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Characterization of EWI-2wint and the mechanism behind its inhibition of HCV infection

Final Report Summary - HCV-EWI-2WINT (Characterization of EWI-2wint and the mechanism behind its inhibition of HCV infection)

There are an estimated 5-10 million individuals living with Hepatitis C virus (HCV) infection within the European community. Of those infected approximately 80% fail to clear the virus, and a significant number of these will go on to develop severe liver diseases such as cirrhosis and even primary liver cancer. In Southern Europe, 80% of liver transplants are performed on individuals with severe liver damage due to chronic HCV infection. There is still a great need to develop new anti-viral drugs for HCV infection.

HCV is a positive strand enveloped RNA virus with two envelope glycoproteins E1 and E2, which form a non-covalently linked heterodimer. This heterodimer is the main component of the HCV envelope and plays a major role in HCV entry into its target cells, the hepatocytes. Several receptors have been found to play a role in HCV entry, among them CD81. The search for additional players in the viral entry led to the discovery of a negative regulator, EWI-2wint in the host institution. This protein, which is a processed variant of EWI-2, is able to block the binding of the HCV envelope proteins to the receptor CD81. Expression of EWI-2wint in cells susceptible to HCV leads to a reduced infection rate. EWI-2 is a ubiquitously expressed type I transmembrane protein with four immunoglobulin (Ig) domains and a very short cytoplasmic tail. EWI-2wint lacks the first Ig-domain.
CD81 belongs to the tetraspanin protein family, which is characterized by four transmembrane domains (TM), a large (LEL) and a small extracellular loop (SEL). Tetraspanins have the unique characteristic that they interact with each other to form tetraspanin enriched microdomains (TEMs) with each other at the cell surface. In these TEMs they incorporate proteins, with which they specifically interact. CD81, as well as CD9 associates in high stoichiometry with EWI-2 and EWI-2wint.
The goal of the project was to characterize the interaction of EWI-2 and EWI-2wint with CD81 and to analyze the effect this interaction has on viral infection. A second part of the project was the production of soluble proteins corresponding to the complete or partial extracellular domain of EWI-2wint to see if they would have the same inhibitory effect, while a third part aims to identify the protease responsible for the cleavage of EWI-2 to EWI-2wint.
In order to be able to identify the protease responsible for the cleavage of EWI-2, cleavage sequence in EWI-2 that leads to the production of EWI-2wint was characterized through an alanine scanning approach. These experiments revealed that cleavage of EWI-2 depends on two basic amino acids at the cleavage site. Further experiments suggested that the protease responsible is not furin, but the use of different protease inhibitors did not reveal the protease and suggest that the protein might be cleaved by different protease under different conditions.
To better understand the effect of EWI-2wint on HCV infection, we characterized the interaction between EWI-2/EWI-2wint and CD81. Through several series of chimeric recombinant proteins, we analyzed the interaction and found that it depends on TM 3 and TM 4 and the LEL in CD81 and on Ig domain 4, the TM and a glycinzipper and the palmitoylation in EWI-2/EWI-2wint. The regulation of interaction by palmitoylation of the partner proteins might be a general principle of regulation in TEMs.
The different proteins were also tested for their effects in HCV infection. These experiments showed that HCV needs more than just the CD81-LEL to successfully infect cells and that EWI-2wint needs to interact with CD81 to inhibit infection.
Due to technical difficulties the production of soluble proteins did not yield enough protein to test their effect on infection or do structural analysis. Currently ongoing projects aim to test these in inducible systems as well as test the effect in a mouse HCV entry model.