HCV entry proteome
Mapping of the CD81-associated HCV entry proteome
Hepatitis C virus (HCV) is a small enveloped RNA virus and the causative agent of hepatitis C. It affects 3% of the world population and can cause severe liver disease including cirrhosis, fibrosis and hepatocellular carcinoma. Consequently, HCV infection is the primary indication for liver transplantation and strategies are needed to block infection of the graft tissue. To successfully intervene with HCV entry into hepatocytes, a clear understanding of the virus internalization process is needed. Moreover, HCV uses a unique and tightly controlled entry pathway. Thus, HCV entry research can reveal important and novel aspects of cellular membrane trafficking and signaling.
HCV entry depends on four critical factors: scavenger receptor B type I (SCARB1), CD81, claudin-1 and occludin. In particular, the tetraspanin CD81 is central to HCV entry as it fulfills multiple functions among them:
1. Binding of the HCV envelope glycoprotein E2
2. Lateral translocation together with virus particles along the plasma membrane
3. Co-endocytosis with HCV
4. Priming of E2 for pH-dependent membrane Fusion
Importantly, CD81 is a tetraspanin, which coordinates membrane proteins in specialized microdomains. We hypothesize that CD81 is the central regulator of HCV entry events coordinating key interactions with membrane proximal signaling adaptors and cytoskeleton regulators.
Our project team aims at analyzing the CD81-host protein interaction network during HCV penetration. To this end, we perform high-end quantitative proteomics. Specifically, we employ two mass spectrometry methods in conjunction with CD81 co-immunoprecipitation. On the one hand, we define the CD81 interactome in resting hepatoma 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 CD81 during HCV entry. Our collaboration partner Prof. Dr. Mann is a leading expert in quantitative mass spectrometry guaranteeing state of the art technical execution.
In follow up experiments, we address the functional role of CD81 interaction partners during HCV entry using RNA silencing and blocking techniques. We further test whether novel entry co-factors are specifically involved in HCV entry by comparison with other enveloped and non-enveloped viruses. Selected candidate proteins, like the recently identified protein SRFBP1, are subsequently analyzed with regards to their mechanism and kinetics. This work holds the promise of identifying the protein network surrounding CD81 during HCV entry. Moreover, this is the first application of quantitative proteomics to study host invasion and thus our work will pioneer future studies using a wide range of pathogens.
Dr. Felix Meissner, Prof. Matthias Mann
Department of Proteomics and Signal Transduction
Max Planck Institute for Biochemistry
Prof. Charles M. Rice
Laboratory of Virology and Infectious Disease
Center for the Study of Hepatitis C
The Rockefeller University
Prof. Lars Kaderali
Department of Statistical Bioinformatics and Systems Biology
University of Technology Dresden