Projects Virus Interaction Proteomics

Characterization of CD81 receptor interactors in hepatitis C virus and Plasmodium liver cell entry

Localization of SRFBP1, a HCV entry factor identified by quantitative proteomics, in human hepatoma cells.

Hepatitis C virus (HCV) and the malaria parasite Plasmodium falciparum both use the human transmembrane protein CD81 to infect liver cells. This is remarkable as both pathogens substantially differ in their molecular makeup, transmission and pathology. A common theme of HCV and the human pathogen Plasmodium falciparum is, however, the narrow host tropism as they naturally only infect human vertebrate hosts. CD81 is one of several determinants for this host tropism. How CD81 guides entry of HCV and Plasmodium remained largely enigmatic. Since CD81 lacks signaling domains, we had hypothesized that it coordinates HCV and Plasmodium uptake through protein-protein interactions (PPI) with membrane proximal signaling adaptors and cytoskeleton regulators. Our previous work identified 33 CD81 PPIs in human hepatoma cells and could show that at least ten of the CD81 interactors are required for HCV and Plasmodium infection.

Among them are the epidermal growth factor receptor, a receptor tyrosine kinase known to aid HCV entry, an ubiquitin ligase and an endopeptidase. Currently, we characterize the molecular function of these host factors in depth. By testing domain mutants of the host factors, we aim to shed light on how the proteins guide pathogen entry. At the same time we address the specificity of the discovered host factors by performing infection assays with other enveloped viruses, with different HCV genotypes and with human and rodent Plasmodium species in CRISPR/Cas9 knockout cells for the respective entry factor. Lastly, our work will determine if the host factors contribute to the narrow tissue and host tropism of HCV and Plasmodium falciparum sporozoites. This work will reveal commonalities and differences in liver entry of two important human pathogens and holds the promise of identifying urgently needed drug targets to combat malaria.

Collaboration partners: Olivier Silvie (INSERM, Paris, France), Felix Meissner (Max Planck Institute of Biochemistry, Martinsried, Germany), Lars Kaderali (University Greifswald, Germany)

Publications

Enveloped virus entry factor discovery

Quantitative proteomics approaches to study virus entry

Mass spectrometry based interaction proteomics has become highly sensitive and quantitative. Using state of the art proteomics, we search for cell surface proteins engaged by human pathogenic viruses. Specifically, we employ label free quantification (LFQ) to define the interactome of known entry factors such as tetraspanin-9 and TIM-1. On the other hand, we use stable isotope labeling of amino acids in cell culture (SILAC) and targeted crosslinking approaches to identify proteins, which interact with viral glycoproteins during cell entry. In follow up experiments, we address the functional role of interaction partners using CRISPR/Cas9 knockout, RNA silencing and blocking techniques.

Our major pathogens of interest are re-emerging and mosquito-borne human pathogens of the alphavirus genus and enteric viruses, including human norovirus. Identified entry factors and receptors will be tested for their specificity to various virus strains and their expression in different human tissues targeted by the virus. Lastly, we will analyze if orthologs in mosquitoes and non-human primates, which serve as transmission and reservoir hosts for alphaviruses, also function as entry factors. We collaborate with Niklas Arnberg (Umea University) and Lennart Svensson (Linköping University) for functional follow up work on norovirus entry factors. The work will shed light on how alphaviruses and enteric viruses enter host cells with putative implications for antiviral strategy development.

Collaboration partners: Margaret Kielian (Albert Einstein College, New York, NY, USA), Pierre-Yves Lozach (Heidelberg University Hospital, Germany), Charles M. Rice (Rockefeller University, New York, NY, USA), Niklas Arnberg (Umea University, Sweden), Lennart Svensson (Linköping University, Sweden)

Publications

HCV – arenavirus co-infection and modulation of innate sensing mechanisms

Innate immune response to HCV and viral antagonism mediated by the NS3/4A protease. (Adapted from Weigel, Bruening, Gerold, Journal of Immunology Research, 2016)

HCV is a small enveloped RNA virus and the causative agent of hepatitis C. It affects 71 million individuals worldwide and can cause severe liver disease including cirrhosis, fibrosis and hepatocellular carcinoma. The virus is underdiagnosed and thought to be highly prevalent in Western African regions, where outbreaks of hemorrhagic fever viruses such as the arenavirus Lassa virus occur. Since chronic HCV infection alters the immune status of the liver, which is also a target organ for arenaviruses, we hypothesized that a co-infection with HCV and arenaviruses may alter the severity of disease. Using cell culture models of hepatoma cells and primary hepatocytes, we address how a co-infection impacts virus propagation, cellular innate immune responses and sensitivity to licensed antiviral drugs. These efforts will allow the assessment of risks associated with underlying chronic hepatitis during arenavirus outbreaks.

This work is funded by the German Liver Foundation (S1 63/10135/2017)

Collaboration partners: Stefan Kunz (CHUV, Lausanne, Switzerland), Jan Münch, Ulm University, Germany

Characterization of CD81 receptor interactors in hepatitis C virus and Plasmodium liver cell entry

Bruening J, Banse P, Kahl S, Vondran FW, Kaderali L, Marinach C, Silvie O, Pietschmann T, Meissner F*, Gerold G* (2018)  Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB. Manuscript accepted for publication at PLOS Pathogens. *these authors contributed equally

Lasswitz L, Chandra N, Arnberg N*, Gerold G* (2018)  Glycomics and proteomics approaches to investigate early adenovirus - host cell interactions. J Mol Biol. doi: 10.1016/j.jmb.2018.04.039. *these authors contributed equally

Banse P, Bruening J, Lasswitz L, Kahl S, Khan AG, Marcotrigiano J, Pietschmann T, Gerold G (2018) CD81 receptor regions outside the large extracellular loop determine hepatitis C virus susceptibility. Viruses doi: 10.3390/v10040207.

Gerold G, Bruening J, Weigel B, Pietschmann T (2017) Protein interactions during the flavivirus and hepacivirus life cycle. Mol Cell Proteomics. pii: mcp.R116.065649.

Gerold G, Meissner F, Bruening J, Welsch K, Perin PM, Thomas F. Baumert, Vondran FW, Kaderali L, Marcotrigiano J, Khan AG, Mann M, Rice CM, Pietschmann T (2015) Quantitative Proteomics Identifies Serum Response Factor Binding Protein 1 as a Host Factor for Hepatitis C Virus Entry. Cell Reports. 4;12(5):864-78.

Funding:  DFG (GE  2145/3-1), DFG (GE  2145/3-2), DFG (SFB900-C7), DZIF (TI 07.003), Deutsche Leberstiftung (S163/10073/2011), HFSPO (LT000048/2009-L).

Virus entry factor discovery: Mosquito-borne viruses and enteric viruses

Fedeli C, Torriani G, Galan-Navarro C, Moraz ML, Moreno H, Gerold G, Kunz S (2018) Axl Can Serve as Entry Factor for Lassa Virus Depending on the Functional Glycosylation of Dystroglycan. J Virol. doi: 10.1128/JVI.01613-17.

von Schaewen M, Dorner M, Hueging K, Foquet L, Gerges S, Hrebikova G, Heller B, Bitzegeio J, Doerrbecker J, Horwitz J, Gerold G, Suerbaum S, Rice CM, Meuleman P, and Pietschmann T, Ploss A (2016) Expanding the host range of hepatitis C virus through viral adaptation. MBio. 8;7(6).

Scull MA, Shi C, de Jong YP, Gerold G, Ries M, von Schaewen M, Donovan BM, Labitt RN, Horwitz JA, Gaska JM, Hrebikova G, Xiao JW, Flatley B, Fung C, Chiriboga L, Walker CM, Evans DT, Rice CM, Ploss A. (2015) Hepatitis C virus infects rhesus macaque hepatocytes and simianized mice. Hepatology. 62(1):57-67.

Gerold G, Bruening J, Pietschmann T. (2015) Decoding protein networks during virus entry by quantitative proteomics. Virus Research. 218:25-39.

Kapoor A, Simmonds P, Gerold G, Qaisar N, Jain K, Henriquez JA, Firth C, Hirschberg DL, Rice CM, Shields S, Lipkin WI. (2011) Characterization of a canine homolog of hepatitis C virus. PNAS. 108(28):11608-13.

Funding:  DFG (GE  2145/3-2), German Liver Foundation (S163/10135/2017), DAAD, ZIB, Friends of the MHH.