The impact of HBx protein on mitochondrial dynamics and associated signaling pathways strongly depends on the hepatitis B virus genotype.

Chronic hepatitis B virus (HBV) infections are strongly associated with liver cirrhosis, inflammation, and hepatocellular carcinoma. In this context, the viral HBx protein is considered as a major factor influencing HBV-associated pathogenesis through deregulation of multiple cellular signaling pathways and is therefore a potential target for prognostic and therapeutic applications. However, HBV-associated pathogenesis differs significantly between genotypes, with the relevant factors and in particular the contribution of the genetic diversity of HBx being largely unknown. To address this question, we studied the specific genotype-dependent impact of HBx on cellular signaling pathways, focusing in particular on morphological and functional parameters of mitochondria. To exclusively investigate the impact of HBx of different genotypes on integrity and function of mitochondria in the absence of additional viral factors, we overexpressed HBx in Huh7 or HepG2 cells. Key signaling pathways were profiled by kinome analysis and correlated with expression levels of mitochondrial and pathogenic markers. Conclusively, HBx of genotypes A and G caused strong disruption of mitochondrial morphology alongside an induction of PTEN-induced putative kinase 1/Parkin-mediated mitophagy. These effects were only moderately dysregulated by genotypes B and E, whereas genotypes C and D exhibit an intermediate effect in this regard. Accordingly, changes in mitochondrial membrane potential and elevated reactive oxygen species production were associated with the HBx-mediated dysfunction among different genotypes. Also, genotype-related differences in mitophagy induction were identified and indicated that HBx-mediated changes in the mitochondria morphology and function strongly depend on the genotype. This indicates a relevant role of HBx in the process of genotype-dependent liver pathogenesis of HBV infections and reveals underlying mechanisms.IMPORTANCEThe hepatitis B virus is the main cause of chronic liver disease worldwide and differs in terms of pathogenesis and clinical outcome among the different genotypes. Furthermore, the viral HBx protein is a known factor in the progression of liver injury by inducing aberrant mitochondrial structures and functions. Consequently, the selective removal of dysfunctional mitochondria is essential to maintain overall cellular homeostasis and cell survival. Consistent with the intergenotypic difference of HBV, our data reveal significant differences regarding the impact of HBx of different genotypes on mitochondrial dynamic and function and thereby on radical oxygen stress levels within the cell. We subsequently observed that the induction of mitophagy differs significantly across the heterogenetic HBx proteins. Therefore, this study provides evidence that HBx-mediated changes in the mitochondria dynamics and functionality strongly depend on the genotype of HBx. This highlights an important contribution of HBx in the process of genotype-dependent liver pathogenesis.

Guanylate-binding protein 1 acts as a pro-viral factor for the life cycle of hepatitis C virus.

Viral infections trigger the expression of interferons (IFNs) and interferon stimulated genes (ISGs), which are crucial to modulate an antiviral response. The human guanylate binding protein 1 (GBP1) is an ISG and exhibits antiviral activity against several viruses. In a previous study, GBP1 was described to impair replication of the hepatitis C virus (HCV). However, the impact of GBP1 on the HCV life cycle is still enigmatic. To monitor the expression and subcellular distribution of GBP1 and HCV we performed qPCR, Western blot, CLSM and STED microscopy, virus titration and reporter gene assays. In contrast to previous reports, we observed that HCV induces the expression of GBP1. Further, to induce GBP1 expression, the cells were stimulated with IFNγ. GBP1 modulation was achieved either by overexpression of GBP1-Wt or by siRNA-mediated knockdown. Silencing of GBP1 impaired the release of viral particles and resulted in intracellular HCV core accumulation, while overexpression of GBP1 favored viral replication and release. CLSM and STED analyses revealed a vesicular distribution of GBP1 in the perinuclear region. Here, it colocalizes with HCV core around lipid droplets, where it acts as assembly platform and thereby favors HCV morphogenesis and release. Collectively, our results identify an unprecedented function of GBP1 as a pro-viral factor. As such, it is essential for viral assembly and release acting through tethering factors involved in HCV morphogenesis onto the surface of lipid droplets.

Relevance of HBx for Hepatitis B Virus-Associated Pathogenesis.

The hepatitis B virus (HBV) counts as a major global health problem, as it presents a significant causative factor for liver-related morbidity and mortality. The development of hepatocellular carcinomas (HCC) as a characteristic of a persistent, chronic infection could be caused, among others, by the pleiotropic function of the viral regulatory protein HBx. The latter is known to modulate an onset of cellular and viral signaling processes with emerging influence in liver pathogenesis. However, the flexible and multifunctional nature of HBx impedes the fundamental understanding of related mechanisms and the development of associated diseases, and has even led to partial controversial results in the past. Based on the cellular distribution of HBx-nuclear-, cytoplasmic- or mitochondria-associated-this review encompasses the current knowledge and previous investigations of HBx in context of cellular signaling pathways and HBV-associated pathogenesis. In addition, particular focus is set on the clinical relevance and potential novel therapeutic applications in the context of HBx.

Presence of Intact Hepatitis B Virions in Exosomes.

BACKGROUND & AIMS: Hepatitis B virus (HBV) was identified as an enveloped DNA virus with a diameter of 42 nm. Multivesicular bodies play a central role in HBV egress and exosome biogenesis. In light of this, it was studied whether intact virions wrapped in exosomes are released by HBV-producing cells. METHODS: Robust methods for efficient separation of exosomes from virions were established. Exosomes were subjected to limited detergent treatment for release of viral particles. Electron microscopy of immunogold labeled ultrathin sections of purified exosomes was performed for characterization of exosomal HBV. Exosome formation/release was affected by inhibitors or Crispr/Cas-mediated gene silencing. Infectivity/uptake of exosomal HBV was investigated in susceptible and non-susceptible cells. RESULTS: Exosomes could be isolated from supernatants of HBV-producing cells, which are characterized by the presence of exosomal and HBV markers. These exosomal fractions could be separated from the fractions containing free virions. Limited detergent treatment of exosomes causes stepwise release of intact HBV virions and naked capsids. Inhibition of exosome morphogenesis impairs the release of exosome-wrapped HBV. Electron microscopy confirmed the presence of intact virions in exosomes. Moreover, the presence of large hepatitis B virus surface antigen on the surface of exosomes derived from HBV expressing cells was observed, which conferred exosome-encapsulated HBV initiating infection in susceptible cells in a , large hepatitis B virus surface antigen/Na+-taurocholate co-transporting polypeptide-dependent manner. The uptake of exosomal HBV with low efficiency was also observed in non-permissive cells. CONCLUSION: These data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV.

Identification of the interferon-inducible GTPase GBP1 as major restriction factor for the Hepatitis E virus.

This study aims to gain deeper insight into HEV-induced innate immunity by characterizing the crosstalk between the virus and the host factor guanylate-binding protein 1 (GBP1). We observe that the amount of GBP1 is elevated upon infection, although number of transcripts is decreased, which is explained by a prolonged protein half-life. Modulation of GBP1 levels via overexpression significantly inhibits the viral life cycle. Use of various GBP-1 mutants revealed that the antiviral effect of GBP-1 on HEV is independent from the GTPase-activity, but depends on the capacity of GBP-1 to form GBP1 homodimers. This connects GBP-1 to the autophagosomal pathway. Indeed, dimerization competent GBP1 targets the viral capsid protein to the lysosomal compartment leading to inactivation of the viral particle. Most importantly, silencing of GBP1 abolishes the antiviral effect of IFNγ on HEV. In IFNγ treated cells the virus is targeted to lysosomal structures and destroyed therein. This process depends in part on GBP1. These observations about the relevance of GBP1 for type II interferon-mediated innate immunity against HEV could be a base for tailoring novel antivirals and improvement of disease management.IMPORTANCE Although HEV represents a worldwide public health problem with 20 million infections and 44.000 death cases per year, there are still no specific antivirals available and many aspects of the viral life cycle are not well understood. Here we identify the guanylate binding protein 1 (GBP1) as a restriction factor affecting life cycle of HEV. Surprisingly, the antiviral effect of GBP1 does not depend on its GTPase function, but on its capacity to homodimerize. We revealed that GBP1 exerts its antiviral activity by targeting HEV to the lysosomal compartment where the virus is inactivated. Most importantly, we observed that the antiviral effect of interferon-γ on HEV strongly depends on GBP1. Our observation that GBP1 impairs HEV and is crucial for the antiviral effect of interferons on HEV extends understanding of host defense-mechanisms. As the interferon-system represents a universal defense-mechanism, our study could help to design novel antivirals targeting.