Identification of Aquarius and Senataxin as Restriction Host Factors for Hepatitis B Virus Infection

© 2020 by the authorsHepatitis B virus (HBV) represents an important human pathogen causing acute and chronic hepatitis. Over 240 million people are chronically infected, many of whom will die due to complications such as liver cirrhosis and hepatocellular carcinoma. Currently approved therapies are very effective in suppressing virus replication and viremia, but they are not curative, because they do not completely eliminate the nuclear episomal DNA form of HBV (cccDNA) that re-establishes infection upon interruption of therapy. Despite our understanding of many aspects of the HBV lifecycle, details of the HBV cccDNA biology remain poorly understood. Our group is pursuing a loss-of-function genetic screening approach, to identify cellular factors regulating HBV infection. A lentivirus-delivered short hairpin RNA (shRNA) library, composed of 384 shRNAs, was used to interrogate the function of 80 DNA damage repair pathway proteins in the establishment of HBV infection. The primary screening identified 10 cellular factors that regulate the HBV infection both positively or negatively. Two of those proteins, aquarius (AQR) and senataxin (SETX), were subsequently validated as factors restricting the HBV infection in independent experiments. Silencing of AQR and SETX led to an increased infection efficiency that was characterized by higher intracellular levels of HBV cccDNA, HBV mRNA, and core protein, and increased HBV e antigen (HBeAg) accumulation in the supernatants of infected cells. The expression level, glycosylation pattern, and localization of the HBV receptor, sodium taurocholate cotransporting polypeptide (NTCP), in AQR- and SETX-downregulated cells was equivalent to that of the control cells. Collectively, our results are compatible with AQR and SETX restricting early steps in the HBV lifecycle and downstream HBV entry, that affect the establishment of the HBV cccDNA pool. Experiments to unravel the function of these proteins in the context of HBV infection are currently underway.This work was supported by grants SAF2016-75169-R (AEI/FEDER, UE) from the Spanish Ministry of Economy, Industry and Competitiveness, and a CTSA Pilot Award (NIH/NCATS/STSI 5UL1 TR001114) to U.G. A.G.M is supported by the Spanish Minitry of Education (FPU17/03424)

Native hepatitis B virions and capsids visualized by electron cryomicroscopy

Hepatitis B virus (HBV) infects more than 350 million people, of which one million will die every year. The infectious virion is an enveloped capsid containing the viral polymerase and double-stranded DNA genome. The structure of the capsid assembled in vitro from expressed core protein has been studied intensively. However, little is known about the structure and assembly of native capsids present in infected cells, and even less is known about the structure of mature virions. We used electron cryomicroscopy (cryo-EM) and image analysis to examine HBV virions (Dane particles) isolated from patient serum and capsids positive and negative for HBV DNA isolated from the livers of transgenic mice. Both types of capsids assembled as icosahedral particles indistinguishable from previous image reconstructions of capsids. Likewise, the virions contained capsids with either T = 3 or T = 4 icosahedral symmetry. Projections extending from the lipid envelope were attributed to surface glycoproteins. Their packing was unexpectedly nonicosahedral but conformed to an ordered lattice. These structural features distinguish HBV from other enveloped viruses

The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection

© 2019 by the authors.Development of hepatitis C virus (HCV) infection cell culture systems has permitted the identification of cellular factors that regulate the HCV life cycle. Some of these cellular factors affect steps in the viral life cycle that are tightly associated with intracellular membranes derived from the endoplasmic reticulum (ER). Here, we describe the discovery of erlin-1 protein as a cellular factor that regulates HCV infection. Erlin-1 is a cholesterol-binding protein located in detergent-resistant membranes within the ER. It is implicated in cholesterol homeostasis and the ER-associated degradation pathway. Silencing of erlin-1 protein expression by siRNA led to decreased infection efficiency characterized by reduction in intracellular RNA accumulation, HCV protein expression and virus production. Mechanistic studies revealed that erlin-1 protein is required early in the infection, downstream of cell entry and primary translation, specifically to initiate RNA replication, and later in the infection to support infectious virus production. This study identifies erlin-1 protein as an important cellular factor regulating HCV infection.This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers RYC-2014-15805 and SAF2016-75169-R (AEI/FEDER, UE) to U.G. and A.G.-M. is supported by an FPU fellowship (FPU17/03424) from the Spanish Ministry of Education, Culture and Sports.Peer reviewe

Short-Range Exosomal Transfer of Viral RNA from Infected Cells to Plasmacytoid Dendritic Cells Triggers Innate Immunity

International audienceViral nucleic acids often trigger an innate immune response in infected cells. Many viruses, including hepatitis C virus (HCV), have evolved mechanisms to evade intracellular recognition. Nevertheless, HCV-permissive cells can trigger a viral RNA-, TLR7-and cell contact-dependent compensatory interferon response in nonpermissive plasmacytoid dendritic cells (pDCs). Here we report that these events are mediated by transfer of HCV RNA-containing exosomes from infected cells to pDCs. The exosomal viral RNA transfer is dependent on the endosomal sorting complex (ESCRT) machinery and on Annexin A2, an RNA-binding protein involved in membrane vesicle trafficking, and it is suppressed by exosome release inhibitors. Further, purified concentrated HCV RNA-containing exosomes are sufficient to activate pDCs. Thus, vesicular sequestration and exosomal export of viral RNA may serve both as a viral strategy to evade pathogen-sensing within infected cells and as a host strategy to induce an unopposed innate response in replicationnonpermissive bystander cells