Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKepsilon-mediated IRF activation

Viruses are detected by different classes of pattern recognition receptors (PRRs), such as Toll-like receptors and RIG-like helicases. Engagement of PRRs leads to activation of interferon (IFN)-regulatory factor 3 (IRF3) and IRF7 through IKKepsilon and TBK1 and consequently IFN-beta induction. Vaccinia virus (VACV) encodes proteins that manipulate host signalling, sometimes by targeting uncharacterised proteins. Here, we describe a novel VACV protein, K7, which can inhibit PRR-induced IFN-beta induction by preventing TBK1/IKKepsilon-mediated IRF activation. We identified DEAD box protein 3 (DDX3) as a host target of K7. Expression of DDX3 enhanced Ifnb promoter induction by TBK1/IKKepsilon, whereas knockdown of DDX3 inhibited this, and virus- or dsRNA-induced IRF3 activation. Further, dominant-negative DDX3 inhibited virus-, dsRNA- and cytosolic DNA-stimulated Ccl5 promoter induction, which is also TBK1/IKKepsilon dependent. Both K7 binding and enhancement of Ifnb induction mapped to the N-terminus of DDX3. Furthermore, virus infection induced an association between DDX3 and IKKepsilon. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKepsilon-mediated IRF activation and Ifnb promoter induction.     

IFN regulatory factor family members differentially regulate the expression of type III IFN (IFN-lambda) genes

Virus replication induces the expression of antiviral type I (IFN-alphabeta) and type III (IFN-lambda1-3 or IL-28A/B and IL-29) IFN genes via TLR-dependent and -independent pathways. Although type III IFNs differ genetically from type I IFNs, their similar biological antiviral functions suggest that their expression is regulated in a similar fashion. Structural and functional characterization of the IFN-lambda1 and IFN-lambda3 gene promoters revealed them to be similar to IFN-beta and IFN-alpha genes, respectively. Both of these promoters had functional IFN-stimulated response element and NF-kappaB binding sites. The binding of IFN regulatory factors (IRF) to type III IFN promoter IFN-stimulated response element sites was the most important event regulating the expression of these genes. Ectopic expression of the components of TLR7 (MyD88 plus IRF1/IRF7), TLR3 (Toll/IL-1R domain-containing adapter-inducing factor), or retinoic acid-inducible gene I (RIG-I) signal transduction pathways induced the activation of IFN-lambda1 promoter, whereas the IFN-lambda3 promoter was efficiently activated only by overexpression of MyD88 and IRF7. The ectopic expression of Pin1, a recently identified suppressor for IRF3-dependent antiviral response, decreased the IFN promoter activation induced by any of these three signal transduction pathways, including the MyD88-dependent one. To conclude, the data suggest that the IFN-lambda1 gene is regulated by virus-activated IRF3 and IRF7, thus resembling that of the IFN-beta gene, whereas IFN-lambda2/3 gene expression is mainly controlled by IRF7, thus resembling those of IFN-alpha genes.     

Nuclear localization of the Nipah virus W protein allows for inhibition of both virus- and toll-like receptor 3-triggered signaling pathways

The Nipah virus V and W proteins, which are encoded by the P gene via RNA editing, have a common N-terminal domain but unique C-terminal domains. They localize to the cytoplasm and nucleus, respectively, and have both been shown to function as inhibitors of JAK/STAT signaling. Here we report that V and W proteins also block virus activation of the beta interferon (IFN-beta) promoter and the IFN regulatory factor 3 (IRF3)-responsive IFN-stimulated gene 54 promoter. Surprisingly, only W protein shows strong inhibition of promoter activation in response to stimulation of Toll-like receptor 3 (TLR3) by extracellular double-stranded RNA. This activity is dependent on the nuclear localization of W protein. Within the unique C-terminal domain of W protein, we have identified a nuclear localization signal (NLS) that requires basic residues at positions 439, 440, and 442. This NLS is responsible for mediating the preferential interaction of W protein with karyopherin-alpha 3 and karyopherin-alpha 4. Nuclear localization of W protein therefore enables it to target both virus and TLR3 pathways, whereas the cytoplasmic V protein is restricted to inhibiting the virus pathway. We propose that this discrepancy is in part due to the V protein being less able to block signaling in response to the kinase, TBK-1, whereas both V and W can prevent promoter activation in response to IKKepsilon. We demonstrate that, when the TLR3 pathway is stimulated, the levels of phosphorylated IRF3 are reduced in the presence of W protein but not V protein, confirming the differential effects of these proteins and illustrating that W protein-mediated inhibition is due to a loss of active IRF3.     

Toll-like receptor 3 mediates a more potent antiviral response than Toll-like receptor 4

We have recently described an IFN regulatory factor 3-mediated antiviral gene program that is induced by both Toll-like receptor (TLR)3 and TLR4 ligands. In our current study, we show that activation of IFN/viral response gene expression in primary macrophage cells is stronger and prolonged with TLR3 stimulation compared with that of TLR4. Our data also reveal that the cytoplasmic tails of both TLR3 and TLR4 can directly interact with myeloid differentiation factor 88 (MyD88). However, although Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like is able to associate with TLR4, we were unable to detect any interaction between Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like and TLR3. By using quantitative real-time PCR assays, we found that TLR3 expression is inducible by both TLR3 and TLR4 ligands, while TLR4 expression is not inducible by these same stimuli. Furthermore, using cells derived from mice deficient in the IFN-alphabetaR, we show that both TLR3 and TLR4 require IFN-beta autocrine/paracrine feedback to induce TLR3 expression and activate/enhance genes required for antiviral activity. More specifically, a subset of antiviral genes is initially induced independent of IFN-beta, yet the cytokine further enhances expression at later time points. This was in contrast to a second set of genes (including TLR3) that is induced only after IFN-beta production. Taken together, our data argue that, despite both TLR3 and TLR4 being able to use IFN-beta to activate/enhance antiviral gene expression, TLR3 uses multiple mechanisms to enhance and sustain the antiviral response more strongly than TLR4.     

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.     

Short duration of elevated vIRF-1 expression during lytic replication of human herpesvirus 8 limits its ability to block antiviral responses induced by alpha interferon in BCBL-1 cells

Human herpesvirus 8 (HHV-8) encodes multiple proteins that disrupt the host antiviral response, including viral interferon (IFN) regulatory factor 1 (vIRF-1). The product of the vIRF-1 gene blocks responses to IFN when overexpressed by transfection, but the functional consequence of vIRF-1 that is expressed during infection with HHV-8 is not known. These studies demonstrate that BCBL-1 cells that were latently infected with HHV-8 expressed low levels of vIRF-1 that were associated with PML bodies, whereas much higher levels of vIRF-1 were transiently expressed during the lytic phase of HHV-8 replication. The low levels of vIRF-1 that were associated with PML bodies were insufficient to block alpha IFN (IFN-alpha)-induced alterations in gene expression, whereas cells that expressed high levels of vIRF-1 were resistant to some changes induced by IFN-alpha, including the expression of the double-stranded-RNA-activated protein kinase. High levels of vIRF-1 were expressed for only a short period during the lytic cascade, so many cells with HHV-8 in the lytic phase responded to IFN-alpha with increased expression of antiviral genes and enhanced apoptosis. Furthermore, the production of infectious virus was severely compromised when IFN-alpha was present early during the lytic cascade. These studies indicate that the transient expression of high levels of vIRF-1 is inadequate to subvert many of the antiviral effects of IFN-alpha so that IFN-alpha can effectively induce apoptosis and block production of infectious virus when present early in the lytic cascade of HHV-8.     

Interferon regulatory factor 7 regulates expression of Epstein-Barr virus latent membrane protein 1: a regulatory circuit

We have shown previously that interferon regulatory factor 7 (IRF7), a multifunctional protein intimately involved in latent Epstein-Barr virus (EBV) infection, is induced as well as activated by EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein. Since the LMP1 promoter (LMP1p) contains an interferon-stimulated response element (ISRE), we hypothesized that IRF7 might be able to regulate LMP1 expression and thus participate in a regulatory circuit between these two genes. In this study, IRF7 was shown first to activate LMP1p in transient transfection assays. Compared with EBV nuclear antigen 2 (EBNA2), the most potent viral transactivator of LMP1p, IRF7 has a lesser effect (approximately 10% that of EBNA2) on induction of LMP1p. Study with IRF7 deletion mutants showed that IRF7 functional domains have similar effects on both the beta interferon (IFN-beta) and LMP1 promoters in BJAB and 293 cells, and study with IRF7 phosphomimetic mutants showed that IRF7 phosphorylation may be involved in the activation of these two promoters. Further, the ISRE in LMP1p responds to IRF7 induction and IRF7 binds to this element. In the EBV-positive cell line P3HR1, which lacks the complete EBNA2 and EBV-encoded leader protein genes and hence expresses low-level LMP1, IRF7 alone can notably increase the endogenous LMP1 mRNA and protein levels. These results indicate that LMP1 is regulated by this host cell gene in addition to the viral factor, EBNA2, and may help to explain how LMP1 is expressed in type II latency in the absence of EBNA2. Moreover, IRF7 can regulate a viral gene in addition to a host cellular gene such as the IFN-beta gene. Together with the previous data that LMP1 can induce IRF7 expression and facilitate IRF7 phosphorylation and nuclear translocation, these results suggest a positive regulatory circuit between IRF7 and LMP1.