Lin R, Lacoste J, Nakhaei P, Sun Q, Yang L, Paz S et al.. Dissociation of a MAVS/IPS-1/VISA/Cardif-IKKepsilon molecular complex from the mitochondrial outer membrane by hepatitis C virus NS3-4A proteolytic cleavage. J Virol 80: 6072-6083

Lady Davis Institute for Medical Research, McGill University, Montreal H3T 1E2, Quebec, Canada.
Journal of Virology (Impact Factor: 4.44). 07/2006; 80(12):6072-83. DOI: 10.1128/JVI.02495-05
Source: PubMed


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 IKKepsilon 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 IKKepsilon in the presence of NS3-4A. These observations provide an outline of the mechanism by which hepatitis C virus evades the interferon antiviral response.

Download full-text


Available from: Rongtuan Lin
  • Source
    • "Later studies showed that the role of A20 goes beyond the control of NF-κB and that A20 is a general inhibitor in innate immune signaling; it protects cells from chronic inflammation, endotoxic shock and plays a role of tumor suppressor [28], [29]. In particular, A20 inhibits IRF3/IRF7 signaling [30], [31]. Similarly as for the NF-κB pathway, it acts upstream of the TBK1–IKKϵ–IKKγ complex regulating negatively retinoic acid-inducible gene I protein (RIG-I) [32], and potentially may act at the level of this complex by binding to IKKγ [31]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: NF-κB is a key transcription factor that regulates innate immune response. Its activity is tightly controlled by numerous feedback loops, including two negative loops mediated by NF-κB inducible inhibitors, IκBα and A20, which assure oscillatory responses, and by positive feedback loops arising due to the paracrine and autocrine regulation via TNFα, IL-1 and other cytokines. We study the NF-κB system of interlinked negative and positive feedback loops, combining bifurcation analysis of the deterministic approximation with stochastic numerical modeling. Positive feedback assures the existence of limit cycle oscillations in unstimulated wild-type cells and introduces bistability in A20-deficient cells. We demonstrated that cells of significant autocrine potential, i.e., cells characterized by high secretion of TNFα and its receptor TNFR1, may exhibit sustained cytoplasmic-nuclear NF-κB oscillations which start spontaneously due to stochastic fluctuations. In A20-deficient cells even a small TNFα expression rate qualitatively influences system kinetics, leading to long-lasting NF-κB activation in response to a short-pulsed TNFα stimulation. As a consequence, cells with impaired A20 expression or increased TNFα secretion rate are expected to have elevated NF-κB activity even in the absence of stimulation. This may lead to chronic inflammation and promote cancer due to the persistent activation of antiapoptotic genes induced by NF-κB. There is growing evidence that A20 mutations correlate with several types of lymphomas and elevated TNFα secretion is characteristic of many cancers. Interestingly, A20 loss or dysfunction also leaves the organism vulnerable to septic shock and massive apoptosis triggered by the uncontrolled TNFα secretion, which at high levels overcomes the antiapoptotic action of NF-κB. It is thus tempting to speculate that some cancers of deregulated NF-κB signaling may be prone to the pathogen-induced apoptosis.
    Full-text · Article · Nov 2013 · PLoS ONE
  • Source
    • "A previous study by Foy et al. showed that the NS3/4A serine protease derived from a subgenomic replicon participates in the suppression of the cellular pathway that activates IRF-3 [12]. The NS3/4A protein of HCV disrupts signaling of the double-stranded RNA (dsRNA) receptors, retinoic acid-inducible gene-I (RIG-I), and Toll-like receptor 3 (TLR3) by inducing proteolysis of interferon promoter stimulator-1 (IPS-1) [13] [14] [15] and Toll/interleukin-1 receptor (TIR) domain-containing adaptor protein inducing IFN-b (TRIF) [16], as well as by suppressing the downstream activation of IFN-b [13]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Interferon regulatory factor-3 (IRF-3), a key transcriptional factor in the type I interferon system, is frequently impaired by hepatitis C virus (HCV), in order to establish persistent infection. However, the exact mechanism by which the virus establishes persistent infection has not been fully understood yet. The present study aimed to investigate the effects of various HCV proteins on IRF-3 activation, and elucidate the underlying mechanisms. To achieve this, full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into HepG2 cells. IFN- induction, plaque formation, and IRF-3 dimerization were elicited by Newcastle disease virus (NDV) infection. The expressions of IRF-3 homodimer and its monomer, Ser386-phosphorylated IRF-3, and HCV core protein were detected by immunofluorescence and western blotting. IFN- mRNA expression was quantified by real-time PCR (RT-PCR), and IRF-3 activity was measured by the levels of IRF-3 dimerization and phosphorylation, induced by NDV infection or polyriboinosinic:polyribocytidylic acid [poly(I:C)]. Switching of the expression of the complete HCV genome as well as the core proteins, E1, E2, and NS2, suppressed IFN- mRNA levels and IRF-3 dimerization, induced by NDV infection. Our study revealed a crucial region of the HCV core protein, basic amino acid region 1 (BR1), to inhibit IRF-3 dimerization as well as its phosphorylation induced by NDV infection and poly ((I:C), thus interfering with IRF-3 activation. Therefore, our study suggests that rescue of the IRF-3 pathway impairment may be an effective treatment for HCV infection.
    Full-text · Article · Oct 2012 · Biochemical and Biophysical Research Communications
  • Source
    • "In agreement with the above conclusions, peptides that represented a potential cleavage site in IPS-1 (503EREVPC-HRPS512), a potential host cell target of NS3/4A proteolysis [28], were not cleaved in our cleavage tests. It is now clear that an insertion of the positively charged P5 Arg between the Glu/Asp-Glu/Asp negatively charged sequence makes the IPS-1 sequence resistant to NS3/4A proteolysis. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The hepatitis C virus (HCV) genome encodes a long polyprotein, which is processed by host cell and viral proteases to the individual structural and non-structural (NS) proteins. HCV NS3/4A serine proteinase (NS3/4A) is a non-covalent heterodimer of the N-terminal, ∼180-residue portion of the 631-residue NS3 protein with the NS4A co-factor. NS3/4A cleaves the polyprotein sequence at four specific regions. NS3/4A is essential for viral replication and has been considered an attractive drug target. Using a novel multiplex cleavage assay and over 2,660 peptide sequences derived from the polyprotein and from introducing mutations into the known NS3/4A cleavage sites, we obtained the first detailed fingerprint of NS3/4A cleavage preferences. Our data identified structural requirements illuminating the importance of both the short-range (P1-P1') and long-range (P6-P5) interactions in defining the NS3/4A substrate cleavage specificity. A newly observed feature of NS3/4A was a high frequency of either Asp or Glu at both P5 and P6 positions in a subset of the most efficient NS3/4A substrates. In turn, aberrations of this negatively charged sequence such as an insertion of a positively charged or hydrophobic residue between the negatively charged residues resulted in inefficient substrates. Because NS5B misincorporates bases at a high rate, HCV constantly mutates as it replicates. Our analysis revealed that mutations do not interfere with polyprotein processing in over 5,000 HCV isolates indicating a pivotal role of NS3/4A proteolysis in the virus life cycle. Our multiplex assay technology in light of the growing appreciation of the role of proteolytic processes in human health and disease will likely have widespread applications in the proteolysis research field and provide new therapeutic opportunities.
    Full-text · Article · Apr 2012 · PLoS ONE
Show more