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.

This work is funded by the DFG (GE  2145/3-2).
Collaboration partners: Olivier Silvie (INSERM, Paris, France), Felix Meissner (Max Planck Institute of Biochemistry, Martinsried, Germany), Lars Kaderali (University Greifswald, Germany)

Enveloped virus entry factor discovery

Quantitative proteomics approaches to study virus entry

Mass spectrometry based interaction proteomics has become highly sensitive and quantitative in the past years. Using our expertise gained during our HCV work we are currently using state of the art proteomics to search for cell surface proteins engaged by enveloped viruses such as alphaviruses. Specifically, we employ two mass spectrometry methods in conjunction with co-immunoprecipitation. On the one hand, we define the interactome of known entry factors such as tetraspanin-9 and TIM-1 in resting cells by label free quantification (LFQ). On the other hand, we use stable isotope labeling of amino acids in cell culture (SILAC) to identify proteins, which interact with viral glycoproteins and receptors during cell entry.

Our collaboration partners Dr. Felix Meissner and Dr. Mann are leading experts in quantitative mass spectrometry guaranteeing state of the art technical execution. In the long run, we strive to set up an attachment factor identification pipeline in an intermediate throughput manner. In follow up experiments, we address the functional role of interaction partners during entry using CRISPR/Cas9 knockout, RNA silencing and blocking techniques, which we streamlined during our HCV work. Our major pathogen of interest is the re-emerging and mosquito-borne human pathogen Chikungunya virus. 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 this zoonotic pathogen, also function as entry factors. The work will shed light on how alphaviruses enter host cells with putative implications for antiviral strategy development. Moreover, we expect that our work will pioneer future studies using a wide range of pathogens.

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)

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