studies have shown that type I IFN induction activated by the host RNA sensors could be inhibited by HBV. However, it remains unknown whether HBV as a DNA virus utilizes evasion mechanisms against foreign DNA-elicited antiviral signaling. In recent years, the cytosolic DNA sensor and key adaptor STING has been demonstrated to be essential in multiple foreign DNA-elicited innate immune signalings. Here, for the first time, we report STING as a new target of HBV to antagonize IFN induction and identify the viral polymerase responsible for the inhibitory effect, thus providing an additional molecular mechanism by which HBV evades the innate immunity; this implies that in addition to its inherent catalytic function, HBV polymerase is a multifunctional immunomodulatory protein.

Although the mechanisms by which innate pathogen-recognition receptors enhance adaptive immune responses are increasingly well understood, whether signaling events from distinct classes of receptors affect each other in modulating adaptive immunity remains unclear. We found here that the activation of cytosolic RIG-I-like receptors (RLRs) resulted in the selective suppression of transcription of the gene encoding the p40 subunit of interleukin 12 (Il12b) that was effectively induced by the activation of Toll-like receptors (TLRs). The RLR-activated transcription factor IRF3 bound dominantly, relative to IRF5, to the Il12b promoter, where it interfered with the TLR-induced assembly of a productive transcription-factor complex. The activation of RLRs in mice attenuated TLR-induced responses of the T helper type 1 cell (T(H)1 cell) and interleukin 17-producing helper T cell (T(H)17 cell) subset types and, consequently, viral infection of mice caused death at sublethal doses of bacterial infection. The innate immune receptor cross-interference we describe may have implications for infection-associated clinical episodes.

IFN regulatory factor 7 (IRF7) is a potent transcription factor of type I IFNs and IFN-stimulated genes and is known as the master regulator of type I IFN-dependent immune responses. Because excessive responses could harm the host, IRF7 itself is delicately regulated at the transcriptional, translational, and posttranslational levels. Modification of IRF7 by small ubiquitin-related modifiers (SUMOs) has been shown to regulate IFN expression and antiviral responses negatively, but the specific E3 ligase needed for IRF7 SUMOylation has remained unknown. As reported in this article, we have identified the tripartite motif-containing protein 28 (TRIM28) as a binding partner of IRF7. We have demonstrated that TRIM28 also interacts with the SUMO E2 enzyme and increases SUMOylation of IRF7 both in vivo and in vitro, suggesting it acts as a SUMO E3 ligase of IRF7. Unlike the common SUMO E3 ligase, protein inhibitor of activated STAT1, the E3 activity of TRIM28 is specific to IRF7, because it has little effect on IRF7's close relative IRF3. TRIM28 is therefore, so far as we know, the first IRF7-specific SUMO E3 reported. TRIM28-mediated SUMOylation of IRF7 is increased during viral infection, and SUMOylation of transcription factors usually results in transcriptional repression. Overexpression of TRIM28 therefore inhibits IRF7 transactivation activity, whereas knockdown of TRIM28 has the opposite effect and potentiates IFN production and antiviral responses. Collectively, our results suggest that TRIM28 is a specific SUMO E3 ligase and negative regulator of IRF7.

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.

Detection of foreign RNA by the innate immune system can trigger the induction of type I interferon (IFN) and apoptosis. Important antiviral defense pathways that result in type I IFN production following the recognition of foreign double-stranded RNA (dsRNA) include the RIG-I family helicases and IPS-1 adaptor cytosolic pathway and the Toll-like receptor 3 and TIR domain-containing adaptor-inducing IFN-beta (TRIF) adaptor membrane-associated pathway, both of which activate IFN regulatory factor 3 (IRF3). In addition to triggering an innate immune response, dsRNAs are widely used to mediate gene-selective silencing in mammalian cells by the RNA interference pathway. We investigated the ability of short interfering RNAs, including T7 phage polymerase-synthesized RNA (PRNA), which like some viral RNAs contains a 5'-triphosphate, to selectively silence gene expression and to cause induction of IFN-beta and apoptosis. We found that PRNA-mediated gene silencing and associated nonspecific pro-apoptotic and IFN-inducing effects were dependent on the cell line and RNA length. Double-stranded PRNAs 50 nucleotides long as well as polyinosinic-polycytidylic acid activated the RNA-dependent protein kinase (PKR) and induced significant levels of IFN-beta and apoptosis, whereas shorter PRNAs and chemically synthesized dsRNAs did not. Effector caspase activation and apoptosis following RNA transfection was enhanced by pretreatment with IFN, and removal of the 5'-phosphate from PRNAs decreased induction of both IFN-beta and apoptosis. PKR, in addition to IPS-1 and IRF3 but not TRIF, was required for maximal type I IFN-beta induction and the induction of apoptosis by both transfected PRNAs and polyinosinic-polycytidylic acid.

TANK-binding kinase (TBK1) is essential for transcription of the interferon (IFN) β gene in response to lipopolysaccharide (LPS) and double-stranded RNA, but the molecular mechanisms that underlie the activation of TBK1 are incompletely understood. Previously, we identified the NF-κB essential modulator (NEMO)-related polyubiquitin-binding protein, optineurin (OPTN), as a novel binding partner of TBK1. To determine whether the ubiquitin-binding function of OPTN is involved in regulating TBK1 and IFNβ production, we generated a mouse in which wild-type optineurin was replaced by the polyubiquitin binding-defective mutant, OPTN(D477N/D477N). In this study, we found that LPS or poly(I:C)-induced TBK1 activity was significantly reduced in bone marrow-derived macrophage (BMDM) from OPTN(D477N/D477N) mice. Consistent with this, the phosphorylation of IFN regulatory factor 3 (IRF3) and the production of IFNβ mRNA and secretion were reduced. Stimulation of BMDMs with LPS triggered the phosphorylation of OPTN, which was reversed by phosphatase treatment and prevented by pharmacological inhibition of both the canonical IκB kinases (IKKα/β) and the IKK-related kinases (TBK1/IKKε). In contrast, LPS-stimulated phosphorylation of OPTN(D477N) was markedly reduced in BMDMs from OPTN(D477N/D477N) mice, and inhibition of the canonical IKKs alone prevented phosphorylation, providing further evidence that ubiquitin binding to OPTN contributes to LPS-induced TBK1 activation. TBK1 and IKKβ phosphorylated OPTN preferentially at Ser-177 and Ser-513, respectively, in vitro. In conclusion, our results suggest that OPTN binds to polyubiquitylated species formed in response to LPS and poly(I:C), enhancing the activation of TBK1 that is required for optimal phosphorylation of IRF3 and production of IFNβ.

Ischemic tolerance can be induced by numerous preconditioning stimuli, including various Toll-like receptor (TLR) ligands. We have shown previously that systemic administration of the TLR4 ligand LPS or the TLR9 ligand unmethylated CpG oligodeoxynucleotide before transient brain ischemia in mice confers substantial protection against ischemic damage. To elucidate the molecular mechanisms of preconditioning, we compared brain genomic profiles in response to preconditioning with these TLR ligands and with preconditioning via exposure to brief ischemia. We found that exposure to the TLR ligands and brief ischemia induced genomic changes in the brain characteristic of a TLR pathway-mediated response. Interestingly, all three preconditioning stimuli resulted in a reprogrammed response to stroke injury that converged on a shared subset of 13 genes not evident in the genomic profile from brains that were subjected to stroke without prior preconditioning. Analysis of the promoter region of these shared genes showed sequences required for interferon regulatory factor (IRF)-mediated transcription. The importance of this IRF gene network was tested using mice deficient in IRF3 or IRF7. Our data show that both transcription factors are required for TLR-mediated preconditioning and neuroprotection. These studies are the first to discover a convergent mechanism of neuroprotection induced by preconditioning--one that potentially results in reprogramming of the TLR-mediated response to stroke and requires the presence of IRF3 and IRF7.

Enterovirus 71 (EV71) is a positive-stranded RNA virus which is capable of inhibiting innate immunity. Among virus-encoded proteins, the 3C protein compromises the type I interferon (IFN-I) response mediated by retinoid acid-inducible gene-I (RIG-I) or Toll-like receptor 3 that activates interferon regulatory 3 (IRF3) and IRF7. In the present study, we report that enterovirus 71 downregulates IRF7 through the 3C protein, which inhibits the function of IRF7. When expressed in mammalian cells, the 3C protein mediates cleavage of IRF7 rather than that of IRF3. This process is insensitive to inhibitors of caspase, proteasome, lysosome, and autophagy. H40D substitution in the 3C active site abolishes its activity, whereas R84Q or V154S substitution in the RNA binding motif has no effect. Furthermore, 3C-mediated cleavage occurs at the Q189-S190 junction within the constitutive activation domain of IRF7, resulting in two cleaved IRF7 fragments that are incapable of activating IFN expression. Ectopic expression of wild-type IRF7 limits EV71 replication. On the other hand, expression of the amino-terminal domain of IRF7 enhances EV71 infection, which correlates with its ability to interact with and inhibit IRF3. These results suggest that control of IRF7 by the 3C protein may represent a viral mechanism to escape cellular responses.

In mouse embryonic fibroblasts (MEFs), the bovine rotavirus (UK strain) but not the simian rhesus rotavirus (RRV) robustly triggers beta interferon (IFN-β) secretion, resulting in an IFN-dependent restriction of replication. We now find that both rotavirus strains trigger antiviral transcriptional responses early during infection and that both transcriptional responses and IFN-β secretion are completely abrogated in MAVS/IPS-1(-/-) MEFs. Replication of UK virus could be rescued in MAVS/IPS-1(-/-) MEFs, and synthesis of viral RNA significantly increased early during virus infection. UK virus induced IFN-β secretion and transcription of IFN-stimulated genes (ISGs) in both RIG-I(-/-) and MDA-5(-/-) MEFs, and neither receptor was essential by itself for the antiviral response to UK rotavirus. However, when receptors RIG-I and MDA-5 were depleted using RNA interference, we found that both contribute to the magnitude of the IFN response. IRF3 was found to be essential for MAVS/IPS-1-directed ISG transcription and IFN-β secretion during rotavirus infection. Interestingly, absence of the double-stranded RNA-dependent protein kinase PKR led to a profound defect in the capacity of host cells to secrete IFN-β in response to virus. Both PKR and IRF3 restricted the early replication of UK as indicated by significant increases in viral RNA in fibroblasts lacking either gene. Despite the loss in IFN-β secretion in PKR(-/-) MEFs, we did not observe decreased IRF3- or NF-κB-dependent early ISG transcription in these cells. Levels of transcripts encoding IFN-α4, IFN-α5, and IFN-β were high in infected PKR(-/-) MEFs, indicating that during rotavirus infection, PKR functions at a stage between IFN gene transcription and subsequent IFN-β secretion. These findings reveal that activation of the antiviral response by rotavirus is dependent on MAVS/IPS-1 and IRF3 and involves both RIG-I and MDA-5 and that IFN-β secretion during rotavirus infection is regulated by PKR.

Intracellular RNA viruses are sensed by receptors retinoic acid-inducible gene I/MDA5, which trigger formation of the mitochondrial antiviral signaling (MAVS) complex on mitochondria. Consequently, this leads to the activation of TNFR-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) and phosphorylation of IFN regulatory factor 3 (IRF3). It remains to be elucidated how MAVS activates TBK1/IRF3. In this study, we report that IFN-induced protein with tetratricopeptide repeats 3 (IFIT3) is significantly induced upon RNA virus infection. Ectopic expression or knockdown of IFIT3 could, respectively, enhance or impair IRF3-mediated gene expression. Mechanistically, the tetratrico-peptide repeat motif (E164/E165) of IFIT3 interacts with the N terminus (K38) of TBK1, thus bridging TBK1 to MAVS on the mitochondrion. Disruption of this interaction markedly attenuates the activation of TBK1 and IRF3. Furthermore, host antiviral responses are significantly boosted or crippled in the presence or absence of IFIT3. Collectively, our study characterizes IFIT3 as an important modulator in innate immunity, revealing a new function of the IFIT family proteins (IFN-induced protein with tetratricopeptide repeats).

Legionella pneumophila, a Gram-negative facultative intracellular bacterium, causes severe pneumonia (Legionnaires' disease). Type I interferons (IFNs) were so far associated with antiviral immunity, but recent studies also indicated a role of these cytokines in immune responses against (intracellular) bacteria. Here we show that wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, or heat-inactivated Legionella induced IFNbeta expression in human lung epithelial cells. We found that factor (IRF)-3 and NF-kappaB-p65 translocated into the nucleus and bound to the IFNbeta gene enhancer after L. pneumophila infection of lung epithelial cells. RNA interference demonstrated that in addition to IRF3, the caspase recruitment domain (CARD)-containing adapter molecule IPS-1 (interferon-beta promoter stimulator 1) is crucial for L. pneumophila-induced IFNbeta expression, whereas other CARD-possessing molecules, such as RIG-I (retinoic acid-inducible protein I), MDA5 (melanoma differentiation-associated gene 5), Nod27 (nucleotide-binding oligomerization domain protein 27), and ASC (apoptosis-associated speck-like protein containing a CARD) seemed not to be involved. Finally, bacterial multiplication assays in small interfering RNA-treated cells indicated that IPS-1, IRF3, and IFNbeta were essential for the control of intracellular replication of L. pneumophila in lung epithelial cells. In conclusion, we demonstrated a critical role of IPS-1, IRF3, and IFNbeta in Legionella infection of lung epithelium.

Glucocorticoids dramatically inhibit cytokine and chemokine production. They act through the glucocorticoid receptor (GR), a ligand-dependent transcription factor that binds to and represses activities of other DNA-bound regulators, activator protein 1 and nuclear factor kappaB, utilizing a p160 GRIP1 as a corepressor. A yeast two-hybrid screen with the GRIP1 corepression domain (RD) yielded interferon (IFN) regulatory factor (IRF)3-a downstream effector of Toll-like receptors (TLR) 3/4 and an essential activator of several IFN and chemokine genes. We defined the GRIP1:IRF3 interface and showed that endogenous GRIP1 and IRF3 interact in mammalian cells. Interestingly, GR and IRF3 competed for GRIP1 binding; GR activation or GRIP1 knockdown in macrophages blocked whereas GRIP1 overexpression rescued IRF3-dependent gene expression. GR interference persisted in MyD88- and IFNA receptor-deficient mice, suggesting a specific disruption of TLR3-IRF3 pathway, not of autocrine IFN signaling. Finally, IRF3-stimulated response elements were necessary and sufficient for TLR3-dependent induction and glucocorticoid inhibition. Thus, GRIP1 plays a cofactor role in innate immunity. Competition with GR for GRIP1 antagonizes IRF3-mediated transcription, identifying the GRIP1:IRF3 interaction as a novel target for glucocorticoid immunosuppression.

The cellular protein IFI16 colocalizes with the herpes simplex virus 1 (HSV-1) ubiquitin ligase ICP0 at early times of infection and is degraded as infection progresses. Here, we report that the factors governing the degradation of IFI16 and its colocalization with ICP0 are distinct from those of promyelocytic leukemia protein (PML), a well-characterized ICP0 substrate. Unlike PML, IFI16 colocalization with ICP0 was dependent on the ICP0 RING finger and did not occur when proteasome activity was inhibited. Expression of ICP0 in the absence of infection did not destabilize IFI16, the degradation occurred efficiently in the absence of ICP0 if infection was progressing efficiently, and IFI16 was relatively stable in wild-type (wt) HSV-1-infected U2OS cells. Therefore, IFI16 stability appears to be regulated by cellular factors in response to active HSV-1 infection rather than directly by ICP0. Because IFI16 is a DNA sensor that becomes associated with viral genomes during the early stages of infection, we investigated its role in the recruitment of PML nuclear body (PML NB) components to viral genomes. Recruitment of PML and hDaxx was less efficient in a proportion of IFI16-depleted cells, and this correlated with improved replication efficiency of ICP0-null mutant HSV-1. Because the absence of interferon regulatory factor 3 (IRF3) does not increase the plaque formation efficiency of ICP0-null mutant HSV-1, we speculate that IFI16 contributes to cell-mediated restriction of HSV-1 in a manner that is separable from its roles in IRF3-mediated interferon induction, but that may be linked to the PML NB response to viral infection.

BACKGROUND

We examined host genetic factors to identify those more common in individuals whose human papillomavirus (HPV) infections were most likely to persist and progress to cervical intraepithelial neoplasia grade 3 (CIN3) and cancer.

METHODS

We genotyped 92 single-nucleotide polymorphisms (SNPs) from 49 candidate immune response and DNA repair genes obtained from 469 women with CIN3 or cancer, 390 women with persistent HPV infections (median duration, 25 months), and 452 random control subjects from the 10,049-woman Guanacaste Costa Rica Natural History Study. We calculated odds ratios and 95% confidence intervals (CIs) for the association of SNP and haplotypes in women with CIN3 or cancer and HPV persistence, compared with random control subjects.

RESULTS

A SNP in the Fanconi anemia complementation group A gene (FANCA) (G501S) was associated with increased risk of CIN3 or cancer. The AG and GG genotypes had a 1.3-fold (95% CI, 0.95-1.8-fold) and 1.7-fold (95% CI, 1.1-2.6-fold) increased risk for CIN3 or cancer, respectively (P(trend) = .008; referent, AA). The FANCA haplotype that included G501S also conferred increased risk of CIN3 or cancer, as did a different haplotype that included 2 other FANCA SNPs (G809A and T266A). A SNP in the innate immune gene IRF3 (S427T) was associated with increased risk for HPV persistence (P(trend) = .009).

CONCLUSIONS

Our results require replication but support the role of FANCA variants in cervical cancer susceptibility and of IRF3 in HPV persistence.

IL-27 is a heterodimeric cytokine composed of EBV-induced gene 3 and p28. Produced by dendritic cells (DCs) in response to TLR ligands, IL-27 recently emerged as a key regulator of inflammatory responses. In this study, we first demonstrate that Toll/IL-1R-containing adaptor inducing IFN-beta and its associated IFN regulatory factor (IRF) 3 transcription factor are critically involved in IL-27p28 expression in mouse DCs stimulated by TLR ligands. We then show that IL-27 serum levels are dramatically reduced in IRF3(-/-) upon LPS injection, indicating a critical role for IRF3 in TLR4-mediated IL-27 production in vivo. We identified an IRF3-binding site within the IL-27p28 promoter region which is required for IL-27p28 gene activation in reporter gene assays. In human DCs, IL-27p28 mRNA was preferentially induced by Toll/IL-1R-containing adaptor inducing IFN-beta-coupled TLR ligands and following CMV infection. Furthermore, chromatin immunoprecipitation studies demonstrate that IRF3 is recruited to the endogenous p28 promoter in TLR4-stimulated human DCs. We conclude that IRF3 activation is a master switch for IL-27 synthesis.

Beta interferon (IFN-beta) gene expression in response to virus infection relies on the dynamic assembly of a multiprotein enhanceosome complex that is initiated by the activation of two inducible transcription factors, interferon regulatory factor 3 (IRF3) and NF-kappaB. Virus or double-stranded RNA-induced activation of IFN-beta gene expression is prevented by the addition of protein deacetylase inhibitors. The isolated IRF-responsive positive regulatory domain was found to require deacetylation for its activity, but IRF3 protein activation leading to its nuclear translocation and DNA binding was not impaired by deacetylase inhibition. In contrast, NF-kappaB activity was not affected by deacetylase inhibitors. RNA interference indicated that several deacetylase enzymes, including histone deacetylase 1 (HDAC1), HDAC8, and HDAC6, influence IFN-beta gene expression with opposing effects. While HDAC1 and HDAC8 repress IFN-beta expression, HDAC6 acts as a coactivator essential for enhancer activity. Virus replication is enhanced in HDAC6-depleted cells, demonstrating HDAC6 is an essential component of innate antiviral immunity.

Dendritic cells (DCs) are key regulators of the immune system; they capture antigens and then can either stimulate an immune response or induce tolerance. Our aim was to activate individual DC signaling pathways to regulate the immune response. We therefore expressed constitutive activators of mitogen-activated protein kinase (MAPK) pathways or the interferon pathway, together with tumor antigens, using lentivectors. Triggering of p38 activated DCs substantially enhanced the antitumor immune response and prolonged survival of tumor-bearing mice. Activation of extracellular signal-regulated kinase (ERK) increased TGF-beta expression while expression of a constitutively activated interferon regulatory factor-3 (IRF3) stimulated IL-10 secretion by DCs. ERK and IRF3 suppressed the immune response and stimulated expansion of regulatory T cells. These results provide a toolkit to regulate immune responses to viral vector or DC immunization; vaccine responses to foreign or tumor antigens can be enhanced and harmful responses to self-antigens or introduced transgenes can be reduced.

Lys63-linked polyubiquitination of RIG-I is essential in antiviral immune defense, yet the molecular mechanism that negatively regulates this critical step is poorly understood. Here, we report that USP21 acts as a novel negative regulator in antiviral responses through its ability to bind to and deubiquitinate RIG-I. Overexpression of USP21 inhibited RNA virus-induced RIG-I polyubiquitination and RIG-I-mediated interferon (IFN) signaling, whereas deletion of USP21 resulted in elevated RIG-I polyubiquitination, IRF3 phosphorylation, IFN-α/β production, and antiviral responses in MEFs in response to RNA virus infection. USP21 also restricted antiviral responses in peritoneal macrophages (PMs) and bone marrow-derived dendritic cells (BMDCs). USP21-deficient mice spontaneously developed splenomegaly and were more resistant to VSV infection with elevated production of IFNs. Chimeric mice with USP21-deficient hematopoietic cells developed virus-induced splenomegaly and were more resistant to VSV infection. Functional comparison of three deubiquitinases (USP21, A20, and CYLD) demonstrated that USP21 acts as a bona fide RIG-I deubiquitinase to down-regulate antiviral response independent of the A20 ubiquitin-editing complex. Our studies identify a previously unrecognized role for USP21 in the negative regulation of antiviral response through deubiquitinating RIG-I.

Here we identify Viperin as a highly inducible gene in response to lipopolysaccharide (LPS), double-stranded RNA (poly(I-C)) or Sendai virus (SV). The only known function of Viperin relates to its ability to inhibit human Cytomegalovirus replication. Very little data are available on the regulation of this gene. In silico analysis of the promoter identified two interferon (IFN)-stimulated response elements (ISRE), which in other genes bind IRF3 or the IFN-stimulated gene factor-3 (ISGF3) complex. LPS and poly(I-C) induce very high levels of Viperin in wild type cells but not in cells deficient in TRIF, TBK1, IRF3, or the type I IFNalpha/betaR. SV-induced Viperin gene expression was mediated independently of Toll-like receptor (TLR) signaling by retinoic acid-inducible gene (RIG-I) and the downstream adapter, mitochondrial anti-viral signaling (MAVS). Virus-induced Viperin expression was not attenuated in macrophages deficient in either TBK1 or IKKepsilon alone. Moreover, IRF3-deficient, but not IFNalpha/betaR deficient, macrophages still induced Viperin in response to SV. Promoter reporter studies combined with DNA immunoprecipitation assays identified the ISGF3 complex as the key regulator of Viperin gene expression. Moreover, positive regulatory domain I-binding factor 1 (PRDI-BF1, also called BLIMP1) binds the ISRE sites and competes with ISGF3 binding in a virus inducible manner to inhibit Viperin transcription. Collectively, these studies identify Viperin as a tightly regulated ISGF3 target gene, which is counter-regulated by PRDI-BF1.

The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe pulmonary disease in humans and represents the second example of a highly pathogenic coronavirus (CoV) following severe acute respiratory syndrome coronavirus (SARS-CoV). Genomic studies revealed that two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), process the polyproteins encoded by the MERS-CoV genomic RNA. We previously reported that SARS-CoV PLpro acts as both deubiquitinase (DUB) and IFN antagonist, but the function of the MERS-CoV PLpro was poorly understood. In this study, we characterized MERS-CoV PLpro, which is a protease and can recognize and process the cleavage sites (CS) of nsp1-2, nsp2-3 and nsp3-4. The LXGG consensus cleavage sites in the N terminus of pp1a/1ab, which is generally essential for CoV PLpro-mediated processing, were also characterized in MERS-CoV. MERS-CoV PLpro, like human SARS-CoV PLpro and NL63-CoV PLP2, is a viral deubiquitinating enzyme. It acts on both K48- and K63-linked ubiquitination and ISG15-linked ISGylation. We confirmed that MERS-CoV PLpro acts as an IFN antagonist through blocking the phosphorylation and nuclear translocation of IFN regulatory factor 3 (IRF3). These findings indicate that MERS-CoV PLpro acts as a viral DUB and suppresses production of IFN-β by an interfering IRF3-mediated signalling pathway, in addition to recognizing and processing the CS at the N terminus of replicase polyprotein to release the non-structural proteins. The characterization of proteolytic processing, DUB and IFN antagonist activities of MERS-CoV PLpro would reveal the interactions between MERS-CoV and its host, and be applicable to develop strategies targeting PLpro for the effective control of MERS-CoV infection.

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.