Orchestrating the interferon antiviral response through the mitochondrial antiviral signaling (MAVS) adapter

Sensing of RNA virus infection by the RIG-I-like receptors (RLRs) engages a complex signaling cascade that utilizes the mitochondrial antiviral signaling (MAVS) adapter protein to orchestrate the innate host response to pathogen, ultimately leading to the induction of antiviral and inflammatory responses mediated by type I interferon (IFN) and NF-κB pathways. MAVS is localized to the outer mitochondrial membrane, and has been associated with peroxisomes, the endoplasmic reticulum and autophagosomes, where it coordinates signaling events downstream of RLRs. MAVS not only plays a pivotal role in the induction of antiviral and inflammatory pathways but is also involved in the coordination of apoptotic and metabolic functions. This review summarizes recent findings related to the MAVS adapter and its essential role in the innate immune response to RNA viruses.     

Activation of the TLR3 pathway regulates IFNbeta production in chickens

Toll-like receptors (TLRs) play key roles in the response to pathogens and in mammals the host response to virus critically relies on TLR3 to detect viral-derived dsRNA. However, in chickens there is a paucity of information about this pathway, and in view of the recent concerns with regard to highly pathogenic avian influenza, there is a clear need for understanding these antiviral pathways. Furthermore, TLR3 engagement is important to the outcome of viral infection because of its role in the induction of interferons (IFNs) and the diverse antiviral effects that these molecules induce. With this in mind, we have investigated the role of TLR3 and its impact on the production of IFNs. We show that in the chicken, poly(I:C), a dsRNA analogue, rapidly induces type 1 IFN similar to that seen in mammals. Furthermore, IFN can activate the upregulation of TLR3, which in some cell types induces them to become responsive to dsRNA. These data highlight the similar function that TLR3 plays in chickens and mammals. To determine the role of chicken TLR3 in response to poly(I:C), we used RNAi-mediated gene silencing to show that poly(I:C)-stimulated IFNbeta expression involves TLR3 signalling. The interrelationship between TLR3 and interferon as well as the observed increase in TLR3 and IFNbeta expression during H5N1 avian influenza infection indicates the importance of these molecules in viral infections in chickens.     

The Toll-IL-1 receptor adaptor family grows to five members

Toll-like receptor (TLR) signal transduction is mediated by an adaptor protein termed MyD88. In the case of TLR2 and TLR4, another adaptor related to MyD88 called Mal also participates in signalling. Two recent papers have added a third adaptor to the family, called Toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-beta (IFN-beta) (TRIF) or TIR-containing adaptor molecule-1 (TICAM-1), which is particularly important for IFN regulatory factor-3 (IRF-3) activation by antiviral TLR3. Two additional adaptors are present in humans, termed Trif-related adaptor molecule (TRAM) and sterile alpha and HEAT-Armadillo motifs (SARM). It is probable that differential use of adaptors will help explain the distinct pathways activated by TLRs during host defence.     

Toll-like receptor 3 activation decreases porcine arterivirus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that initiates infection in pulmonary alveolar macrophages (PAMs), elicits weak immune responses, and establishes a persistent infection. To understand the role of dsRNA intermediates in eliciting host immunity, we sought to determine if toll-like receptor-3 (TLR3), a well-known dsRNA sensor, is involved in the regulation of PRRSV infection. TLR3 gene expression was increased in PAMs of congenitally infected 2-wk-old pigs. Stimulation of PAMs with dsRNA increased gene expression for TLR3 and interferon-beta and suppressed PRRSV infectivity. To investigate activation and signaling parameters, expression constructs of wild-type and functional-domain-truncated porcine TLR3 were used in cell transfection studies. When cells that overexpressed porcine TLR3 were stimulated with dsRNA a rapid and robust calcium influx was induced. Moreover, ligand activation of porcine TLR3 expressed in MARC-145 cells elicited an antiviral response to PRRSV. Conversely, transfection of PAMs with small-interfering RNA targeting porcine TLR3 resulted in up to 80% suppression of TLR3 mRNA expression and an increase in PRRSV infectivity. These data provide fundamental genetic and molecular information for porcine TLR3, and implicate its involvement in PRRSV infection, findings that may suggest new strategies to limit this costly pandemic disease.     

Multi-Step Regulation of Interferon Induction by Hepatitis C Virus

Acute hepatitis C virus (HCV) infection evokes several distinct innate immune responses in host, but the virus usually propagates by circumventing these responses. Although a replication intermediate double-stranded RNA is produced in infected cells, type I interferon (IFN) induction and immediate cell death are largely blocked in infected cells. In vitro studies suggested that type I and III IFNs are mainly produced in HCV-infected hepatocytes if the MAVS pathway is functional, and dysfunction of this pathway may lead to cellular permissiveness to HCV replication and production. Cellular immunity, including natural killer cell activation and antigen-specific CD8 T-cell proliferation, occurs following innate immune activation in response to HCV, but is often ineffective for eradication of HCV. Constitutive dsRNA stimulation differs in output from type I IFN therapy, which has been an authentic therapy for patients with HCV. Host innate immune responses to HCV RNA/proteins may be associated with progressive hepatic fibrosis and carcinogenesis once persistent HCV infection is established in opposition to the IFN system. Hence, innate RNA sensing exerts pivotal functions against HCV genome replication and host pathogenesis through modulation of the IFN system. Molecules participating in the RIG-I and Toll-like receptor 3 pathways are the main targets for HCV, disabling the anti-viral functions of these IFN-inducing molecules. We discuss the mechanisms that abolish type I and type III IFN production in HCV-infected cells, which may contribute to understanding the mechanism of virus persistence and resistance to the IFN therapy.     

Translocase of outer mitochondrial membrane 70 induces interferon response and is impaired by hepatitis C virus NS3

Hepatitis C virus (HCV) elevated expression of the translocase of outer mitochondrial membrane 70 (Tom70). Interestingly, overexpression of Tom70 induces interferon (IFN) synthesis in hepatocytes, and it was impaired by HCV. Here, we addressed the mechanism of this impairment. The HCV NS3/4A protein induced Tom70 expression. The HCV NS3 protein interacted in cells, and cleaved the adapter protein mitochondrial anti-viral signaling (MAVS). Ectopic overexpression of Tom70 could not inhibit this cleavage. As a result, IRF-3 phosphorylation was impaired and IFN-β induction was suppressed. These results indicate that MAVS works upstream of Tom70 and the cleavage of MAVS by HCV NS3 protease suppresses signaling of IFN induction.     

Soluble G protein of respiratory syncytial virus inhibits Toll-like receptor 3/4-mediated IFN-beta induction

Monocyte-derived dendritic cells (mDCs) recognize viral RNA extrinsically by Toll-like receptor (TLR) 3 on the membrane and intrinsically retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm to induce type I IFNs and mDC maturation. When mDCs were treated with live or UV-irradiated respiratory syncytial virus (RSV), early ( approximately 4 h) induction of IFN-beta usually occurs in other virus infections was barely observed. Live RSV subsequently replicated to activate the cytoplasmic IFN-inducing pathway leading to robust type I IFN induction. We found that RSV initial attachment to cells blocked polyI:C-mediated IFN-beta induction, and this early IFN-beta-modulating event was abrogated by antibodies against envelope proteins of RSV, demonstrating the presence of a IFN-regulatory mode by early RSV attachment to host cells. By IFN-stimulated response element (ISRE) reporter analysis in HEK293 cells, polyI:C- or LPS-mediated ISRE activation was dose dependently inhibited by live and inactive RSV to a similar extent. Of the RSV envelope proteins, simultaneously expressed or exogenously added RSV G or soluble G (sG) proteins inhibited TLR3/4-mediated ISRE activation in HEK293 cells. sG proteins expressed in cells did not affect the RIG-I/MDA5 pathway but inhibited the TLR adaptor TRIF/TICAM-1 pathway for ISRE activation. Finally, extrinsically added sG protein suppressed the production of IFN-beta in mDCs. Although the molecular mechanism of this extrinsic functional mode of the RSV G glycoprotein (G protein) remains undetermined, G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TLR4 and may cause insufficient raising cell-mediated immunity against RSV.     

B‐cell‐activating factor expressions in salivary epithelial cells after dsRNA virus infection depends on RNA‐activated protein kinase activation

B‐cell‐activating factor (BAFF) plays a key role in promoting activation of autoimmune B cells. This cytokine may be expressed in and secreted by salivary gland epithelial cells (SGEC) after stimulation with type I IFN or viral or synthetic dsRNA. Because this BAFF expression depends only in part on endosomal TLR and type I IFN, we investigated whether other dsRNA sensors could be implicated in BAFF expression. Using human SGEC, we confirmed the partial dependence of BAFF expression on TLR‐3 by replicating the partial inhibition of BAFF expression observed upon endosomal inhibition using TLR‐3 or Toll/IL‐1R domain‐containing protein inducing IFN‐β silencing mRNA, but not with TLR‐7 silencing mRNA. Melanoma differentiation‐associated gene 5 silencing mRNA had no effect on BAFF expression, but retinoic acid‐inducible gene I silencing mRNA had a slight effect observed following infection with dsRNA reovirus‐1. Inhibition of RNA‐activated protein kinase (PKR) by 2‐aminopurine completely abolished both BAFF mRNA and protein production after reovirus‐1 infection and poly(I:C) stimulation through NF‐κB and p38 MAPK pathways, with the latter implicated only after poly(I:C) stimulation. Thus, PKR is the dsRNA sensor implicated in BAFF induction in SGEC after dsRNA stimulation. In autoimmune diseases, PKR may be an interesting target for preventing BAFF following the induction of innate immunity.     

Cigarette smoke attenuation of poly I:C-induced innate antiviral responses in human PBMC is mainly due to inhibition of IFN-beta production

The cellular response to dsRNA or its synthetic analog polyinosinic-polycytidylic acid (poly I:C) results in IRF-3-, IRF-7- and NF-kB-mediated activation of type 1 IFNs and pro-inflammatory cytokines critical for innate antiviral immune responses. To investigate whether cigarette smoke compromises type 1 IFN signaling in humans, peripheral blood mononuclear cells (PBMCs) from non-smoking individuals were treated with smoke-conditioned media (SCM) and stimulated with poly I:C. We observed a marked attenuation of IRF-3 and NF-kB activation in PBMCs exposed to SCM compared to control PBMCs. Similarly, PBMCs from smokers or splenocytes from smoke-exposed mice also displayed marked reduction of poly I:C-induced antiviral responses compared with either non-smokers or sham-exposed mice. Cigarette smoke was found to block the production of type I IFNs following poly I:C treatment and inhibit subsequent STAT1 activation. Finally, we confirmed that inhibition of IFN-beta, but not IFN-alpha, predominantly contributes to the cigarette smoke-mediated suppression of innate antiviral responses. These findings provide novel mechanistic insights to the susceptibility of cigarette smokers to viral infections.     

Antiviral responses induced by the TLR3 pathway

Antiviral responses are successively induced in virus‐infected animals, and include primary innate immune responses such as type I interferon (IFN) and cytokine production, secondary natural killer (NK) cell responses, and final cytotoxic T lymphocyte (CTL) responses and antibody production. The endosomal Toll‐like receptors (TLRs) and cytoplasmic RIG‐I‐like receptors (RLRs), which recognize viral nucleic acids, are responsible for virus‐induced type I IFN production. RLRs are expressed in most tissues and cells and are primarily implicated in innate immune responses against various viruses through type I IFN production, whereas nucleic acid‐sensing TLRs, TLRs 3, 7, 8 and 9, are expressed on the endosomal membrane of dendritic cells (DCs) and play distinct roles in antiviral immunity. TLR3 recognizes viral double‐stranded RNA taken up into the endosome and serves to protect the host against viral infection by the induction of a range of responses including type I IFN production and DC‐mediated activation of NK cells and CTLs, although the deteriorative role of TLR3 has also been reported in some virus infections. Here, we review the current knowledge on the role of TLR3 during viral infection, and the current understanding of the TLR3‐signalling cascade that operates via the adaptor protein TICAM‐1 (also called TRIF). Copyright © 2011 John Wiley & Sons, Ltd.     

Strain-to-strain difference of V protein of measles virus affects MDA5-mediated IFN-β-inducing potential

Laboratory-adapted and vaccine strains of measles virus (MV) induce type I interferon (IFN) in infected cells to a far greater extent than wild-type strains. We investigated the mechanisms for this differential type I IFN production in cells infected with representative MV strains. The overexpression of the wild-type V protein suppressed melanoma differentiation-associated gene 5 (MDA5)-induced IFN-β promoter activity, while this was not seen in A549 cells expressing CD150 transfected with the V protein of the vaccine strain. The V proteins of the wild-type also suppressed poly I:C-induced IFN regulatory factor 3 (IRF-3) dimerization. The V proteins of the wild-type and vaccine strain did not affect retinoic acid-inducible gene 1 (RIG-I)- or toll-IL-1R homology domain-containing adaptor molecule 1 (TICAM-1)-induced IFN-β promoter activation. We identified an amino acid substitution of the cysteine residue at position 272 (which is conserved among paramyxoviruses) to an arginine residue in the V protein of the vaccine strain. Only the V protein possessing the 272C residue binds to MDA5. The mutation introduced into the wild-type V protein (C272R) was unable to suppress MDA5-induced IRF-3 nuclear translocation and IFN-β promoter activation as seen in the V proteins of the vaccine strain, whereas the mutation introduced in the vaccine strain V protein (R272C) was able to inhibit MDA5-induced IRF-3 and IFN-β promoter activation. The other 6 residues of the vaccine strain V sequence inconsistent with the authentic sequence of the wild-type V protein barely affected the IRF-3 nuclear translocation. These data suggested that the structural difference of vaccine MV V protein hampers MDA5 blockade and acts as a nidus for the spread/amplification of type I IFN induction. Ultimately, measles vaccine strains have two modes of IFN-β-induction for their attenuation: V protein mutation and production of defective interference (DI) RNA.     

Hepatitis C virus NS2 protease inhibits host cell antiviral response by inhibiting IKKε and TBK1 functions

Hepatitis C virus (HCV) encodes for several proteins that can interfere with host cell signaling and antiviral response. Previously, serine protease NS3/4A was shown to block host cell interferon (IFN) production by proteolytic cleavage of MAVS and TRIF, the adaptor molecules of the RIG‐I and TLR3 signaling pathways, respectively. This study shows that another HCV protease, NS2 can interfere efficiently with cytokine gene expression. NS2 and its proteolytically inactive mutant forms were able to inhibit type I and type III IFN, CCL5 and CXCL10 gene promoters activated by Sendai virus infection. However, the CXCL8 gene promoter was not inhibited by NS2. In addition, constitutively active RIG‐I (ΔRIG‐I), MAVS, TRIF, IKKε, and TBK1‐induced activation of IFN‐β promoter was inhibited by NS2. Cotransfection experiments with IKKε or TBK1 together with interferon regulatory factor 3 (IRF3) and HCV expression constructs revealed that NS2 in a dose‐dependent manner inhibited IKKε and especially TBK1‐induced IRF3 phosphorylation. GST pull‐down experiments with GST‐NS2 and in vitro‐translated and cell‐expressed IKKε and TBK1 demonstrated direct physical interactions of the kinases with NS2. Further evidence that the IKKε/TBK1 kinase complex is the target for NS2 was obtained from the observation that the constitutively active form of IRF3 (IRF3‐5D) activated readily IFN‐β promoter in the presence of NS2. The present study identified HCV NS2 as a potent interferon antagonist, and describes an explanation of how NS2 downregulates the major signaling pathways involved in the development of host innate antiviral responses. J. Med. Virol. 85:71–82, 2012. © 2012 Wiley Periodicals, Inc.