Research Projects
I. Cell biology of hepatitis B virus infection: towards curative approaches of chronic hepatitis B
HBV is a pararetrovirus replicating its DNA genome via reverse transcription of an overlength RNA precursor (the pregenome). Hallmarks of HBV are the compact genome organization, the use of internal promoters and the persistence of the viral DNA genome as an episome in the nucleus of infected cells. This covalently closed circular DNA (cccDNA) serves as persistence reservoir and is not affected by existing antiviral therapy. Consequently, these treatments are not curative, but only viro-suppressive and have to be applied life-long.
Schematic of the replication cycles of HCV, HBV and HDV. Upon entry of HCV (top sector), viral RNA is translated at the ER and a membranous replication factory is formed (membranous web). There, viral RNA is amplified and packaged into nucleocapsids that bud into the ER. Enveloped virions are secreted out of the cell. In the case of HBV (lower right sector), upon entry in an NTCP dependent manner, the partially double stranded viral DNA genome is delivered into the nucleus where it is converted into the cccDNA formpersisting as nuclear episome. Viral (v)RNAs are transcribed and used for protein synthesis. Within the nucleocapsids the viral RNA pre-genome is reverse transcribed (the red dot indicates the co-packaged P protein) and virions are formed by budding into the ER lumen. Virions are secreted from the cell, along with subviral particles (SVPs) that lack a nucleocapsid and are therefore non-infectious. SVPs are composed of the same envelope proteins as infectious HBV particles. In the case of HDV (lower left sector), the virus enters hepatocytes probably in the same way as HBV does. The viral RNA genome is delivered into the nucleus and used for the production of mRNAs that are translated in the cytoplasm. Fromthere the two HDV proteins are imported into the nucleus to form RNPs. These are exported into the cytoplasm where they acquire their envelope via budding using HBV envelope glycoproteins. HDV particles are released by the secretory pathway, along with HBV virions and SVPs in case of those cells that are co-infected with both viruses.
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Most HBV infections are asymptomatic and can persist, with the risk of persistence increasing with decreasing age. While more than 90% of perinatal HBV infections persist, this number drops to 5-10% in case of adults, arguing that immunological competence of the infected individual is critical to control virus infection. Although the underlying mechanisms of persistence are not understood, there is increasing evidence that HBV is a "stealth" virus, neither inducing an interferon response, nor being profoundly affected by the antiviral program triggered by this cytokine. In addition, there is solid evidence that an insufficient adaptive immune response accounts for chronicity, most notably a lack of T cell response against the viral surface protein(s). This is due, at least in part, to the massive production of subviral particles (SVPs), which are "empty" virus envelopes containing viral surface glycoproteins, but lacking a capsid and the viral genome. Of note, SVPs are released in ~1,000-fold excess over virus particles. SVPs are non-infectious, but the excessive amount of HBV surface proteins contained therein probably is blunting especially T cell immunity.
To overcome this limitation we attempt to develop strategies to block excessive production of SVPs. To this end, we study the cell biology of SVP production and release by using state-of-the-art infectable cell culture models. By using RNA interference-based screens we want to identify host cell factors and pathways in SVP formation and secretion with the aim to exploit these factors and pathways as targets for the development of curative therapeutic approaches of chronic hepatitis B. In addition, we use proteomic approaches and RNAi-based screening to identify host cell factors of relevance for the HBV replication cycle.
Seitz S, Habjanič J, Schütz AK, Bartenschlager R. 2020. The Hepatitis B Virus Envelope Proteins: Molecular Gymnastics Throughout the Viral Life Cycle. Annu Rev Virol. 7(1):263-288.
Lee J-Y, Cortese M, Haselmann U, Tabata K, Romero-Brey I, Funaya C, Schieber N, Qiang Y, Bartenschlager M, Kallis S, Ritter C, Rohr K, Schwab Y, Ruggieri A, Bartenschlager R. 2019. Spatio-temporal coupling of the hepatitis C virus replication cycle by creation of a lipid droplet proximal membranous replication compartment. Cell Reports, 27(12):3602-3617.
Mutz P, Metz P, Lempp FA, Bender S, Qu B, Schöneweis K, Seitz S, Tu T, Restuccia A, Frankish J, Dächert C, Schusser B, Koschny R, Polychronidis G, Schemmer P, Hoffmann K, Baumert TF, Binder M, Urban S, Bartenschlager R. 2018. HBV Bypasses the Innate Immune Response and Does not Protect HCV From Antiviral Activity of Interferon. Gastroenterology. 154(6):1791-1804.
Bartenschlager R, Baumert TF, Bukh J, Houghton M, Lemon SM, Lindenbach BD, Lohmann V, Moradpour D, Pietschmann T, Rice CM, Thimme R, Wakita T. 2018. Critical challenges and emerging opportunities in hepatitis C virus research in an era of potent antiviral therapy: Considerations for scientists and funding agencies. Virus Res. 248:53-62.
Seitz S, Iancu C, Volz T, Mier W, Dandri M, Urban S, Bartenschlager R. 2016. A slow maturation process renders hepatitis B virus infectious. Cell Host & Microbe, 20(1):25-35.
Lempp FA, Mutz P, Lipps C, Wirth D, Bartenschlager R, Urban S. 2015. Evidence that hepatitis B virus replication in mouse cells is limited by the lack of a host cell dependency factor. Journal of Hepatology 64(3):556-64.
Bartenschlager R, Schaller H. 1992. Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. EMBO J. 1992 Sep;11(9):3413-20.
II. Induction of antiviral immune responses against hepatitis viruses and viral countermeasures
As part of our research activities that are ongoing in our sister unit at the Department of Infectious Diseases, Molecular Virology, in this subproject we aim to understand the strategies how HCV infection is sensed by pattern recognition receptors and how the virus escapes this pathway. We have earlier shown that HCV blocks signaling pathways by cleaving the adaptor protein MAVS and obtained evidence that HCV persistence might be facilitated by the stochastic nature of the interferon response. We now want to dissect further the predominant sensor of HCV RNA and how, in addition to MAVS cleavage, the virus counteracts the interferon response. Moreover, we want to characterize the ISGs responsible for control of HCV infection. Finally, by using co-culture systems we want to study the cross-talk between innate and adaptive immune response. Here we focus on T cell exhaustion, which is a hallmark of HCV and HBV infection and that appears to be caused by mechanisms differing between these two viruses.
Schematic how HCV activates the interferon (IFN) response and points of viral countermeasure that are based on proteolytic cleavage of signaling molecules by the viral NS3/4A protease. IFN released from infected cells induces an antiviral state in infected and bystander cells. It is unclear to what extent HCV blocks Jak/Stat signaling as contradicting reports exist. The important role of IL28 SNPs in the outcome of HCV infection (acute self-limited versus persistence) is indicated. It is also unclear whether sustained IFN production as induced by HCV infection has an effect on T cell response. Figure adapted from Metz et al., 2013.
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HDV is a satellite of HBV that is unable to produce infectious progeny in the absence of the HBV helper virus. HDV usurps the HBV envelope to assemble virus particles and therefore HDV infected patients are HBV infected as well. Co-infected patients have a faster progress of serious liver disease, including liver cancer, as compared to HBV mono-infected individuals. HDV resembles viroids found in many plants. It has a circular single-stranded RNA genome replicating in the nucleus by using RNA polymerase II. Antiviral drugs targeting HBV have no effect on HDV and until recently, HDV infections could not be treated. However, recently, in our sister department “Molecular Virology” at University Hospital Heidelberg we were able to develop the first antiviral drug that has been approved for treatment of chronic hepatitis D. Building on these results, we now aim to understand how HDV overcomes innate antiviral immunity to establish persistence.
Zhang Z, Filzmayer C, Ni Y, Sültmann H, Mutz P, Hiet MS, Vondran FWR, Bartenschlager R, Urban S. 2018. Hepatitis D Virus replication is sensed by MDA5 and induces IFN-β/λ responses in hepatocytes. Journal of Hepatology Jul;69(1):25-35.
Hiet MS, Bauhofer O, Zayas M, Roth H, Tanaka Y, Schirmacher P, Willemsen J, Grünvogel O, Bender S, Binder M, Lohmann V, Lotteau V, Ruggieri A, Bartenschlager R. 2015. Control of temporal activation of hepatitis C virus-induced interferon response by domain 2 of nonstructural protein 5A. Journal of Hepatology 63(4):829-37.
Bender S, Reuter A, Eberle F, Einhorn E, Binder M, Bartenschlager R. 2015. Activation of Type I and III Interferon Response by Mitochondrial and Peroxisomal MAVS and Inhibition by Hepatitis C Virus. PLoS Pathogens 11(11):e1005264.
Bauhofer O, Ruggieri A, Schmid B, Schirmacher P, Bartenschlager R. 2012. Persistance of HCV in Quiescent Hepatic Cells during an Interferon-Induced Antiviral Response. Gastroenterology. 143:429-38.
III. Cell biology of the SARS-CoV-2 replication cycle.
Cortese et al. use integrative imaging techniques to generate a publicly available repository of morphological alterations induced by SARS-CoV-2 in lung cells. Accumulation of ER-derived double-membrane vesicles, the viral replication organelle, occurs concomitantly with cytoskeleton remodeling and Golgi fragmentation. Pharmacological alteration of cytoskeleton dynamics restricts viral replication and spread.
© Cortese et al., 2020, Cell Host & Microbe 28, 853–866 December 9, 2020 © 2020 Elsevier Inc.
Viral and host determinants of SARS-CoV-2 replication: Common pathways as targets for broad-spectrum antiviral therapy
In this project we aim to identify the viral and host cell factors responsible for SARS-CoV-2 replication. A particular focus is put on the formation of the viral replication organelle (RO) and its genome amplification machinery. We will decipher those factors that are commonly used by other RNA viruses forming analogous ROs (e.g. other coronaviruses). Conserved factors will be tested for suitability as targets for broad-spectrum antiviral drugs to be employed e.g. as pan-coronavirus inhibitors, in line with the Transfer mission of COVIPA. Complementary to that we will study the morphological alterations induced by SARS-CoV-2 in infected cells and how they contribute to cytopathogenicity.
Neufeldt C, Cerikan B, Cortese M, Frankish J, Lee J_Y, Plociennikowska A, Heigwer F, Prasad V, Joecks S, Burkart S, Zander D, Subramanian B, Gimi R, Padmanabhan S, Iyer R, Gendarme M, El Debs B, Halama N, Merle U, Boutros M, Binder M, Bartenschlager R. 2022. SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-κB. Communication Biology
Twu WI, Lee JY, Kim H, Prasad V, Cerikan B, Haselmann U, Tabata K, Bartenschlager R. 2021. Contribution of autophagy machinery factors to HCV and SARS-CoV-2 replication organelle formation. Cell Rep. 37(8):110049.
Tabata K, Prasad V, Paul D, Lee JY, Pham MT, Twu WI, Neufeldt CJ, Cortese M, Cerikan B, Stahl Y, Joecks S, Tran CS, Lüchtenborg C, V'kovski P, Hörmann K, Müller AC, Zitzmann C, Haselmann U, Beneke J, Kaderali L, Erfle H, Thiel V, Lohmann V, Superti-Furga G, Brügger B, Bartenschlager R. 2021. Convergent use of phosphatidic acid for hepatitis C virus and SARS-CoV-2 replication organelle formation. Nat Commun. 12(1):7276.
Ke Z, Oton J, Qu K, Cortese M, Zila V, McKeane L, Nakane T, Zivanov J, Neufeldt CJ, Cerikan B, Lu JM, Peukes J, Xiong X, Kräusslich HG, Scheres SHW, Bartenschlager R, Briggs JAG. 2020. Structures and distributions of SARS-CoV-2 spike proteins on intact virions. Nature 2020 Aug 17.
Cortese M, Lee YL, Cerikan B, Neufeldt CJ, Oorschot VMJ, Köhrer S, Hennies J, Schieber NL, Ronchi P, Mizzon G, Romero-Brey I, Santarella-Mellwig R, Schorb M, Boermel M, Mocaer K, Beckwith MS, Templin RM, Gross V, Pape C, Tischer C, Frankish J, Horvat NK, Laketa V, Stanifer M, Boulant S, Ruggieri A, Chatel-Chaix L, Schwab Y, Bartenschlager R. 2020. Integrative imaging reveals SARS-CoV-2 induced reshaping of subcellular morphologies. Cell Host & Microbe, 2020 Nov 17:S1931-3128(20)30620-X.