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How Hepatitis C Short-Circuits the Immune System
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Newswise — To find an effective treatment for hepatitis C, which chronically infects nearly 200 million people worldwide, researchers need to understand how the virus is able to avoid destruction by the immune system. Hepatitis C persists in the body for decades after an initial infection, often causing so much liver damage that liver transplantation may be a patient's only chance for survival.
Now, scientists at the University of Texas Medical Branch at Galveston (UTMB) and the University of Texas Southwestern Medical Center in Dallas have figured out two key parts of hepatitis C's strategy for evading the human immune response. In papers to be published online today in the Proceedings of the National Academy of Sciences (PNAS), UTMB and UT-Southwestern researchers define two critical elements of the immune response shut down by a hepatitis C virus protein called NS3/4A. These virus-caused "short circuits" prevent the production of signaling molecules that mobilize cells' antiviral defenses.
In an additional paper, appearing in the current Journal of Virology and available now online, the UT Southwestern and UTMB researchers demonstrate the critical role played by one of the "circuits," known as the RIG-I pathway, in cellular defense against hepatitis C virus.
The discoveries are particularly important in light of recent promising results from early clinical trials of new investigational drugs that target NS3/4A and both block the reproduction of hepatitis C and nullify its ability to dodge immune defenses, according to Stanley M. Lemon, a senior author on the papers and director of UTMB's NIH-funded hepatitis C research center. "At least one protease inhibitor has had extraordinary activity against hepatitis C in human clinical trials, but we're going to need to improve on it in a number of ways, including reducing the potential for the virus to become resistant to it," Lemon said. "A better understanding of how NS3/4A does its work in blocking the immune response will help make that possible."
Both articles are reported below.
Lemon's group, including lead author and assistant professor of microbiology and immunology Kui Li, postdoctoral fellows Josephine C. Ferreon, Mitsuyasu Nakamura, Allan C.M. Ferreon and Masanori Ikeda, collaborated with Michael Gale and Eileen Foy of UT Southwestern on one PNAS paper, "Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF." Lemon and Li also collaborated with the UT-Southwestern team led by Gale on the second PNAS paper ("Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling"), on which Foy is the lead author, and Rhea Sumpter, Jr., Yueh-Ming Loo, Cynthia L. Johnson, Chunfu Wang, and Penny Mar-Fish are co-authors.
Regulating Intracellular Antiviral Defense and Permissiveness to Hepatitis C Virus RNA Replication through a Cellular RNA Helicase, RIG-I
Journal of Virology, March 2005
Rhea Sumpter Jr.,1 Yueh-Ming Loo,1 Eileen Foy,1 Kui Li,2 Mitsutoshi Yoneyama,3 Takashi Fujita,3 Stanley M. Lemon,2 and Michael Gale Jr.1*
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas,1 Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas,2 Department of Tumor Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Tokyo, Japan3
ABSTRACT
Virus-responsive signaling pathways that induce alpha/beta interferon production and engage intracellular immune defenses influence the outcome of many viral infections. The processes that trigger these defenses and their effect upon host permissiveness for specific viral pathogens are not well understood. We show that structured hepatitis C virus (HCV) genomic RNA activates interferon regulatory factor 3 (IRF3), thereby inducing interferon in cultured cells. This response is absent in cells selected for permissiveness for HCV RNA replication. Studies including genetic complementation revealed that permissiveness is due to mutational inactivation of RIG-I, an interferon-inducible cellular DExD/H box RNA helicase. Its helicase domain binds HCV RNA and transduces the activation signal for IRF3 by its caspase recruiting domain homolog. RIG-I is thus a pathogen receptor that regulates cellular permissiveness to HCV replication and, as an interferon-responsive gene, may play a key role in interferon-based therapies for the treatment of HCV infection.
INTRODUCTION
Hepatitis C virus (HCV) is a major public health problem, infecting nearly 200 million people worldwide and causing hepatic fibrosis, end-stage cirrhosis, and hepatocellular carcinoma (16). A member of the Flaviviridae, HCV's positive-sense RNA genome contains highly structured 5' and 3' nontranslated regions (NTRs) flanking a large open reading frame (ORF) encoding a polyprotein that is processed into both structural (core-E2) and nonstructural (NS) proteins (Fig. 1A). The NS3-NS5B proteins support viral genome replication, which is also dependent upon conserved RNA sequences within the 5'NTR and 3'NTR that are highly structured and required for both protein translation and RNA replication (15, 20). HCV infection is treated with alpha interferon (IFN-{alpha})-based therapy, but treatment is effective at best in only 50% of patients (10). The nearly unique ability of HCV to establish persistent infections in humans has been attributed, in part, to a variety of strategies to evade host immune and IFN-induced defenses (12). Epidemiological studies suggest that 25 to 50% of all persons resolve acute HCV infection without treatment (16), however, indicating that innate and/or adaptive immune responses are indeed capable of controlling the outcome of HCV infection. Processes that regulate innate intracellular antiviral responses may therefore serve as pivotal points of control, potentially limiting host permissiveness for HCV replication and favorably modulating subsequent adaptive immune responses.
Virus-induced production of IFN-{alpha} and IFN-ß and the subsequent expression of IFN-stimulated genes (ISGs) are central to these antiviral defenses (22). This host response is initiated by cellular recognition of a pathogen-associated molecular pattern (PAMP) presented by the infection, in which a host protein receptor is engaged by the PAMP ligand and signals downstream components to activate intracellular immune defenses. In mammalian cells, replicating viral RNAs present features of nucleic acid sequence or structure that are recognized as distinct PAMPs by membrane-spanning Toll-like receptors (TLRs) or intracellular proteins coupled to signaling pathways that induce interferon production. Double-stranded RNA (dsRNA) and GU-rich single-stranded RNA (ssRNA) are recognized by TLR3 and TLR7/8, respectively (18). However, viral dsRNA can also initiate cellular responses through an intracellular, TLR3-independent mechanism (5, 32), thereby activating a set of latent transcription factors, including IFN-regulatory factor 3 (IRF3) and NF-{kappa}B that coordinately assemble onto the IFN-ß promoter and induce IFN expression, ISG production and an intracellular antiviral state. Among these, IRF3 is essential for IFN production (21). Its activation occurs through redundant actions of TBK1 or IKK{varepsilon} protein kinases, which catalyze its carboxyl-terminal phosphorylation and result in nuclear translocation, DNA binding, and transcription-effector actions (6, 23). The IRF3-mediated induction of IFNs and ISGs, initiated by the recognition of an HCV-specific PAMP, is likely to impose intracellular restrictions to viral replication that limit host cell permissiveness (7, 25). To define how this host response influences HCV infection, we investigated the relationship between innate antiviral responses and cellular permissiveness for HCV RNA replication in a cell culture model.
AUTHOR DISCUSSION
RIG-I is a signal transducer and HCV RNA PAMP receptor. Our results define the cellular RNA helicase, RIG-I, as a transducer of signals that activate innate antiviral defenses limiting HCV RNA replication. In cultured hepatocytes, RIG-I plays an essential role as a TLR-independent PAMP receptor that specifically binds dsRNA and structured regions within the HCV genome to signal IRF3 activation, IFN production, and ISG expression. This response is an important component of the acute hepatic antiviral defenses that are triggered in vivo within days of exposure to HCV (2) and is therefore likely to contribute to the resolution of acute infection (26). Our data also demonstrate that RIG-I is essential for triggering IRF3 activation in response to SenV or VSV infection in hepatocytes (Fig. 2B and 3E), a finding consistent with its role as a viral PAMP receptor. We speculate that secondary or tertiary RNA structures, often present in viral RNAs (24), present a specific PAMP that is engaged by RIG-I. RIG-I specifically bound the structured 5'NTR and 3'NTR segments of the HCV genome but not the predicted nonstructured viral RNAs (Fig. 4B and E). The RIG-I-based cellular response exploits the need for HCV to conserve unique structured terminal RNA segments that are essential to viral replication. This interaction signals the induction of IRF3 responsive genes and other ISGs, including ISG56, that have been shown to suppress HCV RNA replication (7, 29). The binding of RIG-I to viral RNA thus induces critical antiviral effectors impacting cellular permissiveness for HCV through processes that limit viral RNA replication.
CARD signaling activates downstream IRF3. CARD-containing proteins mediate signaling events in response to a variety of intracellular and extracellular pathogen-related stimuli that direct NF-{kappa}B activation via TLR-independent mechanisms (4). RIG-I and other CARD-containing DExD/H box helicase proteins thus appear to represent a family of proteins that activate a TLR3-independent response against a variety of viral pathogens (32). The RIG-I CARD-homology domain is capable of inducing IRF3 activation. Sequence differences, which distinguish the RIG-I homolog from other CARD proteins (4, 32), may provide for recruitment of unique signaling components that activate IRF3 through processes that depart from the canonical CARD-CARD homotypic interaction pathways. The kinase TBK1, which does not contain a CARD, is essential for dsRNA or virus-induced IRF3 activation (17), suggesting that it is required for RIG signaling to IRF3. TBK1 does not directly associate with RIG-I (E. Foy and M. Gale, Jr., unpublished observations). The most likely scenario for RIG-I-mediated IRF3 activation involves interaction with downstream adaptor(s) or CARD-containing proteins that ultimately results in TBK1/IRF3 activation. In this regard, the location of the T55I mutation in the first CARD domain of the Huh7.5 RIG-I mutant is of particular interest. It uncoupled signal transduction from viral RNA binding. Huh7.5 cells are derived from and are isogenic to Huh7 cells (3). Among other potential lesions in antiviral processes within Huh7-derived cells, the hyperpermissive phenotype of Huh7.5 cells can be attributed to the RIG-I T55I mutation that abolishes PAMP signaling to IRF3. The IRF3 response contributes to control of HCV RNA replication (7). Thus, activation of IRF3 is a critical determinant of cellular permissiveness for HCV RNA replication, although defects in other, RIG-I-independent innate antiviral defense pathways could contribute to the overall level of cellular permissiveness for HCV.
A new model for virus activation of IFN-{alpha}/ß and ISGs. We propose a model in which within the infected hepatocyte RIG-I binding to the HCV RNA PAMP induces events that are regulated by its helicase domain and alter its conformation sufficiently to recruit or interact with signaling partners that direct the downstream phosphorylation of IRF3 and expression of antiviral effector genes (Fig. 5D). This response is likely to be triggered by the HCV RNA PAMP immediately upon introduction of the viral genome into the host cell cytoplasm (see Fig. 5A). Moreover, recently published observations demonstrate that HCV RNA replication can trigger IRF3 activation (25), implying that RIG-I engages HCV during the process of viral replication. Since RIG-I is strongly IFN inducible (Fig. S3 in the supplemental material), one of the effects of IFN therapy is an enhanced sensing of virus replication, which initiates a cascade of antiviral events. The importance of the RIG-I pathway for cellular permissiveness to viral replication is underscored by the variety of ways that viruses antagonize downstream IRF3 actions (12). Indeed, HCV has the potential to block virus triggering of IRF3 phosphorylation through the actions of its NS3/4A serine protease (7). The results from recent work now indicate that this regulation is attributed to blockade of RIG-I-dependent signaling imposed by the protease function of NS3/4A (7a), and it is likely that upon its accumulation NS3/4A antagonizes RIG-I signaling in the infected cell. Therapeutic approaches to control RIG-I and NS3/4A function may provide novel strategies to limit HCV infection by modulating cellular permissiveness for virus replication.
Immune evasion by hepatitis C virus NS3 4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF
Kui Li*†, Eileen Foy‡, Josephine C. Ferreon*†, Mitsuyasu Nakamura*†, Allan C. M. Ferreon§, Masanori Ikeda*†,
Stuart C. Ray¶, Michael Gale, Jr.‡, and Stanley M. Lemon*†
*Departments of Microbiology and Immunology and §Human Biological Chemistry and Genetics and †Institute for Human Infections and Immunity,
University of Texas Medical Branch, Galveston, TX 77555-1019; ‡Department of Microbiology, University of Texas Southwestern Medical Center,
Dallas, TX 75390-9048; and ¶Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
ABSTRACT
Toll-like receptors (TLRs) bind pathogen-specific ligands early in infection, initiating signaling pathways that lead to expression of multiple protective cellular genes. Many viruses have evolved strategies that block the effector mechanisms induced through these signaling pathways, but viral interference with critical proximal receptor interactions has not been described. We show here that the NS3 4A serine protease of hepatitis C virus (HCV), a virus notorious for its ability to establish persistent intrahepatic infection, causes specific proteolysis of Toll-IL-1 receptor domaincontaining adaptor inducing IFN- (TRIF or TICAM-1), an adaptor protein linking TLR3 to kinases responsible for activating IFN regulatory factor 3 (IRF-3) and NF- B, transcription factors controlling a multiplicity of antiviral defenses. NS3 4A-mediated cleavage of TRIF reduces its abundance and inhibits polyI:C-activated signaling through the TLR3 pathway before its bifurcation to IRF-3 and NF- B. This uniquely broad mechanism of immune evasion potentially limits expression of multiple host defense genes, thereby promoting persistent infections with this medically important virus.
AUTHOR DISCUSSION
Strong IFN responses have been demonstrated in the livers of chimpanzees during acute HCV infection (34, 35). These responses may be explained by the fact that NS3 4A is capable of inhibiting viral activation of IRF-3 and NF- B only within infected cells. Nonetheless, IRF-3 activation limits the replication of HCV RNA in cultured hepatocytes (3). NS3 4A disruption of signaling pathways that lead to IRF-3 activation may thus contribute to HCV persistence by inhibiting expression of type 1 IFNs and ISGs that restrict viral replication (11, 12). In addition, an impairment in the virus-induced expression of type 1 IFNs and other cytokines could suppress or delay subsequent adaptive CD8 T cell responses required for elimination of HCV (36, 37). Disruption of virus-activated NF- B-mediated responses may also promote viral persistence (38). Although the pathways leading to activation of IRF-3 are likely to be redundant in vivo (7, 8), mice defective for the murine homolog of TRIF are compromised in their response to infection with murine cytomegalovirus (26), demonstrating the potential importance of the TLR3--TRIF pathway for control of virus infection.
The cleavage of TRIF by NS3 4A represents a uniquely broad mechanism of viral immune evasion. Poxvirus A52R protein blocks TLR3-mediated activation of NF- B by associating with IL-1 receptor-associated kinase 2 and TRAF6, key proteins within this TLR3 signaling pathway (39). However, NS3 4A targeting of TRIF represents a more proximal attack on the pathway and inhibits both NF- B and IRF-3 activation through TLR3. The targeting of TRIF by NS3 4A thus has the potential to exert a crippling effect on a multiplicity of protective cellular defense mechanisms. Nonetheless, the cleavage of TRIF is unlikely to account for the NS3 4A inhibition of Sendai virus activation of IRF-3 that we observed previously in Huh7 cells (3). These cultured hepatoma cells are TLR3-deficient (Fig. 9B), perhaps explaining why they are relatively permissive for HCV RNA replication (28). Thus, although we found that NS3 4A expressed from replicating HCV RNA suppresses TLR3 signaling in HeLa cells, further studies will be needed to determine the status of this pathway in infected hepatocytes. Sendai virus activates IRF-3 in Huh7 cells through the RIG-I pathway independently of TRIF, leading us to speculate that the NS3 4A disruption of this pathway represents proteolytic targeting of yet a second signaling molecule (40).
The capacity of the protease active site to accommodate unique substrate interactions with TRIF, and, possibly, a second cellular protein, represents a remarkable example of RNA virus evolution and may account for the unusually shallow active site conformation that distinguishes NS3 4A from other viral proteases (6). It may also explain, in part, the exceptionally potent antiviral efficacy of a specific inhibitor of the NS3 4A protease, BILN 2061, in recent clinical trials (41). Inhibition of the protease may not only suppress replication of the virus, but also restore innate antiviral defenses to the virus in infected hepatocytes.
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