Functional and therapeutic analysis of hepatitis C virus NS3.4A protease control of antiviral immune defense

Chronic hepatitis C virus (HCV) infection is a major global public health problem. HCV infection is supported by viral strategies to evade the innate antiviral response wherein the viral NS3.4A protease complex targets and cleaves the interferon promoter stimulator-1 (IPS-1) adaptor protein to ablate signaling of interferon alpha/beta immune defenses. Here we examined the structural requirements of NS3.4A and the therapeutic potential of NS3.4A inhibitors to control the innate immune response against virus infection. The structural composition of NS3 includes an amino-terminal serine protease domain and a carboxyl-terminal RNA helicase domain. NS3 mutants lacking the helicase domain retained the ability to control virus signaling initiated by retinoic acid-inducible gene-I (RIG-I) or melanoma differentiation antigen 5 and suppressed the downstream activation of interferon regulatory factor-3 (IRF-3) and nuclear factor kappaB (NF-kappaB) through the targeted proteolysis of IPS-1. This regulation was abrogated by truncation of the NS3 protease domain or by point mutations that ablated protease activity. NS3.4A protease control of antiviral immune signaling was due to targeted proteolysis of IPS-1 by the NS3 protease domain and minimal NS4A cofactor. Treatment of HCV-infected cells with an NS3 protease inhibitor prevented IPS-1 proteolysis by the HCV protease and restored RIG-I immune defense signaling during infection. Thus, the NS3.4A protease domain can target IPS-1 for cleavage and is essential for blocking RIG-I signaling to IRF-3 and NF-kappaB, whereas the helicase domain is dispensable for this action. Our results indicate that NS3.4A protease inhibitors have immunomodulatory potential to restore innate immune defenses to HCV infection.     

Convergent evolution of escape from hepaciviral antagonism in primates

The ability to mount an interferon response on sensing viral infection is a critical component of mammalian innate immunity. Several viruses directly antagonize viral sensing pathways to block activation of the host immune response. Here, we show that recurrent viral antagonism has shaped the evolution of the host protein MAVS--a crucial component of the viral-sensing pathway in primates. From sequencing and phylogenetic analyses of MAVS from 21 simian primates, we found that MAVS has evolved under strong positive selection. We focused on how this positive selection has shaped MAVS' susceptibility to Hepatitis C virus (HCV). We functionally tested MAVS proteins from diverse primate species for their ability to resist antagonism by HCV, which uses its protease NS3/4A to cleave human MAVS. We found that MAVS from multiple primates are resistant to inhibition by the HCV protease. This resistance maps to single changes within the protease cleavage site in MAVS, which protect MAVS from getting cleaved by the HCV protease. Remarkably, most of these changes have been independently acquired at a single residue 506 that evolved under positive selection. We show that "escape" mutations lower affinity of the NS3 protease for MAVS and allow it to better restrict HCV replication. We further show that NS3 proteases from all other primate hepaciviruses, including the highly divergent GBV-A and GBV-C viruses, are functionally similar to HCV. We conclude that convergent evolution at residue 506 in multiple primates has resulted in escape from antagonism by hepaciviruses. Our study provides a model whereby insights into the ancient history of viral infections in primates can be gained using extant host and virus genes. Our analyses also provide a means by which primates might clear infections by extant hepaciviruses like HCV.     

Control of innate immune signaling and membrane targeting by the Hepatitis C virus NS3/4A protease are governed by the NS3 helix α0

Hepatitis C virus (HCV) infection is sensed in the host cell by the cytosolic pathogen recognition receptor RIG-I. RIG-I signaling is propagated through its signaling adaptor protein MAVS to drive activation of innate immunity. However, HCV blocks RIG-I signaling through viral NS3/4A protease cleavage of MAVS on the mitochondrion-associated endoplasmic reticulum (ER) membrane (MAM). The multifunctional HCV NS3/4A serine protease is associated with intracellular membranes, including the MAM, through membrane-targeting domains within NS4A and also at the amphipathic helix α(0) of NS3. The serine protease domain of NS3 is required for both cleavage of MAVS, a tail-anchored membrane protein, and processing the HCV polyprotein. Here, we show that hydrophobic amino acids in the NS3 helix α(0) are required for selective cleavage of membrane-anchored portions of the HCV polyprotein and for cleavage of MAVS for control of RIG-I pathway signaling of innate immunity. Further, we found that the hydrophobic composition of NS3 helix α(0) is essential to establish HCV replication and infection. Alanine substitution of individual hydrophobic amino acids in the NS3 helix α(0) impaired HCV RNA replication in cells with a functional RIG-I pathway, but viral RNA replication was rescued in cells lacking RIG-I signaling. Therefore, the hydrophobic amphipathic helix α(0) of NS3 is required for NS3/4A control of RIG-I signaling and HCV replication by directing the membrane targeting of both viral and cellular substrates.     

丙型肝炎病毒感染过程中其NS3/4A蛋白酶切割线粒体抗病毒信号蛋白的动态过程

已知丙型肝炎病毒(hepatitis C virus,HCV)可通过其蛋白酶NS3/4A切割线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS)来逃逸天然免疫识别,但尚不清楚其切割动力学及切割在抑制干扰素中的作用。本研究旨在细胞模型中探讨HCV感染过程中病毒复制建立及病毒NS3/4A切割MAVS的动态过程,探究NS3/4A切割MAVS对病毒逃逸宿主天然免疫建立感染的贡献。首先构建基于绿色荧光蛋白(green fluorescent protein,GFP)的MAVS切割报告系统(GFP-NLS-MAVS-TM462),用 HCV Jc1-Gluc 感染Huh7.5/GFP-NLS-MAVS-TM462细胞。结果显示,病毒复制早期MAVS切割效率较低;NS3/4A高效切割MAVS发生于HCV复制晚期,且其切割效率与NS3蛋白水平相关。利用带有GFP ypet的HCV报告病毒Jc1-378-1感染Huh7.5/RFP-NLS-MAVS-TM462细胞,在单细胞水平观察HCV感染阳性细胞中MAVS被切割情况,发现HCV复制细胞中仅部分细胞MAVS被切割。进一步研究发现,不同基因型NS3/4A切割MAVS的效率仅与NS3表达水平相关。以上结果提示,HCV蛋白酶NS3/4A切割MAVS依赖NS3/4A蛋白在病毒复制过程中的累积,对在病毒复制早期逃逸宿主天然免疫建立感染可能无显著贡献。     

Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A. Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B. The minimum cofactor region that supported GBV-B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity. Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function.     

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

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.     

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.     

Canine Hepacivirus NS3 Serine Protease Can Cleave the Human Adaptor Proteins MAVS and TRIF

Canine hepacivirus (CHV) was recently identified in domestic dogs and horses. The finding that CHV is genetically the virus most closely related to hepatitis C virus (HCV) has raised the question of whether HCV might have evolved as the result of close contact between dogs and/or horses and humans. The aim of this study was to investigate whether the NS3/4A serine protease of CHV specifically cleaves human mitochondrial antiviral signaling protein (MAVS) and Toll-IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF). The proteolytic activity of CHV NS3/4A was evaluated using a bacteriophage lambda genetic screen. Human MAVS- and TRIF-specific cleavage sites were engineered into the lambda cI repressor. Upon infection of Escherichia coli cells coexpressing these repressors and a CHV NS3/4A construct, lambda phage replicated up to 2000-fold more efficiently than in cells expressing a CHV protease variant carrying the inactivating substitution S139A. Comparable results were obtained when several HCV NS3/4A constructs of genotype 1b were assayed. This indicates that CHV can disrupt the human innate antiviral defense signaling pathway and suggests a possible evolutionary relationship between CHV and HCV.     

GB virus B and hepatitis C virus NS3 serine proteases share substrate specificity.

GB virus B (GBV-B) is a recently discovered virus responsible for hepatitis in tamarins (Saguinus species). GBV-B belongs to the Flaviviridae family and is closely related to the human pathogen hepatitis C virus (HCV). Nonstructural protein 3 (NS3) of HCV has been shown to encompass a serine protease domain required for viral maturation. GBV-B and HCV share only about 30% of the amino acid sequence within the NS3 protease domain. The catalytic triad is conserved, and the residue Phe-154, presumed to be a crucial amino acid for determining the S1 specificity pocket of the HCV NS3 protease, is also conserved. We have expressed a synthetic gene encoding the GBV-B NS3 protease domain in Escherichia coli and have characterized the purified recombinant protein for its activity on HCV substrates. We have shown that the NS3 region of the GBV-B genome actually encodes a serine protease that, despite the low sequence homology, shares substrate specificity with the HCV NS3 protease.     

Identification of a novel sequence at the 3' end of the GB virus B genome

GB virus B (GBV-B) is a virus of the family Flaviviridae that infects small primates (Saguinus sp. [tamarins]) and shows similarities to hepatitis C virus (HCV) in genome organization, protein function, tissue tropism, and pathogenicity. This suggests the possibility of using tamarins infected by GBV-B or GBV-B/HCV chimeric viruses as a surrogate animal model of HCV infection. To achieve the construction of such chimeric viruses, it is essential to produce a complete and infectious GBV-B genomic RNA. We have identified a novel sequence at the 3' end of the GBV-B genome and show that it can be arranged in a secondary structure resembling that of the 3' end of the HCV genome, which is known to be essential for infectivity.     

Activation of Type I and III Interferon Response by Mitochondrial and Peroxisomal MAVS and Inhibition by Hepatitis C Virus

Sensing viruses by pattern recognition receptors (PRR) triggers the innate immune system of the host cell and activates immune signaling cascades such as the RIG-I/IRF3 pathway. Mitochondrial antiviral-signaling protein (MAVS, also known as IPS-1, Cardif, and VISA) is the crucial adaptor protein of this pathway localized on mitochondria, peroxisomes and mitochondria-associated membranes of the endoplasmic reticulum. Activation of MAVS leads to the production of type I and type III interferons (IFN) as well as IFN stimulated genes (ISGs). To refine the role of MAVS subcellular localization for the induction of type I and III IFN responses in hepatocytes and its counteraction by the hepatitis C virus (HCV), we generated various functional and genetic knock-out cell systems that were reconstituted to express mitochondrial (mito) or peroxisomal (pex) MAVS, exclusively. Upon infection with diverse RNA viruses we found that cells exclusively expressing pexMAVS mounted sustained expression of type I and III IFNs to levels comparable to cells exclusively expressing mitoMAVS. To determine whether viral counteraction of MAVS is affected by its subcellular localization we employed infection of cells with HCV, a major causative agent of chronic liver disease with a high propensity to establish persistence. This virus efficiently cleaves MAVS via a viral protease residing in its nonstructural protein 3 (NS3) and this strategy is thought to contribute to the high persistence of this virus. We found that both mito- and pexMAVS were efficiently cleaved by NS3 and this cleavage was required to suppress activation of the IFN response. Taken together, our findings indicate comparable activation of the IFN response by pex- and mitoMAVS in hepatocytes and efficient counteraction of both MAVS species by the HCV NS3 protease.     

IFN-α/β and autophagy

Hepatitis C virus (HCV) infects approximately 130 million people worldwide. The clinical sequelae of this chronic disease include cirrhosis, functional failure and carcinoma of the liver. HCV induces autophagy, a fundamental cellular process for maintaining homeostasis and mediating innate immune response, and also inhibits autophagic protein degradation and suppresses antiviral immunity. In addition to this ploy, the HCV serine protease composed of the viral non-structural proteins 3/4A (NS3/4A) can enzymatically digest two cellular proteins, mitochondria-associated anti-viral signaling protein (MAVS) and Toll/interleukin-1 receptor domain containing adaptor inducing IFN-β (TRIF). Since these two proteins are the adaptor molecules in the retinoic acid-inducible gene I (RIG-I) and TLR3 pathways, respectively, their cleavage has been suggested as a pivotal mechanism by which HCV blunts the IFN-α/β signaling and antiviral responses. Thus far, how HCV perturbs autophagy and copes with IFN-α/β in the liver remains unclear.     

IFN-α/β and autophagy: tug-of-war between HCV and the host

Hepatitis C virus (HCV) infects approximately 130 million people worldwide. The clinical sequelae of this chronic disease include cirrhosis, functional failure and carcinoma of the liver. HCV induces autophagy, a fundamental cellular process for maintaining homeostasis and mediating innate immune response, and also inhibits autophagic protein degradation and suppresses antiviral immunity. In addition to this ploy, the HCV serine protease composed of the viral non-structural proteins 3/4A (NS3/4A) can enzymatically digest two cellular proteins, mitochondria-associated anti-viral signaling protein (MAVS) and Toll/interleukin-1 receptor domain containing adaptor inducing IFN-β (TRIF). Since these two proteins are the adaptor molecules in the retinoic acid-inducible gene I (RIG-I) and TLR3 pathways, respectively, their cleavage has been suggested as a pivotal mechanism by which HCV blunts the IFN-α/β signaling and antiviral responses. Thus far, how HCV perturbs autophagy and copes with IFN-α/β in the liver remains unclear.     

A Distinct Role of Riplet-Mediated K63-Linked Polyubiquitination of the RIG-I Repressor Domain in Human Antiviral Innate Immune Responses

The innate immune system is essential for controlling viral infections, but several viruses have evolved strategies to escape innate immunity. RIG-I is a cytoplasmic viral RNA sensor that triggers the signal to induce type I interferon production in response to viral infection. RIG-I activation is regulated by the K63-linked polyubiquitin chain mediated by Riplet and TRIM25 ubiquitin ligases. TRIM25 is required for RIG-I oligomerization and interaction with the IPS-1 adaptor molecule. A knockout study revealed that Riplet was essential for RIG-I activation. However the molecular mechanism underlying RIG-I activation by Riplet remains unclear, and the functional differences between Riplet and TRIM25 are also unknown. A genetic study and a pull-down assay indicated that Riplet was dispensable for RIG-I RNA binding activity but required for TRIM25 to activate RIG-I. Mutational analysis demonstrated that Lys-788 within the RIG-I repressor domain was critical for Riplet-mediated K63-linked polyubiquitination and that Riplet was required for the release of RIG-I autorepression of its N-terminal CARDs, which leads to the association of RIG-I with TRIM25 ubiquitin ligase and TBK1 protein kinase. Our data indicate that Riplet is a prerequisite for TRIM25 to activate RIG-I signaling. We investigated the biological importance of this mechanism in human cells and found that hepatitis C virus (HCV) abrogated this mechanism. Interestingly, HCV NS3-4A proteases targeted the Riplet protein and abrogated endogenous RIG-I polyubiquitination and association with TRIM25 and TBK1, emphasizing the biological importance of this mechanism in human antiviral innate immunity. In conclusion, our results establish that Riplet-mediated K63-linked polyubiquitination released RIG-I RD autorepression, which allowed the access of positive factors to the RIG-I protein.     

Hepatitis C virus NS3‐4A inhibits the peroxisomal MAVS‐dependent antiviral signalling response

Hepatitis C virus (HCV) is the cause of one of the most prevalent viral infections worldwide. Upon infection, the HCV genome activates the RIG‐I‐MAVS signalling pathway leading to the production of direct antiviral effectors which prevent important steps in viral propagation. MAVS localizes at peroxisomes and mitochondria and coordinate the activation of an effective antiviral response: peroxisomal MAVS is responsible for a rapid but short‐termed antiviral response, while the mitochondrial MAVS is associated with the activation of a stable response with delayed kinetics. The HCV NS3‐4A protease was shown to specifically cleave the mitochondrial MAVS, inhibiting the downstream response. In this study, we have analysed whether HCV NS3‐4A is also able to cleave the peroxisomal MAVS and whether this would have any effect on the cellular antiviral response. We show that NS3‐4A is indeed able to specifically cleave this protein and release it into the cytosol, a mechanism that seems to occur at a similar kinetic rate as the cleavage of the mitochondrial MAVS. Under these conditions, RIG‐I‐like receptor (RLR) signalling from peroxisomes is blocked and antiviral gene expression is inhibited. Our results also show that NS3‐4A is able to localize at peroxisomes in the absence of MAVS. However, mutation studies have shown that this localization pattern is preferred in the presence of a fully cleavable MAVS. These findings present evidence of a viral evasion strategy that disrupts RLR signalling on peroxisomes and provide an excellent example of how a single viral evasion strategy can block innate immune signalling from different organelles.     

MAVS Dimer Is a Crucial Signaling Component of Innate Immunity and the Target of Hepatitis C Virus NS3/4A Protease

The mitochondrial antiviral signaling (MAVS) protein plays a central role in innate antiviral immunity. Upon recognition of a virus, intracellular receptors of the RIG-I-like helicase family interact with MAVS to trigger a signaling cascade. In this study, we investigate the requirement of the MAVS structure for enabling its signaling by structure-function analyses and resonance energy transfer approaches in live cells. We now report the essential role of the MAVS oligomer in signal transduction and map the transmembrane domain as the main determinant of dimerization. A combination of mutagenesis and computational methods identified a cluster of residues making favorable van der Waals interactions at the MAVS dimer interface. We also correlated the activation of IRF3 and NF-κB with MAVS oligomerization rather than its mitochondrial localization. Finally, we demonstrated that MAVS oligomerization is disrupted upon expression of HCV NS3/4A protease, suggesting a mechanism for the loss of antiviral signaling. Altogether, our data suggest that the MAVS oligomer is essential in the formation of a multiprotein membrane-associated signaling complex and enables downstream activation of IRF3 and NF-κB in antiviral innate immunity.     

Ribavirin induces error-prone replication of GB virus B in primary tamarin hepatocytes

GB virus B (GBV-B) is the closest relative of hepatitis C virus (HCV) and is an attractive surrogate model for HCV antiviral studies. GBV-B induces an acute, resolving hepatitis in tamarins. Utilizing primary cultures of tamarin hepatocytes, we have previously developed a tissue culture system that exhibits high levels of GBV-B replication. In this report, we have extended the utility of this system for testing antiviral compounds. Treatment with human interferon provided only a marginal antiviral effect, while poly(I-C) yielded >3 and 4 log units of reduction of cell-associated and secreted viral RNA, respectively. Interestingly, treatment of GBV-B-infected hepatocytes with ribavirin resulted in an approximately 4-log decrease in viral RNA levels. Guanosine blocked the antiviral effect of ribavirin, suggesting that inhibition of IMP dehydrogenase (IMPDH) and reduction of intracellular GTP levels were essential for the antiviral effect. However, mycophenolic acid, another IMPDH inhibitor, had no antiviral effect. Virions harvested from ribavirin-treated cultures exhibited a dramatically reduced specific infectivity. These data suggest that incorporation of ribavirin triphosphate induces error-prone replication with concomitant reduction in infectivity and that reduction of GTP pools may be required for incorporation of ribavirin triphosphate. In contrast to the in vitro studies, no significant reduction in viremia was observed in vivo following treatment of tamarins with ribavirin during acute infection with GBV-B. These findings are consistent with the observation that ribavirin monotherapy for HCV infection decreases liver disease without a significant reduction in viremia. Our data suggest that nucleoside analogues that induce error-prone replication could be an attractive approach for the treatment of HCV infection if administered at sufficient levels to result in efficient incorporation by the viral polymerase.