Functional evolution of the TICAM-1 pathway for extrinsic RNA sensing

Summary:  The type I interferon (IFN) is a host defense factor against microbial pathogens in vertebrates. In mammals, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm are regarded as sensors for double-stranded RNA (dsRNA) and trigger IFN regulatory factor-3 (IRF-3) activation followed by type I IFN induction through the mitochondrial antiviral signaling (MAVS) adapter. This intrinsic pathway appears to link the main protective responses against RNA virus infection in mammals. On the other hand, human Toll-like receptor 3 (TLR3) is localized in the endosomal membrane or cell surface and signals the presence of extrinsic dsRNA. In response to RNA stimulation, TLR3 recruits the Toll-interleukin 1 receptor domain (TIR)-containing adapter molecule 1 (TICAM-1) adapter and induces IRF-3 activation followed by IFN-β promoter activation. Human TLR3 is localized limitedly extent in myeloid dendritic cells, fibroblasts, and epithelial cells. The TICAM-1 and cytoplasmic MAVS pathways converge at the IRF-3-activating kinase in human cells. The reason for the involvement of this extrinsic mode of IFN-inducing pathways in the dsRNA response remains unknown. In fish, two TLRs, i.e. endoplasmic TLR3 and cell surface TLR22, participate in teleost IFN production without the activation of IRF-3. TLR22 is distinct from mammalian TLR3 in terms of cellular localization, ligand selection, and tissue distribution. TLR22 may be a functional substitute for human cell surface TLR3 and may serve as a surveillance molecule for detecting dsRNA virus infection and alerting the immune system for antiviral protection in fish. In this review, we discuss the fundamentals of the extrinsic dsRNA recognition system, which has evolved to induce cellular effectors to cope with dsRNA virus infection across different vertebrate species.     

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.     

Crimean-Congo hemorrhagic fever virus delays activation of the innate immune response

As a first line of defence against virus infection, mammalian cells elicit an innate immune response, characterized by secretion of type I interferons and the up-regulation of interferon stimulated genes. Many viruses down-regulate the innate immune responses in order to enhance their virulence. Crimean-Congo hemorrhagic fever virus (CCHFV), a Nairovirus of the family Bunyaviridae is the causative agent of severe hemorrhagic fever in humans with high mortality. Knowledge regarding the innate immune response against CCHFV is most limited. Interestingly, in this study it is shown that replicating CCHFV delays substantially the IFN response, possibly by interfering with the activation pathway of IRF-3. In addition, it is demonstrated that CCHFV replication is almost insensitive to subsequent treatment with interferon-alpha. Once the virus is replicating, virus replication is more or less insensitive to the antiviral effects induced by the interferon. By using an interferon bioassay, it is shown that infected cells secrete interferon relatively late after infection, that is, 48 hr post-infection. In summary, the results suggest the presence of a virulence factor encoded by CCHFV that delays the host defence in order to allow rapid viral spread in the host.     

Regulation of HIV-1 transcription in activated monocyte macrophages

Sendai virus infection strongly induces interferon (IFN) production and has recently been shown to interdict the subsequent IFN signaling through the Jak/Stat pathway. This anti-IFN activity of SeV is due to its "C" proteins, a nested set of four proteins (C', C, Y1, Y2) that carry out a nested set of functions in countering the innate immune response. We previously reported that all four C proteins interact with Stat1 to prevent IFN signaling through the Jak/Stat pathway. Nevertheless, only the longer C proteins reduced Stat1 levels and prevented IFN from inducing an antiviral (VSV) state, or apoptosis, in IFN-competent murine cells. Here, we investigate the mechanism by which the various C proteins differentially affect the host antiviral defenses. All four C proteins were found to physically associate with Stat1 during cell culture infections, and in vitro in the absence of other viral gene products (as evidenced by co-immunoprecipitation). In addition, the inability of a null mutant (C(F170S)) to bind Stat1 suggests that this interaction is physiologically relevant. We have also shown that the proteasomal inhibitor MG132 can prevent the C protein-induced dismantling of the antiviral (VSV) state in murine cells; thus, the turnover of Stat1 correlates with the C protein-mediated counteraction of the antiviral (VSV) state. The C protein-induced instability of Stat1 was accompanied by a clear increase in the level of mono-ubiquinated Stat1, an unexpected hallmark of protein degradation. Finally, we show that a rSeV with mutant C proteins but wild-type Y proteins (CDelta10-15, that does not counteract the endogenous antiviral (VSV) state of MEFs even though their C proteins bind Stat1 and prevent its activity) is also unable to decrease bulk Stat1 levels or to increase the level of ubiquinated Stat1.     

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.     

Calling in the troops: regulation of inflammatory cell trafficking through innate cytokine/chemokine networks

The recruitment of immune effector cells to localized sites of infection is crucial for the effective delivery of innate immune mechanisms. Under the conditions of infections with murine cytomegalovirus (MCMV), a herpesvirus with pathogenic potential, early immune functions are essential in the control of virus replication and virus-induced pathology. Our studies have demonstrated that the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) is critical for natural killer (NK) cell inflammation and delivery of interferon (IFN)-gamma to mediate downstream protective responses against MCMV infection in liver. Moreover, IFN-alpha/beta-dependent mechanisms promote MIP-1alpha production and subsequently the accumulation of NK cells in liver. Taken together, the studies highlighted in this review define a unique in vivo pathway mediated by innate cytokines in regulating chemokine responses that are essential in the promotion of NK cell inflammation for localized antiviral defense. In addition, the downstream consequences of these events in enhancing endogenous adaptive immune responses will also be discussed. Overall, the innate cytokine/chemokine networks that are described emphasize the emerging importance of chemokine functions for protective immune responses during infection with viruses.     

Mx genes show weaker primary response to virus than other interferon-regulated genes.

Some interferon (IFN)-regulated genes are induced as a primary response to virus as well as secondarily through virus-induced IFN. Here we investigated whether this dual control mechanism would also regulate the activity of the human and mouse Mx genes that encode proteins with intrinsic antiviral potentials. To distinguish between a primary response to virus and a secondary response to virus-induced IFN, we studied virus-induced Mx gene expression in cell lines that lack a functional IFN system and in cells with blocked protein synthesis. In contrast to the two IFN-regulated human genes ISG56 and ISG15, the human MxA gene showed almost no primary response to Newcastle disease virus (NDV) or influenza virus. Similarly, direct activation of the mouse Mx1 gene by NDV or influenza virus was not significant in mouse embryo cells or explanted peritoneal macrophages. A moderate primary Mx1 response to NDV was observed in the permanent cell line L1210. Lack of a strong IFN-independent Mx response to virus indicates that this mode of gene regulation does not play a significant role in Mx-mediated resistance to viral disease.     

FBXO6 regulates the antiviral immune responses via mediating alveolar macrophages survival

Inducing early apoptosis in alveolar macrophages is one of the strategies influenza A virus (IAV) evolved to subvert host immunity. Correspondingly, the host mitochondrial protein nucleotide-binding oligomerization domain-like receptor (NLR)X1 is reported to interact with virus polymerase basic protein 1-frame 2 (PB1-F2) accessory protein to counteract virus-induced apoptosis. Herein, we report that one of the F-box proteins, FBXO6, promotes proteasomal degradation of NLRX1, and thus facilitates IAV-induced alveolar macrophages apoptosis and modulates both macrophage survival and type I interferon (IFN) signaling. We observed that FBXO6-deficient mice infected with IAV exhibited decreased pulmonary viral replication, alleviated inflammatory-associated pulmonary dysfunction, and less mortality. Analysis of the lungs of IAV-infected mice revealed markedly reduced leukocyte recruitment but enhanced production of type I IFN in Fbxo6^−/− mice. Furthermore, increased type I IFN production and decreased viral replication were recapitulated in FBXO6 knockdown macrophages and associated with reduced apoptosis. Through gain- and loss-of-function studies, we found lung resident macrophages but not bone marrow-derived macrophages play a key role in the differences FBXO6 signaling pathway brings in the antiviral immune response. In further investigation, we identified that FBXO6 interacted with and promoted the proteasomal degradation of NLRX1. Together, our results demonstrate that FBXO6 negatively regulates immunity against IAV infection by enhancing the degradation of NLRX1 and thus impairs the survival of alveolar macrophages and antiviral immunity of the host.     

Innate immune response to influenza A virus in differentiated human alveolar type II cells

Alveolar Type II (ATII) cells are important targets for seasonal and pandemic influenza. To investigate the influenza-induced innate immune response in those cells, we measured the global gene expression profile of highly differentiated ATII cells infected with the influenza A virus at a multiplicity of infection of 0.5 at 4 hours and 24 hours after inoculation. Infection with influenza stimulated a significant increase in the mRNA concentrations of many host defense-related genes, including pattern/pathogen recognition receptors, IFN, and IFN-induced genes, chemokines, and suppressors of cytokine signaling. We verified these changes by quantitative real-time RT-PCR. At the protein level, we detected a robust virus-induced secretion of the three glutamic acid-leucine-arginine (ELR)-negative chemokines CXCL9, CXCL10, and CXCL11, according to ELISA. The ultraviolet inactivation of virus abolished the chemokine and cytokine response. Viral infection did not appear to alter the differentiation of ATII cells, as measured by cellular mRNA and concentrations of surfactant proteins. However, viral infection significantly reduced the secretion of surfactant protein (SP)-A and SP-D. In addition, influenza A virus triggered a time-dependent activation of phosphatidylinositol 3-kinase signaling in ATII cells. The inhibition of this pathway significantly decreased the release of infectious virus and the chemokine response, but did not alter virus-induced cell death. This study provides insights into influenza-induced innate immunity in differentiated human ATII cells, and demonstrates that the alveolar epithelium is a critical part of the initial innate immune response to influenza.     

Activation of the interferon-beta promoter during hepatitis C virus RNA replication

In this study we examined the impact of hepatitis C virus (HCV) RNA replication on the innate antiviral response of the host cell. Replication of an HCV subgenomic replicon stimulated the activation of the interferon (IFN)-beta promoter and the production of IFN in human hepatoma cells. Using a variety of functional assays, we found that HCV RNA replication induced the activation and DNA-binding activity of NFkappaB and interferon regulatory factor (IRF)-1. In addition, microscopy experiments revealed a higher frequency of cells containing the nuclear-localized, active form of IRF-3 in HCV replicon cultures versus control cultures. Consistent with these observations, cells harboring the HCV replicon exhibited high basal level expression of a subset of IFN-stimulated antiviral genes. Our results indicate that HCV RNA replication can stimulate cellular antiviral programs that contribute to the assembly and activation of the IFN-beta enhanceosome complex and initiation of the antiviral state. Stable HCV RNA replication in the face of the host antiviral response suggests that HCV may encode one or more proteins capable of overcoming specific antiviral processes, thereby supporting persistent infection.