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

Abstract

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.

Flexibility and intrinsic disorder are conserved features of hepatitis C virus E2 glycoprotein

“…It is, therefore, very likely that certain regions of E2 are buried in the heterodimer and/or oligomer. Although there is no molecular structure for the E1E2 complex, various mutagenesis and evolutionary analyses has provided consistent reports on the E1-E2 interface, which is likely to include elements of HVR-2, the backlayer and the E2 stalk (the latter is not included in the ectodomain models) [9,[72][73][74][75]. Therefore, although HVR-2 exhibits high flexibility in our simulations, it is likely to be constrained within the E1E2 complex.…”

HCV Glycoprotein Structure and Implications for B-Cell Vaccine Development

“…However, a complete structural picture of the E1E2 heterodimer is still missing due to an incomplete biochemical characterization in the context of infectious virions and difficulties in the expression of a recombinant, natively folded heterodimer. In the absence of a high-resolution structure biologically validated computational models [ 96 , 97 , 98 ] and a low-resolution TEM reconstruction [ 99 ] provide first insights into E1E2 heterodimerization. A comparison of the computational models reveals similarities, especially in the E2 core, but also major differences, concerning the relative orientation of E1 to E2, the presence of an intermolecular disulfide bond, and the conformation and orientation of HVR1 (reviewed in [ 100 ]).…”

“…Nevertheless, tested constructs could serve as a template for more detailed structural studies of the ectodomain and rational antigen design. Hence, in order to obtain E1E2 heterodimer ectodomains for electron microscopy studies earlier approaches were picked up and modified leading to a suitable soluble heterodimer with either an IgG Fc fragment (Flag- and His 6 -tagged) or a de novo designed dimerization-tag expressed in insect cells [ 99 ]. Both heterodimer constructs were obtained as higher oligomers but also as monomers with the latter ones yielding monodisperse particles on negative stain grids.…”

“…Both heterodimer constructs were obtained as higher oligomers but also as monomers with the latter ones yielding monodisperse particles on negative stain grids. sE1E2-Fc particles feature a head and a tail region in the 2D averaged classes with the head region corresponding to the E1E2 heterodimer and the tail region formed by the Fc homodimer [ 99 ]. Similarly, 2D averaging for negative stain images collected for the second construct revealed a doughnut-shaped head and a tail, which correspond to the E1E2 heterodimer and the dimerization tag, respectively.…”

“…A comparison of the recombinant monomeric E1E2 heterodimer under reducing and non-reducing conditions on the SDS-PAGE gel suggests that one or more intermolecular disulfide bonds are formed, which is consistent with virion-associated E1 and E2 [ 101 ]. The heterodimer constructs were also used for expression in mammalian cells with a similar experimental outcome although an increased heterogeneity of the purified sample likely due to larger and rather inhomogeneous glycosylation was observed [ 99 ]. Interestingly, monomers as well as higher oligomers were bound by conformational E1 and E2-specific nAbs as well as CD81, suggesting that both species contain at least partially correctly exposed epitopes and a functional receptor binding site.…”