Functional expression and characterization of the envelope glycoprotein E1E2 heterodimer of hepatitis C virus

Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae . HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV. Hepatitis C virus (HCV) is an enveloped virus that infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV has two envelope proteins, E1 and E2, which form heterodimers on viral surface and are critical for HCV cell entry. However, current studies of HCV E1E2 are often limited by the poor quality of the in vitro expressed E1E2 heterodimers. Here we express the ectodomains of HCV E1E2 with different tags, and are able to isolate soluble E1E2 ectodomains suitable for structural and functional studies. Then we generate the 3D reconstruction of E1E2 heterodimer by electron microscopy and also model the E1E2 structure by the coevolution based strategy with Rosetta, showing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the HCV cellular receptors, neutralizing antibodies as well as the inhibition of HCV infection. These results suggest that the expressed E1E2 heterodimer would be a promising target for both viral studies and vaccination against HCV.