Interference of Hepatitis B Virus with Cellular Signaling

The presence of hepatitis B virus (HBV) proteins leads to changes in the cellular gene expression. As a consequence, the cellular signaling processes are influenced by the actions of HBV proteins. It has been shown that HBV nucleocapsid protein and the amino-terminal part of polymerase termed as terminal protein (TP) could inhibit interferon signaling. Further, the global gene expression profiles differ in hepatoma cells with and without HBV gene expression and replication. The expression of interferon (IFN) stimulated genes (ISGs) was differently regulated in cells with HBV replication and could be modulated by antiviral treatments. The HBV TP has been found to modulate the ISG expression and enhance the HBV replication. The modulation of the cellular signaling processes by HBV may have significant implications for pathogenesis.     

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.     

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.     

The Antiviral Activities of ISG15

Post-translational protein modification is an important strategy for the regulation of the cell proteome independent of the need for new gene expression. Ubiquitin and ubiquitin-like modifiers mediate the regulation of protein levels, signaling pathways, vesicular trafficking, and many other cellular processes through their covalent conjugation to proteins. Interferon stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I interferon. In addition to conjugating to potentially hundreds of target proteins, ISG15 can be found in an unconjugated form both inside of the cell and released from interferon stimulated cells into the extracellular environment. Due to its robust expression after type I interferon stimulation and the broad panel of proteins that it targets, ISG15 has drawn much attention as a potential regulator of the immune response and has been shown to mediate protection in a number of different viral infection models. Here we will review the current state of the field of ISG15, the viruses against which ISG15 mediates protection, and the mechanisms by which ISG15 exerts antiviral activity.     

Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host RNA and protein synthesis

The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced inhibition of host gene expression. It has been proposed that the inhibition of host gene expression by M protein is responsible for suppressing activation of host interferon gene expression. Most wild-type (wt) strains of VSV induce little if any interferon gene expression. Interferon-inducing mutants of VSV have been isolated previously, many of which contain mutations in their M proteins. However, it was not known whether these M protein mutations were responsible for the interferon-inducing phenotype of these viruses. Alternatively, mutations in other genes besides the M gene may enhance the ability of VSV to induce interferons. These hypotheses were tested by transfecting cells with mRNA expressing wt and mutant M proteins in the absence of other viral components and determining their ability to inhibit interferon gene expression. The M protein mutations were the M51R mutation originally found in the tsO82 and T1026R1 mutant viruses, the double substitution V221F and S226R found in the TP3 mutant virus, and the triple substitution E213A, V221F, and S226R found in the TP2 mutant virus. wt M proteins suppressed expression of luciferase from the simian virus 40 promoter and from the beta interferon (IFN-beta) promoter, while M proteins of interferon-inducing viruses were unable to inhibit luciferase expression from either promoter. The M genes of the interferon-inducing mutants of VSV were incorporated into the wt background of a recombinant VSV infectious cDNA clone. The resulting recombinant viruses were tested for their ability to activate interferon gene expression and for their ability to inhibit host RNA and protein synthesis. Each of the recombinant viruses containing M protein mutations induced expression of a luciferase reporter gene driven by the IFN-beta promoter and induced production of interferon bioactivity more effectively than viruses containing wt M proteins. Furthermore, the M protein mutant viruses were defective in their ability to inhibit both host RNA synthesis and host protein synthesis. These data support the idea that wt M protein suppresses interferon gene expression through the general inhibition of host RNA and protein synthesis.     

Indoleamine 2,3-dioxygenase mediates the antiviral effect of gamma interferon against hepatitis B virus in human hepatocyte-derived cells

Alpha interferon (IFN-α) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-γ) is a key mediator of host innate and adaptive antiviral immunity against hepatitis B virus (HBV) infection in vivo. In an effort to elucidate the antiviral mechanism of these cytokines, 37 IFN-stimulated genes (ISGs), which are highly inducible in hepatocytes, were tested for their ability to inhibit HBV replication upon overexpression in human hepatoma cells. One ISG candidate, indoleamine 2,3-dioxygenase (IDO), an IFN-γ-induced enzyme catalyzing tryptophan degradation, efficiently reduced the level of intracellular HBV DNA without altering the steady-state level of viral RNA. Furthermore, expression of an enzymatically inactive IDO mutant did not inhibit HBV replication, and tryptophan supplementation in culture completely restored HBV replication in IDO-expressing cells, indicating that the antiviral effect elicited by IDO is mediated by tryptophan deprivation. Interestingly, IDO-mediated tryptophan deprivation preferentially inhibited viral protein translation and genome replication but did not significantly alter global cellular protein synthesis. Finally, tryptophan supplementation was able to completely restore HBV replication in IFN-γ- but not IFN-α-treated cells, which strongly argues that IDO is the primary mediator of IFN-γ-elicited antiviral response against HBV in human hepatocyte-derived cells.     

Mechanism of inhibiting type I interferon induction by hepatitis B virus X protein

Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.     

Rac1 GTPase is activated by hepatitis B virus replication--involvement of HBX

Hepatitis B virus (HBV) is a causative agent for liver diseases including hepatocellular carcinoma. Understanding its interactions with cellular proteins is critical in the elucidation of the mechanisms of disease progression. Using a cell-based HBV replication system, we showed that HBV replication in HepG2 cells resulted in a cellular morphological changes displaying membrane rufflings and lamellipodia like structures reminiscent of cells expressing constitutively activated Rac1. We also showed that activated Rac1 resulted in increased viral replication. HBV replication specifically activated wild type Rac1, but not Cdc42. The Rac1 activation by HBV replication also resulted in the phosphorylation of ERK1/2 and AKT, the downstream targets of Rac1 signaling cascade. The smallest HBV viral protein, HBX, was able to activate the endogenous Rac1 and induce membrane ruffling when transfected into cells. Significantly, HBX was found to directly interact with a Rac1 nucleotide exchange factor (betaPIX) through a SH3 binding motif. Taken together, we have shown the interaction of HBV with the Rho GTPase, affecting cell morphology through the Rac1 activation pathway. HBV may possibly make use of an activated Rac1 signaling pathway for increased replication and resultant metastatic effects.     

真菌次级代谢产物11’-deoxyverticillin A（C42）通过降低自噬基因的表达抑制肝炎病毒（HBV）复制

C42属于epipolythiodioxopiperazines（ETPs）二酮呱嗪类化合物,具有多种生物学活性,包括抑制病毒复制;不过其对hepatitis B virus（HBV）复制的影响及机理鲜有报道。自噬是广泛存在于真核细胞、通过溶酶体降解长半衰期蛋白的现象,参与多种生理、病理过程。有研究发现自噬对HBV的复制至关重要。C42是否通过改变自噬来影响此病毒的复制目前还未见报道。在该研究中,我们发现表达HBV基因组的HepG2.215细胞较原始的HepG2细胞,自噬体明显增加并伴随着Akt磷酸化的增高。C42可以降低自噬基因LC3-II和p62的水平,同时会影响Akt信号通路。氯喹是一种自噬抑制剂,它的存在可以抑制C42导致的LC3-II降低,表明C42可以引起该细胞的自噬。敲降自噬基因和抑制Akt磷酸化均可以减少HBV-X蛋白表达。而利用氯喹抑制自噬体与溶酶体的融合却提高了HBV-X蛋白水平。由于HBV-X对该病毒的复制至关重要,因此,我们认为,C42通过自噬和Akt信号通路来抑制HBV的复制。     

Adhesion contact kinetics of HepG2 cells during Hepatitis B virus replication: Involvement of SH3-binding motif in HBX

It has been shown that Hepatitis B virus (HBV) replication directly alters the expression of key cytoskeleton-associated proteins which play key roles in mechanochemical signal transduction. Nevertheless, little is known on the correlation between HBV replication and the subsequent adhesion mechanism of HBV-replicating cells. In this study, it is demonstrated that the lag time of adhesion contact evolution of HepG2 cells with HBV replication is significantly increased by two times compared to that of normal HepG2 cell on collagen coated substrate. During the initial 20 min of cell seeding, only diffuse forms of vinculin was detected in HBV replicating cells while vinculin-associated focal complexes were found in normal and control cells. Similar delay in cell adhesion in HBV-replicating cells was observed in cells transfected with HBX, the smallest HBV protein, suggesting its involvement in this cellular process. In addition, a proline rich region found in many SH3 binding proteins was identified in HBX. HBX was found to interact with the focal adhesion protein, vinexin-beta, through the SH3 binding. Furthermore, HepG2 cells with HBV replication showed evidence of cell rounding up, possibly resulting from cytoskeletal reorganizations associated with interaction between HBX and vinexin-beta. Taken together, our results suggest that HBX is involved in the cytoskeletal reorganization in response to HBV replication.     

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.     

2’,3’-Cyclic Nucleotide 3’-Phosphodiesterases Inhibit Hepatitis B Virus Replication

2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) is a member of the interferon-stimulated genes, which includes isoforms CNP1 and CNP2. CNP1 is locally expressed in the myelin sheath but CNP2 is additionally expressed at low levels outside the nervous system. CNPs regulate multiple cellular functions and suppress protein production by association with polyadenylation of mRNA. Polyadenylation of Hepatitis B virus (HBV) RNAs is crucial for HBV replication. Whether CNPs interact with polyadenylation signal of HBV RNAs and interfere HBV replication is unknown. In this study, we evaluated expressions of CNP isoforms in hepatoma cell lines and their effects on HBV replication. We found that CNP2 is moderately expressed and gently responded to interferon treatment in HepG2, but not in Huh7 cells. The CNP1 and CNP2 potently inhibited HBV production by blocking viral proteins synthesis and reducing viral RNAs, respectively. In chronic hepatitis B patients, CNP was expressed in most of HBV-infected hepatocytes of liver specimens. Knockdown of CNP expression moderately improved viral production in the HepG2.2.15 cells treated with IFN-α. In conclusion, CNP might be a mediator of interferon-induced response against HBV.