Peyman Nakhaei

Negative Regulation of the Retinoic Acid-inducible Gene I-induced Antiviral State by the Ubiquitin-editing Protein A20

Activation of the interferon regulatory factors (IRFs) 3 and 7 transcription factors is essential for the induction of type I interferon (IFN) and development of the innate antiviral response. Retinoic acid-inducible gene I has been shown to contribute to virus-induced IFN production independent of the Toll-like receptor pathways in response to a variety of RNA viruses and double-stranded RNA. In the present study, we demonstrate that the NF-κB-inducible, anti-apoptotic protein A20 efficiently blocks RIG-I-mediated activation of NF-κB-, IRF-3-, and IRF-7-dependent promoters but only weakly interferes with TRIF-TLR-3-mediated IFN activation. Expression of A20 completely blocked CARD domain containing ΔRIG-I-induced IRF-3 Ser-396 phosphorylation, homodimerization, and DNA binding. The level of A20 inhibition was upstream of the TBK1/IKKϵ kinases that phosphorylate IRF3 and IRF7 and paradoxically, A20 selectively degraded the TRIF protein but not RIG-I. A20 possesses two ubiquitin-editing domains, an N-terminal deubiquitination domain and a C-terminal ubiquitin ligase domain consisting of seven zinc finger domains. Deletion of the N-terminal de-ubiquitination domain had no significant effect on the inhibitory effect of A20, whereas deletion or mutation of zinc finger motif 7 ablated the inhibitory function of A20 on IRF- or NF-κB-mediated gene expression. Furthermore, cells stably expressing the active form of RIG-I induced an antiviral state that interfered with replication of vesicular stomatitis virus, an effect that was reversed by stable co-expression of A20. These results suggest that the virus-inducible, NF-κB-dependent activation of A20 functions as a negative regulator of RIG-I-mediated induction of the antiviral state.

Dissociation of a MAVS/IPS-1/VISA/Cardif-IKKepsilon molecular complex from the mitochondrial outer membrane by hepatitis C virus NS3-4A proteolytic cleavage

ABSTRACT Intracellular RNA virus infection is detected by the cytoplasmic RNA helicase RIG-I that plays an essential role in signaling to the host antiviral response. Recently, the adapter molecule that links RIG-I sensing of incoming viral RNA to downstream signaling and gene activation events was characterized by four different groups; MAVS/IPS-1-1/VISA/Cardif contains an amino-terminal CARD domain and a carboxyl-terminal mitochondrial transmembrane sequence that localizes to the mitochondrial membrane. Furthermore, the hepatitis C virus NS3-4A protease complex specifically targets MAVS/IPS-1/VISA/Cardif for cleavage as part of its immune evasion strategy. With a novel search program written in python, we also identified an uncharacterized protein, KIAA1271 (K1271), containing a single CARD-like domain at the N terminus and a Leu-Val-rich C terminus that is identical to that of MAVS/IPS-1/VISA/Cardif. Using a combination of biochemical analysis, subcellular fractionation, and confocal microscopy, we now demonstrate that NS3-4A cleavage of MAVS/IPS-1/VISA/Cardif/K1271 results in its dissociation from the mitochondrial membrane and disrupts signaling to the antiviral immune response. Furthermore, virus-induced IKKε kinase, but not TBK1, colocalized strongly with MAVS at the mitochondrial membrane, and the localization of both molecules was disrupted by NS3-4A expression. Mutation of the critical cysteine 508 to alanine was sufficient to maintain mitochondrial localization of MAVS/IPS-1/VISA/Cardif and IKKε in the presence of NS3-4A. These observations provide an outline of the mechanism by which hepatitis C virus evades the interferon antiviral response.

RIG-I-Mediated Antiviral Signaling Is Inhibited in HIV-1 Infection by a Protease-Mediated Sequestration of RIG-I

ABSTRACT The rapid induction of type I interferon (IFN) is essential for establishing innate antiviral responses. During infection, cytoplasmic viral RNA is sensed by two DExD/H box RNA helicases, RIG-I and MDA5, ultimately driving IFN production. Here, we demonstrate that purified genomic RNA from HIV-1 induces a RIG-I-dependent type I IFN response. Both the dimeric and monomeric forms of HIV-1 were sensed by RIG-I, but not MDA5, with monomeric RNA, usually found in defective HIV-1 particles, acting as a better inducer of IFN than dimeric RNA. However, despite the presence of HIV-1 RNA in the de novo infection of monocyte-derived macrophages, HIV-1 replication did not lead to a substantial induction of IFN signaling. We demonstrate the existence of an evasion mechanism based on the inhibition of the RIG-I sensor through the action of the HIV-1 protease (PR). Indeed, the ectopic expression of PR resulted in the inhibition of IFN regulatory factor 3 (IRF-3) phosphorylation and decreased expression of IFN and interferon-stimulated genes. A downregulation of cytoplasmic RIG-I levels occurred in cells undergoing a single-cycle infection with wild-type provirus BH10 but not in cells transfected with a protease-deficient provirus, BH10-PR−. Cellular fractionation and confocal microscopy studies revealed that RIG-I translocated from the cytosol to an insoluble fraction during the de novo HIV-1 infection of monocyte-derived macrophages, in the presence of PR. The loss of cytoplasmic RIG-I was prevented by the lysosomal inhibitor E64, suggesting that PR targets RIG-I to the lysosomes. This study reveals a novel PR-dependent mechanism employed by HIV-1 to counteract the early IFN response to viral RNA in infected cells.

The E3 ubiquitin ligase Triad3A negatively regulates the RIG-I/MAVS signaling pathway by targeting TRAF3 for degradation.

The primary role of the innate immune response is to limit the spread of infectious pathogens, with activation of Toll-like receptor (TLR) and RIG-like receptor (RLR) pathways resulting in a pro-inflammatory response required to combat infection. Limiting the activation of these signaling pathways is likewise essential to prevent tissue injury in the host. Triad3A is an E3 ubiquitin ligase that interacts with several components of TLR signaling and modulates TLR activity. In the present study, we demonstrate that Triad3A negatively regulates the RIG-I RNA sensing pathway through Lys48-linked, ubiquitin-mediated degradation of the tumor necrosis factor receptor-associated factor 3 (TRAF3) adapter. Triad3A was induced following dsRNA exposure or virus infection and decreased TRAF3 levels in a dose-dependent manner; moreover, Triad3A expression blocked IRF-3 activation by Ser-396 phosphorylation and inhibited the expression of type 1 interferon and antiviral genes. Lys48-linked ubiquitination of TRAF3 by Triad3A increased TRAF3 turnover, whereas reduction of Triad3A expression by stable shRNA expression correlated with an increase in TRAF3 protein expression and enhancement of the antiviral response following VSV or Sendai virus infection. Triad3A and TRAF3 physically interacted together, and TRAF3 residues Y440 and Q442—previously shown to be important for association with the MAVS adapter—were also critical for Triad3A. Point mutation of the TRAF-Interacting-Motif (TIM) of Triad3A abrogated its ability to interact with TRAF3 and modulate RIG-I signaling. TRAF3 appears to undergo sequential ubiquitin “immuno-editing” following virus infection that is crucial for regulation of RIG-I-dependent signaling to the antiviral response. Thus, Triad3A represents a versatile E3 ubiquitin ligase that negatively regulates RIG-like receptor signaling by targeting TRAF3 for degradation following RNA virus infection.

Functional analysis of a dominant negative mutation of interferon regulatory factor 5.

Background Interferon regulatory factor (IRF) family members have been implicated as critical transcription factors that function in immune response, hematopoietic differentiation and cell growth regulation. Activation of IRF-5 results in the production of pro-inflammatory cytokines such as TNFα, IL6 and IL12p40, as well as type I interferons. Methodology/Principal Findings In this study, we identify a G202C (position relative to translation start codon) missense-mutation transcript of IRF-5 in transformed B and T cell lines, which were either infected or non-infected by viruses, and peripheral blood from ATL or CLL patients. The mutated transcript encodes a novel protein in which the sixty-eighth amino acid, Alanine, is substituted by Proline (IRF-5P68) in the DNA binding domain of IRF-5. IRF-5P68 phenotype results in a complete loss of its DNA-binding activity and functions as a dominant negative molecule through interacting with wild type IRF-5. Co-expression of IRF-5P68 inhibits MyD88-mediated IRF-5 transactivation. Moreover, Toll-like receptor (TLR)-dependent IL6 and IL12P40 production induced by lipopolysaccharide (LPS), R837 or CpG ODN 1826 was reduced in IRF-5 (P68) expressing cells as compared to the control cells. Conclusion IRF-5P68 acts as a dominant negative regulator that interferes with IRF-5-mediated production of pro-inflammatory cytokines. The functional characterization of the novel IRF-5 mutant in transformed B and T cell lines and in ATL and CLL patients may lead to a better understanding of the role of these transcriptional regulators in hematopoietic malignancies.

Vesicular Stomatitis Virus Oncolysis of T Lymphocytes Requires Cell Cycle Entry and Translation Initiation

ABSTRACT Vesicular stomatitis virus (VSV) is a candidate oncolytic virus that replicates and induces cell death in cancer cells while sparing normal cells. Although defects in the interferon antiviral response facilitate VSV oncolysis, other host factors, including translational and growth regulatory mechanisms, also appear to influence oncolytic virus activity. We previously demonstrated that VSV infection induces apoptosis in proliferating CD4+ T lymphocytes from adult T-cell leukemia samples but not in resting T lymphocytes or primary chronic lymphocytic leukemia cells that remain arrested in G0. Activation of primary CD4+ T lymphocytes with anti-CD3/CD28 is sufficient to induce VSV replication and cell death in a manner dependent on activation of the MEK1/2, c-Jun NH2-terminal kinase, or phosphatidylinositol 3-kinase pathway but not p38. VSV replication is specifically impaired by the cell cycle inhibitor olomoucine or rapamycin, which induces early G1 arrest, but not by aphidicolin or Taxol, which blocks at the G11S or G21M phase, respectively; this result suggests a requirement for cell cycle entry for efficient VSV replication. The relationship between increased protein translation following G0/G1 transition and VSV permissiveness is highlighted by the absence of mTOR and/or eIF4E phosphorylation whenever VSV replication is impaired. Furthermore, VSV protein production in activated T cells is diminished by small interfering RNA-mediated eIF4E knockdown. These results demonstrate that VSV replication in primary T lymphocytes relies on cell cycle transition from the G0 phase to the G1 phase, which is characterized by a sharp increase in ribogenesis and protein synthesis.

STING-ing the Antiviral Pathway

The cytosolic DNA sensing pathway has remained poorly defined thus far. A recent study by Ishikawa et al. demonstrates that STING is essential for DNA-mediated type I IFN production and host defence against DNA pathogens.

IκB Kinase ε-Dependent Phosphorylation and Degradation of X-Linked Inhibitor of Apoptosis Sensitizes Cells to Virus-Induced Apoptosis

ABSTRACT X-linked inhibitor of apoptosis (XIAP) is a potent antagonist of caspase 3-, 7-, and 9-dependent apoptotic activities that functions as an E3 ubiquitin ligase, and it targets caspases for degradation. In this study, we demonstrate that Sendai virus (SeV) infection results in the IKKε- or TBK1-mediated phosphorylation of XIAP in vivo at Ser430, resulting in Lys48-linked autoubiquitination at Lys322/328 residues, followed by the subsequent proteasomal degradation of XIAP. Interestingly, IKKε expression and XIAP turnover increases SeV-triggered mitochondrion-dependent apoptosis via the release of caspase 3, whereas TBK1 expression does not increase apoptosis. Interestingly, phosphorylation also regulates XIAP interaction with the transcription factor IRF3, suggesting a role in IRF3-Bax-mediated apoptosis. Our findings reveal a novel function of IKKε as a regulator of the virus-induced triggering of apoptosis via the phosphorylation-dependent turnover of XIAP.