Cytokine-sensitive replication of hepatitis B virus in immortalized mouse hepatocyte cultures

We have previously shown that alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) inhibit hepatitis B virus (HBV) replication by eliminating pregenomic RNA containing viral capsids from the hepatocyte. We have also shown that HBV-specific cytotoxic T lymphocytes that induce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) in the liver can inhibit HBV gene expression by destabilizing preformed viral mRNA. In order to further study the antiviral activity of IFN-alpha/beta, IFN-gamma, and TNF-alpha at the molecular level, we sought to reproduce these observations in an in vitro system. Accordingly, hepatocytes were derived from the livers of HBV-transgenic mice that also expressed the constitutively active cytoplasmic domain of the human hepatocyte growth factor receptor (c-Met). Here, we show that the resultant well-differentiated, continuous hepatocyte cell lines (HBV-Met) replicate HBV and that viral replication in these cells is efficiently controlled by IFN-alpha/beta or IFN-gamma, which eliminate pregenomic RNA-containing capsids from the cells as they do in the liver. Furthermore, we demonstrate that IFN-gamma, but not IFN-alpha/beta, is capable of inhibiting HBV gene expression in this system, especially when it acts synergistically with TNF-alpha. These cells should facilitate the analysis of the intracellular signaling pathways and effector mechanisms responsible for these antiviral effects.     

Inhibition of hepatitis B virus replication by interferon requires proteasome activity

Hepatitis B virus (HBV) replication is inhibited in a noncytopathic manner by alpha/beta interferon (IFN-alpha/beta) and IFN-gamma. We demonstrate here that inhibitors of cellular proteasome activity can block this antiviral effect. These results suggest that a critical component of the IFN-induced antiviral response may be the proteasome-dependent degradation of viral or cellular proteins that are required for HBV replication.     

Inhibition of Hepatitis B Virus Replication during Adenovirus and Cytomegalovirus Infections in Transgenic Mice

We have previously demonstrated that hepatitis B virus (HBV) replication and gene expression are abolished in the livers of HBV transgenic mice by cytotoxic T lymphocytes (CTLs) and during lymphocytic choriomeningitis virus (LCMV) infection, stimuli that trigger the production of alpha/beta interferon, gamma interferon, and tumor necrosis factor alpha in the liver. We now report that hepatic HBV replication and gene expression are inhibited by the local induction of these cytokines during adenovirus- and murine cytomegalovirus (MCMV)-induced hepatitis. Further, we show that MCMV also blocks HBV replication and gene expression in the proximal convoluted tubules of the kidney by causing interstitial nephritis and inducing the same cytokines in the renal parenchyma. These results suggest that inflammatory cytokines probably contribute to viral clearance during acute viral hepatitis in humans, and they imply that induction of these cytokines in the liver and other infected tissues of chronically infected patients might have therapeutic value.     

Inhibition of hepatitis B virus replication by MyD88 is mediated by nuclear factor-kappaB activation

In our previous paper, we reported that myeloid differential primary response protein (MyD88), a key adaptor in the signaling cascade of the innate immune response, inhibits hepatitis B virus (HBV) replication. The MyD88 activated nuclear factor-kappaB (NF-kappaB) signaling pathway and the intracellular upregulation of NF-kappaB signaling can induce an antiviral effect. Therefore, the association between the inhibition of HBV replication by MyD88 and NF-kappaB activation was investigated further. The results show that NF-kappaB activation was moderately increased after MyD88 expression. The strong activation of NF-kappaB by the IkappaB kinase complex IKKalpha/IKKbeta dramatically suppressed HBV replication; the MyD88 dominant negative mutant that abrogated NF-kappaB activity did not inhibit HBV replication. Furthermore, the IkappaBalpha dominant negative mutant restored the inhibition of HBV replication by MyD88. These results support a role for NF-kappaB activation in the inhibition of HBV replication and suggest a novel mechanism for the inhibition of HBV replication by MyD88 protein.     

Interleukin-2 and alpha/beta interferon down-regulate hepatitis B virus gene expression in vivo by tumor necrosis factor-dependent and -independent pathways.

We have recently reported that administration of recombinant tumor necrosis factor alpha (TNF-alpha) to hepatitis B virus (HBV) transgenic mice reduces the hepatic steady-state content of HBV-specific mRNA by up to 80% in the absence of liver cell injury. In the current study, we analyzed the regulatory effects of several other inflammatory cytokines in the same transgenic model system. Hepatic HBV mRNA content was reduced by up to 90% following administration of a single noncytopathic dose (100,000 U) of interleukin 2 (IL-2). Comparable effects were produced by administration of alpha and beta interferons (IFN-alpha and IFN-beta), but only after multiple injections of at least 500,000 U per mouse. Importantly, the regulatory effect of IL-2 was completely blocked by the prior administration of antibodies to tumor necrosis factor alpha (TNF-alpha), which did not block the effect of IFN-alpha or IFN-beta. In contrast to these observations, recombinant IFN-gamma, IL-1, IL-3, IL-6, TNF-beta, transforming growth factor beta, and granulocyte-monocyte colony-stimulating factor were inactive in this system. These results suggest that selected inflammatory cytokines can down-regulate HBV gene expression in vivo by at least two pathways, one that is dependent on TNF-alpha and another that is not. These results imply that antigen-nonspecific products of the intrahepatic HBV-specific inflammatory response may contribute to viral clearance or persistence during HBV infection.     

Alpha/beta interferon differentially modulates the clearance of cytoplasmic encapsidated replication intermediates and nuclear covalently closed circular hepatitis B virus (HBV) DNA from the livers of hepatocyte nuclear factor 1alpha-null HBV transgenic mice

Treatment with alpha interferon is a standard therapy for patients with chronic hepatitis B virus (HBV) infections. This treatment can reduce virus load and ameliorate disease symptoms. However, in the majority of cases, alpha interferon therapy fails to resolve the chronic HBV infection. The reason alpha interferon therapy is inefficient at resolving chronic HBV infections is assumed to be because it fails to eliminate covalently closed circular (CCC) HBV DNA from the nuclei of infected hepatocytes. In an attempt to address this issue, the stability of HBV CCC DNA in response to alpha/beta interferon induction was examined in HNF1alpha-null HBV transgenic mice. Alpha/beta interferon induction by polyinosinic-polycytidylic acid [poly(I-C)] treatment efficiently eliminated encapsidated cytoplasmic HBV replication intermediates while only modestly reducing nuclear HBV CCC DNA. These observations indicate that nuclear HBV CCC DNA is more stable than cytoplasmic replication intermediates in response to alpha/beta interferon induction. Consequently it appears that for therapies to resolve chronic HBV infection efficiently, they will have to target the elimination of the most stable HBV replication intermediate, nuclear HBV CCC DNA.     

Hepatitis B virus regulatory HBx protein binds to adaptor protein IPS-1 and inhibits the activation of beta interferon

Hepatitis B virus (HBV) encodes the regulatory HBx protein, which is required for virus replication, although its specific role(s) in the replication cycle remains under investigation. An immunoprecipitation/mass spectrometry approach was used to identify four novel HBx binding proteins from the cytoplasmic fraction of HBx transgenic mouse livers. One of these HBx binding partners is beta interferon promoter stimulator 1 (IPS-1), an adaptor protein that plays a critical role in mediating retinoic acid-inducible gene I (RIG-I) signaling, which leads to the activation of beta interferon (IFN-β). The HBx-IPS-1 protein interaction was confirmed in plasmid-transfected HepG2 cells by reciprocal coimmunoprecipitation and Western blotting. We hypothesized that HBx might alter IPS-1 function since proteins of hepatitis C virus and hepatitis A virus similarly bind IPS-1 and target it for inactivation. The effect of HBx on IPS-1-mediated IFN-β signaling was tested in transfected 293T and HepG2 cells, and we show that HBx inhibits double-stranded DNA (dsDNA)-mediated IFN-β activation in a dose-dependent manner when expressed either alone or within the context of HBV replication. However, HBx does not inhibit poly(I:C)-activated IFN-β signaling. These results demonstrate that HBx interferes with the RIG-I pathway of innate immunity. Hepatitis B virus now joins hepatitis C virus and hepatitis A virus in targeting the same innate immune response pathway, presumably as a shared strategy to benefit replication of these viruses in the liver.     

Activation of pattern recognition receptor-mediated innate immunity inhibits the replication of hepatitis B virus in human hepatocyte-derived cells

Recognition of virus infections by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I), and melanoma differentiation associated gene 5 (MDA5), activates signaling pathways, leading to the induction of inflammatory cytokines that limit viral replication. To determine the effects of PRR-mediated innate immune response on hepatitis B virus (HBV) replication, a 1.3mer HBV genome was cotransfected into HepG2 or Huh7 cells with plasmid expressing TLR adaptors, myeloid differentiation primary response gene 88 (MyD88), and TIR-domain-containing adaptor-inducing beta interferon (TRIF), or RIG-I/MDA5 adaptor, interferon promoter stimulator 1 (IPS-1). The results showed that expressing each of the three adaptors dramatically reduced the levels of HBV mRNA and DNA in both HepG2 and Huh7 cells. However, HBV replication was not significantly affected by treatment of HBV genome-transfected cells with culture media harvested from cells transfected with each of the three adaptors, indicating that the adaptor-induced antiviral response was predominantly mediated by intracellular factors rather than by secreted cytokines. Analyses of involved signaling pathways revealed that activation of NF-κB is required for all three adaptors to elicit antiviral response in both HepG2 and Huh7 cells. However, activation of interferon regulatory factor 3 is only essential for induction of antiviral response by IPS-1 in Huh7 cells, but not in HepG2 cells. Furthermore, our results suggest that besides NF-κB, additional signaling pathway(s) are required for TRIF to induce a maximum antiviral response against HBV. Knowing the molecular mechanisms by which PRR-mediated innate defense responses control HBV infections could potentially lead to the development of novel therapeutics that evoke the host cellular innate antiviral response to control HBV infections.     

Role of immunoproteasome catalytic subunits in the immune response to hepatitis B virus

Inhibition of hepatitis B virus (HBV) replication and viral clearance from an infected host requires both the innate and adaptive immune responses. Expression of interferon (IFN)-inducible proteasome catalytic and regulatory subunits correlates with the IFN-alpha/beta- and IFN-gamma-mediated noncytopathic inhibition of HBV in transgenic mice and hepatocytes, as well as with clearance of the virus in acutely infected chimpanzees. The immunoproteasome catalytic subunits LMP2 and LMP7 alter proteasome specificity and influence the pool of peptides available for presentation by major histocompatibility complex class I molecules. We found that these subunits influenced both the magnitude and specificity of the CD8 T-cell response to the HBV polymerase and envelope proteins in immunized HLA-A2-transgenic mice. We also examined the role of LMP2 and LMP7 in the IFN-alpha/beta- and IFN-gamma-mediated inhibition of virus replication using HBV transgenic mice and found that they do not play a direct role in this process. These results demonstrate the ability of the IFN-induced proteasome catalytic subunits to shape the HBV-specific CD8 T-cell response and thus potentially influence the progression of infection to acute or chronic disease. In addition, these studies identify a potential key role for IFN in regulating the adaptive immune response to HBV through alterations in viral antigen processing.     

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.     

Role for bovine viral diarrhea virus Erns glycoprotein in the control of activation of beta interferon by double-stranded RNA

Production of alpha/beta interferon in response to viral double-stranded RNA (dsRNA) produced during viral replication is a first line of defense against viral infections. Here we demonstrate that the Erns glycoprotein of the pestivirus bovine viral diarrhea virus can act as an inhibitor of dsRNA-induced responses of cells. This effect is seen whether Erns is constitutively expressed in cells or exogenously added to the culture medium. The Erns effect is specific to dsRNA since activation of NF-kappaB in cells infected with Semliki Forest virus or treated with tumor necrosis factor alpha was not affected. We also show that Erns contains a dsRNA-binding activity, and its RNase is active against dsRNA at a low pH. Both the dsRNA binding and RNase activities are required for the inhibition of dsRNA signaling, and we discuss here a model to account for these observations.     

Cytopathic and noncytopathic interferon responses in cells expressing hepatitis C virus subgenomic replicons

Hepatitis C virus (HCV) is the only known positive-stranded RNA virus that causes persistent lifelong infections in humans. Accumulation of HCV RNA can be inhibited with alpha interferon (IFN-alpha) in vivo and in culture cells. We used cell-based assay systems to investigate the mechanisms responsible for the cytokine-induced inhibition of HCV replication. The results showed that IFN-alpha could suppress the accumulation of viral RNA by a noncytopathic pathway and could also induce apoptosis of virally infected cells in a concentration- and cell line-dependent fashion. Whereas the noncytopathic IFN-alpha response depended on a functional Jak-STAT signal transduction pathway, it did not appear to require double-stranded RNA-dependent pathways. Moreover, we found that functional proteasomes were required for establishment of the IFN-alpha response against HCV. Based on the results described in this study we propose a model for the mechanism by which IFN-alpha therapy suppresses HCV replication in chronic infections by both cytopathic and noncytopathic means.