Interferons Accelerate Decay of Replication-Competent Nucleocapsids of Hepatitis B Virus

Alpha interferon (IFN-α) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-γ) is a key mediator of host antiviral immunity against hepatitis B virus (HBV) infection in vivo. However, the molecular mechanism by which these antiviral cytokines suppress HBV replication remains elusive. Using an immortalized murine hepatocyte (AML12)-derived cell line supporting tetracycline-inducible HBV replication, we show in this report that both IFN-α and IFN-γ efficiently reduce the amount of intracellular HBV nucleocapsids. Furthermore, we provide evidence suggesting that the IFN-induced cellular antiviral response is able to distinguish and selectively accelerate the decay of HBV replication-competent nucleocapsids but not empty capsids in a proteasome-dependent manner. Our findings thus reveal a novel antiviral mechanism of IFNs and provide a basis for a better understanding of HBV pathobiology.Hepatitis B virus (HBV) is a noncytopathic hepatotropic DNA virus which belongs to the family Hepadnaviridae (11, 44). Despite the fact that most adulthood HBV infections are transient, approximately 5 to 10% of infected adults and more than 90% of infected neonates fail to clear the virus and develop a lifelong persistent infection, which may progress to chronic hepatitis, cirrhosis, and primary hepatocellular carcinoma (4, 33, 34). It has been shown by several research groups that resolution of HBV and other animal hepadnavirus infection in vivo depends on both killing of infected hepatocytes by viral antigen-specific cytotoxic T lymphocytes and noncytolytic suppression of viral replication, which is most likely mediated by inflammatory cytokines, such as gamma interferon (IFN-γ) and tumor necrosis factor α (TNF-α) (10, 12, 15, 20, 26, 27, 48). Moreover, together with five nucleoside or nucleotide analogs that inhibit HBV DNA polymerase, alpha IFN (IFN-α) and pegylated IFN-α are currently available antiviral medications for the management of chronic hepatitis B. Compared to the viral DNA polymerase inhibitors, the advantages of IFN-α therapy include a lack of drug resistance, a finite and defined treatment course, and an increased likelihood for hepatitis B virus surface antigen (HBsAg) clearance (8, 39). However, only approximately 30% of treated patients achieve a sustained virological response to a standard 48-month pegylated IFN-α therapy (6, 32). Thus far, the antiviral mechanism of IFN-α and IFN-γ and the parameters determining the success or failure of IFN-α therapy in chronic hepatitis B remain elusive. Elucidation of the mechanism by which the cytokines suppress HBV replication represents an important step toward understanding the pathobiology of HBV infection and the molecular basis of IFN-α therapy of chronic hepatitis B.Considering the mechanism by which IFNs noncytolytically control HBV infection in vivo, it is possible that the cytokines either induce an antiviral response in hepatocytes to directly limit HBV replication or modulate the host antiviral immune response to indirectly inhibit the virus infection. However, due to the fact that IFN-α and -γ do not inhibit or only modestly inhibit HBV replication in human hepatoma-derived cell lines (5, 22, 23, 30), the direct antiviral effects of the cytokines and their antiviral mechanism against HBV have been studied with either an immortalized hepatocyte cell line derived from HBV transgenic mice or duck hepatitis B virus (DHBV) infection of primary duck hepatocytes (37, 53). While these studies revealed that IFN treatment significantly reduced the amount of encapsidated viral pregenomic RNA (pgRNA) in both mouse and duck hepatocytes, further mechanistic analyses suggested that IFN-α inhibited the formation of pgRNA-containing nucleocapsids in murine hepatocytes (52) but shortened the half-life of encapsidated pgRNA in DHBV-replicating chicken hepatoma cells (21). Moreover, the fate of viral DNA replication intermediates or nucleocapsids in the IFN-treated hepatocytes was not investigated in the previous studies.To further define the target(s) of IFN-α and -γ in the HBV life cycle and to create a robust cell culture system for the identification of IFN-stimulated genes (ISGs) that mediate the antiviral response of the cytokines (25), we established an immortalized murine hepatocyte (AML-12)-derived stable cell line that supported a high level of HBV replication in a tetracycline-inducible manner. Consistent with previous reports, we show that both IFN-α and IFN-γ potently inhibited HBV replication in murine hepatocytes (37, 40). With the help of small molecules that inhibit HBV capsid assembly (Bay-4109) (7, 47) and prevent the incorporation of pgRNA into nucleocapsids (AT-61) (9, 29), we obtained evidence suggesting that the IFN-induced cellular antiviral response is able to distinguish and selectively accelerate the decay of HBV replication-competent nucleocapsids but not empty capsids in a proteasome-dependent manner. Our findings provide a basis for further studies toward better understanding of IFN′s antiviral mechanism, which might ultimately lead to the development of strategies to improve the efficacy of IFN therapy of chronic hepatitis B.     

Lambda Interferon Renders Epithelial Cells of the Respiratory and Gastrointestinal Tracts Resistant to Viral Infections

Virus-infected cells secrete a broad range of interferons (IFN) which confer resistance to yet uninfected cells by triggering the synthesis of antiviral factors. The relative contributions of the various IFN subtypes to innate immunity against virus infections remain elusive. IFN-α, IFN-β, and other type I IFN molecules signal through a common, universally expressed cell surface receptor, whereas type III IFN (IFN-λ) uses a distinct cell-type-specific receptor complex for signaling. Using mice lacking functional receptors for type I IFN, type III IFN, or both, we found that IFN-λ plays an important role in the defense against several human pathogens that infect the respiratory tract, such as influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, and severe acute respiratory syndrome (SARS) coronavirus. These viruses were more pathogenic and replicated to higher titers in the lungs of mice lacking both IFN receptors than in mice with single IFN receptor defects. In contrast, Lassa fever virus, which infects via the respiratory tract but primarily replicates in the liver, was not influenced by the IFN-λ receptor defect. Careful analysis revealed that expression of functional IFN-λ receptor complexes in the lung and intestinal tract is restricted to epithelial cells and a few other, undefined cell types. Interestingly, we found that SARS coronavirus was present in feces from infected mice lacking receptors for both type I and type III IFN but not in those from mice lacking single receptors, supporting the view that IFN-λ contributes to the control of viral infections in epithelial cells of both respiratory and gastrointestinal tracts.The interferon (IFN) system represents a major element of the innate immune response against viral infections (10, 13, 14). Virus-induced IFN is a complex mixture of biologically active molecules, which includes type I and type III IFN. Type I IFN consists of 14 different IFN-α subtypes in the mouse as well as IFN-β, IFN-κ, IFN-ɛ, and limitin, which all signal through the same universally expressed cell surface receptor complex (IFNAR) (30). Type III IFN includes IFN-λ1, IFN-λ2, and IFN-λ3 (21, 28), of which only the latter two are encoded by genes that are expressed in the mouse (22). Type III IFN uses a distinct receptor complex (IL28R) for signaling (21, 28), which appears to be expressed on only a few cell types, including epithelial cells (29). Binding of type I IFN and type III IFN to their cognate receptor complexes triggers signaling cascades that result in the activation of a large number of genes, many of which encode antiviral proteins (10, 32). Type I IFN and type III IFN trigger highly similar gene expression profiles in responsive cells, suggesting that both IFN types might serve similar functions. However, it has to date been largely unclear to which extent IFN-λ might contribute to innate immunity.Using knockout mouse strains that lack receptors for type I IFN (IFNAR1^0/0), type III IFN (IL28Rα^0/0), or both (IFNAR1^0/0IL28Rα^0/0), we have recently shown that IFN-λ contributes to resistance against influenza A virus (FLUAV) (26). Here, we used the same mouse strains to investigate the relative contribution of IFN-λ in resistance against additional viral pathogens that infect the respiratory and gastrointestinal tract and to visualize IFN-λ-responsive cells. We found that the double-knockout mice showed enhanced susceptibility to various viruses that primarily replicate in lung epithelial cells. Our analysis further revealed that epithelial cells of both lung and gastrointestinal tracts can strongly respond to IFN-λ and that IFN-λ inhibited the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in both lung and gastrointestinal tracts.     

The Interferon Stimulator Mitochondrial Antiviral Signaling Protein Facilitates Cell Death by Disrupting the Mitochondrial Membrane Potential and by Activating Caspases

Interferon (IFN) signaling is initiated by the recognition of viral components by host pattern recognition receptors. Dengue virus (DEN) triggers IFN-β induction through a molecular mechanism involving the cellular RIG-I/MAVS signaling pathway. Here we report that the MAVS protein level is reduced in DEN-infected cells and that caspase-1 and caspase-3 cleave MAVS at residue D429. In addition to its well-known function in IFN induction, MAVS is also a proapoptotic molecule that triggers disruption of the mitochondrial membrane potential and activation of caspases. Although different domains are required for the induction of cytotoxicity and IFN, caspase cleavage at residue 429 abolished both functions of MAVS. The apoptotic role of MAVS in viral infection and double-stranded RNA (dsRNA) stimulation was demonstrated in cells with reduced endogenous MAVS expression induced by RNA interference. Even though IFN-β promoter activation was largely suppressed, DEN production was not affected greatly in MAVS knockdown cells. Instead, DEN- and dsRNA-induced cell death and caspase activation were delayed and attenuated in the cells with reduced levels of MAVS. These results reveal a new role of MAVS in the regulation of cell death beyond its well-known function of IFN induction in antiviral innate immunity.In the battle of hosts and microbes, the innate immune system uses pathogen recognition receptors (PRRs) to sense pathogen-associated molecular patterns (23). There are several functionally distinct classes of PRRs, such as the transmembrane (TM) Toll-like receptors (TLRs) and the intracellular retinoic acid-inducible gene I (RIG-I)-like helicase (RLH) receptors (15, 23, 25, 38). RLHs, including RIG-I and melanoma differentiation-associated gene 5 (MDA5), comprise an N-terminal caspase recruitment domain (CARD), a middle DEXD/H box RNA helicase domain, and a C-terminal domain. RLHs sense intracellular viral RNA and initiate an antiviral interferon (IFN) response (1, 43). RIG-I binding to viral RNA triggers conformational changes that expose the CARD for subsequent signaling (42). The adaptor molecule providing a link between RIG-I and downstream events was identified independently by four research groups as a mitochondrial CARD-containing protein, which was named mitochondrial antiviral signaling protein (MAVS) (34), IFN-β promoter stimulator 1 (IPS-1) (12), virus-induced signaling adaptor (VISA) (40), and CARD adaptor-inducing IFN-β (Cardif) (24). We refer to this adaptor as MAVS in this paper. MAVS transduces signals from RIG-I through CARD-CARD interactions, which then lead to interferon regulatory factor 3 (IRF-3) and NF-κB activation of IFN-β induction through a signaling cascade involving IKKα/β/γ, IKKɛ, and TBK1 (15). Recently, a protein termed STING (11) or MITA (47) was identified as a mediator that acts downstream of RIG-I and MAVS and upstream of TBK1.MAVS protein contains an N-terminal CARD required for signaling, a proline-rich domain that interacts with TRAF3, and a C-terminal TM region that targets MAVS to the mitochondrial outer membrane (29). Several cellular and viral proteins target MAVS in the attenuation of the IFN induction pathway. Cleavage of MAVS by hepatitis C virus (HCV) and hepatitis A virus (HAV) proteases, at residues C508 (18, 24) and Q428 (41), respectively, results in the loss of MAVS mitochondrial localization, thereby disrupting its function in IFN induction. Another mitochondrial outer membrane protein, NLRX1, can sequester MAVS from its association with RIG-I and act as a negative regulator of the IFN pathway (28). MAVS was recently found to be cleaved and inactivated by caspases during apoptosis (31, 33).The caspases are a well-known family of cysteinyl aspartate-specific proteases. The diverse roles of caspases in the cell cycle, proliferation, differentiation, cytokine production, innate immune regulation, and microbial infection suggest various functions of caspases beyond apoptosis (13, 14). The caspases can be separated into two subfamilies, namely, the cell death and inflammation subfamilies. In response to apoptotic stimuli, the initiators caspase-2, -8, -9, and -10 and effectors caspase-3, -6, and -7 mediate cell death events. Caspase-1, -4, -5, and -12 are known as the inflammatory caspases. Caspase-1 is involved in the cleavage and maturation of cytokines (8, 17). Caspase-8 and -10 were discovered as essential components that mediate antiviral signaling (37). Caspase-1 and -3 are activated in innate immune signaling (32). These findings indicate that caspases are involved in the regulation of innate immunity, in addition to their well-known apoptotic role. However, the details of how caspases are activated, the role of caspase activation, and how caspases manipulate the signaling pathways in innate immunity are still obscure.The family Flaviviridae contains three genera: Hepacivirus, Flavivirus, and Pestivirus. Infections with flaviviruses, such as dengue virus (DEN), Japanese encephalitis virus, and West Nile virus, are emerging worldwide. DEN triggers IFN-β through a molecular mechanism involving the RIG-I/MAVS signaling pathway (5, 20). In this study, we found that MAVS is cleaved during DEN serotype 2 (DEN-2) infection, in a caspase-dependent manner; this contrasts with viral protease-dependent cleavage of MAVS during infection with HCV and HAV. In a cell-free caspase assay system, MAVS was cleaved at residue D429 by caspase-1 and caspase-3. Cleaved MAVS failed to induce IFN production and caspase activation, and overexpression of MAVS also triggered caspase activation, which then negatively regulated its own function. Importantly, the role of MAVS in viral infection was verified by knockdown of MAVS expression. We discuss the possible regulatory mechanisms of MAVS and the biological significance of this cleavage event by caspases in the context of understanding how these apoptosis-related proteases might achieve cross talk with the innate immune pathway during viral infection.