Induction and Evasion of Innate Antiviral Responses by Hepatitis C Virus

Persistent hepatitis C virus infection is associated with progressive hepatic fibrosis and liver cancer. Acute infection evokes several distinct innate immune responses, but these are partially or completely countered by the virus. Hepatitis C virus proteins serve dual functions in replication and immune evasion, acting to disrupt cellular signaling pathways leading to interferon synthesis, subvert Jak-STAT signaling to limit expression of interferon-stimulated genes, and block antiviral activities of interferon-stimulated genes. The net effect is a multilayered evasion of innate immunity, which negatively influences the subsequent development of antigen-specific adaptive immunity, thereby contributing to virus persistence and resistance to therapy

The Chimpanzee Model of Viral Hepatitis: Advances in Understanding the Immune Response and Treatment of Viral Hepatitis

Chimpanzees (Pan troglodytes) have contributed to diverse fields of biomedical research due to their close genetic relationship to humans and in many instances due to the lack of any other animal model. This review focuses on the contributions of the chimpanzee model to research on hepatitis viruses where chimpanzees represented the only animal model (hepatitis B and C) or the most appropriate animal model (hepatitis A). Research with chimpanzees led to the development of vaccines for HAV and HBV that are used worldwide to protect hundreds of millions from these diseases and, where fully implemented, have provided immunity for entire generations. More recently, chimpanzee research was instrumental in the development of curative therapies for hepatitis C virus infections. Over a span of 40 years, this research would identify the causative agent of NonA,NonB hepatitis, validate the molecular tools for drug discovery, and provide safety and efficacy data on the therapies that now provide a rapid and complete cure of HCV chronic infections. Several cocktails of antivirals are FDA approved that eliminate the virus following 12 weeks of once-per-day oral therapy. This represents the first cure of a chronic viral disease and, once broadly implemented, will dramatically reduce the occurrence of cirrhosis and liver cancer. The recent contributions of chimpanzees to our current understanding of T cell immunity for HCV, development of novel therapeutics for HBV, and the biology of HAV are reviewed. Finally, a perspective is provided on the events leading to the cessation of the use of chimpanzees in research and the future of the chimpanzees previously used to bring about these amazing breakthroughs in human healthcare

trans-Complementation of an NS2 Defect in a Late Step in Hepatitis C Virus (HCV) Particle Assembly and Maturation

Recent studies using cell culture infection systems that recapitulate the entire life cycle of hepatitis C virus (HCV) indicate that several nonstructural viral proteins, including NS2, NS3, and NS5A, are involved in the process of viral assembly and release. Other recent work suggests that Ser-168 of NS2 is a target of CK2 kinase–mediated phosphorylation, and that this controls the stability of the genotype 1a NS2 protein. Here, we show that Ser-168 is a critical determinant in the production of infectious virus particles. Substitution of Ser-168 with Ala (or Gly) ablated production of infectious virus by cells transfected with a chimeric viral RNA (HJ3-5) containing core-NS2 sequences from the genotype 1a H77 virus within the background of genotype 2a JFH1 virus. An S168A substitution also impaired production of virus by cells transfected with JFH1 RNA. This mutation did not alter polyprotein processing or genome replication. This defect in virus production could be rescued by expression of wt NS2 in trans from an alphavirus replicon. The trans-complementing activities of NS2 from genotypes 1a and 2a demonstrated strong preferences for rescue of the homologous genotype. Importantly, the S168A mutation did not alter the association of core or NS5A proteins with host cell lipid droplets, nor prevent the assembly of core into particles with sedimentation and buoyant density properties similar to infectious virus, indicating that NS2 acts subsequent to the involvement of core, NS5A, and NS3 in particle assembly. Second-site mutations in NS2 as well as in NS5A can rescue the defect in virus production imposed by the S168G mutation. In aggregate, these results indicate that NS2 functions in trans, in a late-post assembly maturation step, perhaps in concert with NS5A, to confer infectivity to the HCV particle

Class A scavenger receptor 1 (MSR1) restricts hepatitis C virus replication by mediating toll-like receptor 3 recognition of viral RNAs produced in neighboring cells

Persistent infections with hepatitis C virus (HCV) may result in life-threatening liver disease, including cirrhosis and cancer, and impose an important burden on human health. Understanding how the virus is capable of achieving persistence in the majority of those infected is thus an important goal. Although HCV has evolved multiple mechanisms to disrupt and block cellular signaling pathways involved in the induction of interferon (IFN) responses, IFN-stimulated gene (ISG) expression is typically prominent in the HCV-infected liver. Here, we show that Toll-like receptor 3 (TLR3) expressed within uninfected hepatocytes is capable of sensing infection in adjacent cells, initiating a local antiviral response that partially restricts HCV replication. We demonstrate that this is dependent upon the expression of class A scavenger receptor type 1 (MSR1). MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. RNAi-mediated knockdown of MSR1 expression blocks TLR3 sensing of HCV in infected hepatocyte cultures, leading to increased cellular permissiveness to virus infection. Exogenous expression of Myc-MSR1 restores TLR3 signaling in MSR1-depleted cells with subsequent induction of an antiviral state. A series of conserved basic residues within the carboxy-terminus of the collagen superfamily domain of MSR1 are required for binding and transport of dsRNA, and likely facilitate acidification-dependent release of dsRNA at the site of TLR3 expression in the endosome. Our findings reveal MSR1 to be a critical component of a TLR3-mediated pattern recognition receptor response that exerts an antiviral state in both infected and uninfected hepatocytes, thereby limiting the impact of HCV proteins that disrupt IFN signaling in infected cells and restricting the spread of HCV within the liver

Mechanisms of HCV-induced liver cancer: What did we learn from in vitro and animal studies?

Hepatitis C virus (HCV) is a cause of liver diseases that range from steatohepatitis, to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The challenge of understanding the pathogenesis of HCV-associated liver cancer is difficult as most standard animal models used in biomedical research are not permissive to HCV infection. Herein, we provide an overview of a number of creative in vivo, mostly in the mouse, and in vitro models that have been developed to advance our understanding of the molecular and cellular effects of HCV on the liver, specifically with their relevance to HCC

Innate immune responses in hepatitis C virus infection

Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and hepatocellular carcinoma worldwide and thus poses a significant public health threat. A hallmark of HCV infection is the extraordinary ability of the virus to persist in a majority of infected people. Innate immune responses represent the front line of defense of the human body against HCV immediately after infection. They also play a crucial role in orchestrating subsequent HCV-specific adaptive immunity that is pivotal for viral clearance. Accumulating evidence suggests that the host has evolved multifaceted innate immune mechanisms to sense HCV infection and elicit defense responses, while HCV has developed elaborate strategies to circumvent many of these. Defining the interplay of HCV with host innate immunity reveals mechanistic insights into hepatitis C pathogenesis and informs approaches to therapy. In this review, we summarize recent advances in understanding innate immune responses to HCV infection, focusing on induction and effector mechanisms of the interferon antiviral response as well as the evasion strategies of HCV

Antigenic structure of human hepatitis A virus defined by analysis of escape mutants selected against murine monoclonal antibodies.

We examined the antigenic structure of human hepatitis A virus (HAV) by characterizing a series of 21 murine monoclonal-antibody-resistant neutralization escape mutants derived from the HM175 virus strain. The escape phenotype of each mutant was associated with reduced antibody binding in radioimmunofocus assays. Neutralization escape mutations were identified at the Asp-70 and Gln-74 residues of the capsid protein VP3, as well as at Ser-102, Val-171, Ala-176, and Lys-221 of VP1. With the exception of the Lys-221 mutants, substantial cross-resistance was evident among escape mutants tested against a panel of 22 neutralizing monoclonal antibodies, suggesting that the involved residues contribute to epitopes composing a single antigenic site. As mutations at one or more of these residues conferred resistance to 20 of 22 murine antibodies, this site appears to be immunodominant in the mouse. However, multiple mutants selected independently against any one monoclonal antibody had mutations at only one or, at the most, two amino acid residues within the capsid proteins, confirming that there are multiple epitopes within this antigenic site and suggesting that single-amino-acid residues contributing to these epitopes may play key roles in the binding of individual antibodies. A second, potentially independent antigenic site was identified by three escape mutants with different substitutions at Lys-221 of VP1. These mutants were resistant only to antibody H7C27, while H7C27 effectively neutralized all other escape mutants. These data support the existence of an immunodominant neutralization site in the antigenic structure of hepatitis A virus which involves residues of VP3 and VP1 and a second, potentially independent site involving residue 221 of VP1

Dissecting the Roles of the 5′ Exoribonucleases Xrn1 and Xrn2 in Restricting Hepatitis C Virus Replication

ABSTRACT The replication of hepatitis C virus (HCV) is uniquely dependent on a host microRNA, miR-122. Previous studies using genotype 1a H77S.3 virus demonstrated that miR-122 acts in part by protecting the RNA genome from 5′ decay mediated by the cytoplasmic 5′ exoribonuclease, Xrn1. However, this finding has been challenged by a recent report suggesting that a predominantly nuclear exoribonuclease, Xrn2, mediates the degradation of genotype 2a JFH1 RNA. Here, we dissect the roles of these two 5′ exoribonucleases in restricting the replication of different HCV strains and mediating the decay of HCV RNA. Small interfering RNA (siRNA) depletion experiments indicated that Xrn1 restricts replication of all HCV strains tested: JFH1, H77S.3, H77D (a robustly replicating genotype 1a variant), and HJ3-5 (a genotype 1a/2a chimeric virus). In contrast, the antiviral effects of Xrn2 were limited to JFH1 and H77D viruses. Moreover, such effects were not apparent in cells infected with a JFH1 luciferase reporter virus. Whereas Xrn1 depletion significantly slowed decay of JFH1 and HJ3-5 RNAs, Xrn2 depletion marginally enhanced the JFH1 RNA half-life and had no effect on HJ3-5 RNA decay. The positive effects of Xrn1 depletion on JFH1 replication were largely redundant and nonadditive with those of exogenous miR-122 supplementation, whereas Xrn2 depletion acted additively and thus independently of miR-122. We conclude that Xrn1 is the dominant 5′ exoribonuclease mediating decay of HCV RNA and that miR-122 provides protection against it. The restriction of JFH1 and H77D replication by Xrn2 is likely indirect in nature and possibly linked to cytopathic effects of these robustly replicating viruses. IMPORTANCE HCV is a common cause of liver disease both within and outside the United States. Its replication is dependent upon a small, liver-specific noncoding RNA, miR-122. Although this requirement has been exploited for the development of an anti-miR-122 antagomir as a host-targeting antiviral, the molecular mechanisms underpinning the host factor activity of miR-122 remain incompletely defined. Conflicting reports suggest miR-122 protects the viral RNA against decay mediated by distinct cellular 5′ exoribonucleases, Xrn1 and Xrn2. Here, we compare the roles of these two exoribonucleases in HCV-infected cells and confirm that Xrn1, not Xrn2, is primarily responsible for decay of RNA in cells infected with multiple virus strains. Our results clarify previously published research and add to the current understanding of the host factor requirement for miR-122

miR-122 and the Hepatitis C RNA genome: More than just stability

MicroRNA-122 (miR-122) plays a key role in hepatitis C virus (HCV) replication, but understanding exactly how it functions in the viral lifecycle has been elusive. HCV is a positive-strand virus with a messenger-sense RNA genome, to which miR-122 binds in a non-canonical fashion at two sites near the 5′ end. Recent studies show that miR-122 recruits Ago-2 to the genomic RNA, stabilizing it and slowing its decay in infected cells. This led us to investigate decay pathways that mediate degradation of the viral RNA. We found HCV RNA is degraded primarily by the cytoplasmic 5′ exonuclease Xrn1 in infected cells. miR-122 lost its stabilizing effect when cells were depleted of Xrn1 using an RNAi strategy, providing strong evidence that miR-122 acts to protect the viral RNA from Xrn1-mediated 5′ exonucleolytic decay. However, Xrn1 depletion did not rescue replication of a viral mutant defective in miR-122 binding, indicating that there is much more to miR-122’s actions than prevention of Xrn1 decay. Here, we consider the role of miR-122 in the viral lifecycle, and explore the possibility that it might function directly in viral RNA synthesis

Reassessing immune control of hepatitis A virus

There is renewed interest in hepatitis A virus (HAV) pathogenesis and immunity after 2–3 decades of limited progress. From a public health perspective, the average age at infection has increased in developing countries, resulting in more severe hepatitis that is poorly understood mechanistically. More fundamentally, there is interest in comparing immunity to HAV and hepatitis C virus (HCV): small, positive-strand RNA viruses with very different infection outcomes. Here, we review evidence that circulating HAV virions are cloaked in membranes, with consequences for induction of innate immunity and antibody-mediated neutralization. We also consider the contribution of CD4+ helper versus CD8+ cytotoxic T cells to antiviral immunity and liver injury, and present a model of non-cytotoxic immune control of HAV infection