CD81 Receptor Regions outside the Large Extracellular Loop Determine Hepatitis C Virus Entry into Hepatoma Cells.

Pia Banse,Abdul Khan,Joseph Marcotrigiano,Janina Bruening,Thomas Pietschmann,Sina Kahl,Gisa Gerold,Lisa Lasswitz,Rebecca Moeller

doi:10.3390/v10040207

Abstract

Hepatitis C virus (HCV) enters human hepatocytes using four essential entry factors, one of which is human CD81 (hCD81). The tetraspanin hCD81 contains a large extracellular loop (LEL), which interacts with the E2 glycoprotein of HCV. The role of the non-LEL regions of hCD81 (intracellular tails, four transmembrane domains, small extracellular loop and intracellular loop) is poorly understood. Here, we studied the contribution of these domains to HCV susceptibility of hepatoma cells by generating chimeras of related tetraspanins with the hCD81 LEL. Our results show that non-LEL regions in addition to the LEL determine susceptibility of cells to HCV. While closely related tetraspanins (X. tropicalis CD81 and D. rerio CD81) functionally complement hCD81 non-LEL regions, distantly related tetraspanins (C. elegans TSP9 amd D. melanogaster TSP96F) do not and tetraspanins with intermediate homology (hCD9) show an intermediate phenotype. Tetraspanin homology and susceptibility to HCV correlate positively. For some chimeras, infectivity correlates with surface expression. In contrast, the hCD9 chimera is fully surface expressed, binds HCV E2 glycoprotein but is impaired in HCV receptor function. We demonstrate that a cholesterol-coordinating glutamate residue in CD81, which hCD9 lacks, promotes HCV infection. This work highlights the hCD81 non-LEL regions as additional HCV susceptibility-determining factors.

Highlights

Virus infection of cells is initiated by interaction with cell surface receptors
Chimeric tetraspanins were designed by replacing the large extracellular loop (LEL) of human CD9, human CD82, human TSN32, Danio rerio CD81, Xenopus tropicalis CD81, Caenorhabditis elegans TSP9 and Drosophila melanogaster TSP96F with the human CD81 (hCD81)
The resulting hLEL tetraspanin chimeras were named according to the origin of the respective tetraspanin backbone domain and displayed amino acid sequence percent identities to parental full-length hCD81 of 90%, 87%, 73%, 64%, 61%, 60%, and 58% (Figure 1B and Table 1)

Summary

Introduction

Virus infection of cells is initiated by interaction with cell surface receptors. Receptors bind virus particles and trigger an uptake program, which can include receptor clustering, lateral membrane movement, signaling, internalization, trafficking, membrane penetration and uncoating [1].Mechanistically, protein–protein interactions (PPI) and protein–lipid interactions guide these steps [2,3,4,5].While the receptors for some viruses are known, we typically lack knowledge on the post-binding function of virus receptors, i.e., how they mechanistically induce virus uptake.Viruses 2018, 10, 207; doi:10.3390/v10040207 www.mdpi.com/journal/virusesHepatitis C virus (HCV) is a member of the Flaviviridae family and as such a small enveloped positive strand RNA virus. Virus infection of cells is initiated by interaction with cell surface receptors. Receptors bind virus particles and trigger an uptake program, which can include receptor clustering, lateral membrane movement, signaling, internalization, trafficking, membrane penetration and uncoating [1]. Protein–protein interactions (PPI) and protein–lipid interactions guide these steps [2,3,4,5]. While the receptors for some viruses are known, we typically lack knowledge on the post-binding function of virus receptors, i.e., how they mechanistically induce virus uptake. Hepatitis C virus (HCV) is a member of the Flaviviridae family and as such a small enveloped positive strand RNA virus. It is the causative agent of chronic hepatitis C, which affects approximately

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