RACK1 attenuates RLR antiviral signaling by targeting VISA-TRAF complexes

Virus-induced signaling adaptor (VISA), which mediates the production of type I interferon, is crucial for the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. Upon viral infection, RIG-I recognizes double-stranded viral RNA and interacts with VISA to mediate antiviral innate immunity. However, the mechanisms underlying RIG/VISA-mediated antiviral regulation remain unclear. In this study, we confirmed that receptor for activated C kinase 1 (RACK1) interacts with VISA and attenuates the RIG/VISA-mediated antiviral innate immune signaling pathway. Overexpression of RACK1 inhibited the interferon-β (IFN-β) promoter; interferon-stimulated response element (ISRE); nuclear factor kappa B (NF-κB) activation; and dimerization of interferon regulatory factor 3 (IRF3) mediated by RIG-I, VISA, and TANK-binding kinase 1 (TBK1). A reduction in RACK1 expression level upon small interfering RNA knockdown increased RIG/VISA-mediated antiviral transduction. Additionally, RACK1 disrupted formation of the VISA-tumor necrosis factor receptor-associated factor 2 (TRAF2), VISA-TRAF3, and VISA-TRAF6 complexes during RIG-I/VISA-mediated signal transduction. Additionally, RACK1 enhanced K48-linked ubiquitination of VISA, attenuated its K63-linked ubiquitination, and decreased VISA-mediated antiviral signal transduction. Together, these results indicate that RACK1 interacts with VISA to repress downstream signaling and downregulates virus-induced IFN-β production in the RIG-I/VISA signaling pathway.     

Crystal structure of human IPS-1/MAVS/VISA/Cardif caspase activation recruitment domain

Background  

IPS-1/MAVS/VISA/Cardif is an adaptor protein that plays a crucial role in the induction of interferons in response to viral infection. In the initial stage of the intracellular antiviral response two RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-association gene 5 (MDA5), are independently able to bind viral RNA in the cytoplasm. The 62 kDa protein IPS-1/MAVS/VISA/Cardif contains an N-terminal caspase activation and recruitment (CARD) domain that associates with the CARD regions of RIG-I and MDA5, ultimately leading to the induction of type I interferons. As a first step towards understanding the molecular basis of this important adaptor protein we have undertaken structural studies of the IPS-1 MAVS/VISA/Cardif CARD region.     

Modulation of the interferon antiviral response by the TBK1/IKKi adaptor protein TANK

Induction of type I interferons can be triggered by viral components through Toll-like receptors or intracellular viral receptors such as retinoic acid-inducible gene I. Here, we demonstrate that the TRAF (tumor necrosis factor receptor-associated factor) family member-associated NF-kappaB activator (TANK) plays an important role in interferon induction through both retinoic acid-inducible gene I- and Toll-like receptor-dependent pathways. TANK forms complexes with both upstream signal mediators, such as Cardif/MAVS/IPS-1/VISA, TRIF (Toll-interleukin-1 receptor domain-containing adaptor inducing interferon-beta), and TRAF3 and downstream mediators TANK-binding kinase 1, inducible IkappaB kinase, and interferon regulatory factor 3. In addition, it synergizes with these signaling components in interferon induction. Specific knockdown of TANK results in reduced type I interferon production, increased viral titers, and enhanced cell sensitivity to viral infection. Thus, TANK may be a critical adaptor that regulates the assembly of the TANK-binding kinase 1-inducible IkappaB kinase complex with upstream signaling molecules in multiple antiviral pathways.     

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.     

MasterCARD: a priceless link to innate immunity

Intracellular viral infection is detected by the cytoplasmic RNA helicase RIG-I, which has an essential role in initiating the host antiviral response. The adaptor molecule that connects RIG-I sensing of incoming viral RNA to downstream signaling and gene activation has recently been elucidated by four independent research groups, and has been ascribed four different names: MAVS, IPS-1, VISA and Cardif. The fact that MAVS/IPS-1/VISA/Cardif localizes to the mitochondrial membrane suggests a link between viral infection, mitochondrial function and development of innate immunity. Furthermore, the hepatitis C virus NS3/4A protease specifically cleaves MAVS/IPS-1/VISA/Cardif as part of its immune-evasion strategy. These studies highlight a novel role for the mitochondria and for caspase activation and recruitment domain (CARD)-containing proteins in coordinating immune and apoptotic responses.     

HIV-1 Tat binds to SH3 domains: Cellular and viral outcome of Tat/Grb2 interaction

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.     

MAVS-Mediated Apoptosis and Its Inhibition by Viral Proteins

Background

Host responses to viral infection include both immune activation and programmed cell death. The mitochondrial antiviral signaling adaptor, MAVS (IPS-1, VISA or Cardif) is critical for host defenses to viral infection by inducing type-1 interferons (IFN-I), however its role in virus-induced apoptotic responses has not been elucidated.

Principal Findings

We show that MAVS causes apoptosis independent of its function in initiating IFN-I production. MAVS-induced cell death requires mitochondrial localization, is caspase dependent, and displays hallmarks of apoptosis. Furthermore, MAVS^−/− fibroblasts are resistant to Sendai virus-induced apoptosis. A functional screen identifies the hepatitis C virus NS3/4A and the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) nonstructural protein (NSP15) as inhibitors of MAVS-induced apoptosis, possibly as a method of immune evasion.

Significance

This study describes a novel role for MAVS in controlling viral infections through the induction of apoptosis, and identifies viral proteins which inhibit this host response.     

The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins.

Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex.     

RNF123 has an E3 ligase‐independent function in RIG‐I‐like receptor‐mediated antiviral signaling

Retinoic acid‐inducible gene I (RIG‐I) and melanoma differentiation‐associated gene 5 (MDA5) are cytoplasmic sensors crucial for recognizing different species of viral RNAs, which triggers the production of type I interferons (IFNs) and inflammatory cytokines. Here, we identify RING finger protein 123 (RNF123) as a negative regulator of RIG‐I and MDA5. Overexpression of RNF123 inhibits IFN‐β production triggered by Sendai virus (SeV) and encephalomyocarditis picornavirus (EMCV). Knockdown or knockout of endogenous RNF123 potentiates IFN‐β production triggered by SeV and EMCV, but not by the sensor of DNA viruses cGAS. RNF123 associates with RIG‐I and MDA5 in both endogenous and exogenous cases in a viral infection‐inducible manner. The SPRY and coiled‐coil, but not the RING, domains of RNF123 are required for the inhibitory function. RNF123 interacts with the N‐terminal CARD domains of RIG‐I/MDA5 and competes with the downstream adaptor VISA/MAVS/IPS‐1/Cardif for RIG‐I/MDA5 CARD binding. These findings suggest that RNF123 functions as a novel inhibitor of innate antiviral signaling mediated by RIG‐I and MDA5, a function that does not depend on its E3 ligase activity.     

Aging impairs IFN regulatory factor 7 up-regulation in plasmacytoid dendritic cells during TLR9 activation

Plasmacytoid dendritic cells (pDCs) are innate sensors that produce IFN-alpha in response to viral infections. Determining how aging alters the cellular and molecular function of these cells may provide an explanation of increased susceptibility of older people to viral infections. Hence, we examined whether aging critically impairs pDC function during infection with HSV-2, a viral pathogen that activates TLR9. We found that impaired IFN-alpha production by aged murine pDCs led to impaired viral clearance with aging. Upon TLR9 activation, aged pDCs displayed defective up-regulation of IFN-regulatory factor 7, a key adaptor in the type I IFN pathway, as compared with younger counterparts. Aged pDCs had more oxidative stress, and reducing oxidative stress in aged pDCs partly recovered the age-induced IFN-alpha defect during TLR9 activation. In sum, aging impairs the type I IFN pathway in pDCs, and this alteration may contribute to the increased susceptibility of older people to certain viral infections.     

A conserved dileucine motif mediates clathrin and AP-2-dependent endocytosis of the HIV-1 envelope protein

During the assembly of enveloped viruses viral and cellular components essential for infectious particles must colocalize at specific membrane locations. For the human and simian immunodeficiency viruses (HIV and SIV), sorting of the viral envelope proteins (Env) to assembly sites is directed by trafficking signals located in the cytoplasmic domain of the transmembrane protein gp41 (TM). A membrane proximal conserved GYxx? motif mediates endocytosis through interaction with the clathrin adaptor AP-2. However, experiments with SIV(mac239) Env indicate the presence of additional signals. Here we show that a conserved C-terminal dileucine in HIV(HxB2) also mediates endocytosis. Biochemical and morphological assays demonstrate that the C-terminal dileucine motif mediates internalization as efficiently as the GYxx? motif and that both must be removed to prevent Env internalization. RNAi experiments show that depletion of the clathrin adaptor AP-2 leads to increased plasma membrane expression of HIV Env and that this adaptor is required for efficient internalization mediated by both signals. The redundancy of conserved endocytosis signals and the role of the SIV(mac239) Env GYxx? motif in SIV pathogenesis, suggest that these motifs have functions in addition to endocytosis, possibly related to Env delivery to the site of viral assembly and/or incorporation into budding virions.