Cholesterol sensing by CD81 is important for hepatitis C virus entry

Abstract

a These authors contributed equally to this work CD81 is a multifunctional protein that plays a central role in both physiological and pathological processes. Recent structural analysis of CD81 indicates that it adopts a compact fold with its large extracellular loop sitting close to the plasma membrane; however, there is evidence that CD81 can undergo a conformational switch where the extracellular loop flips into an extended open conformation. We investigated the relevance of CD81 conformational switching to its function as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis was used to generate CD81 variants with altered conformational switching. Each variant maintained proper protein folding and expression, and retained a basic function: the ability to chaperone CD19 to the cell surface. We then used multiple systems to evaluate HCV receptor activity; in these experiments 'open' CD81 variants exhibited consistently poor receptor function, whereas a 'closed' CD81 displayed enhanced ability to support HCV entry. We sought to corroborate these findings by exploring CD81 structural flexibility using molecular dynamics simulations. These experiments were in excellent agreement with our in vitro work, with 'open' CD81 variants having a propensity to undergo conformational switching. Taken together, we provide evidence for a conformational switch in CD81 that regulates its HCV receptor activity. This work furthers our understanding of the molecular mechanism of HCV entry and is relevant to CD81's other functions in health and disease.

A Multiscale study of phosphorylcholine driven cellular phenotypic targeting

“…Despite the interaction of the intramembrane helices with the lipids, the extensive allosteric rearrangements observed in the presence of PMPC are not present. The TM4-EC2 angle explores two conformations, 130 ° and 145 ° , though the second has a lower population ( Figure S5b ), which is compatible with the previously investigated closed structured 18, 32 . While in the presence of PMPC, the observed values for this angle are above 155 ° .…”

“…Final membrane curvature (e). conformations, 130 ∘ and 145 ∘ , though the second has a lower population (Figure S5b), which is compatible with the previously investigated closed structured 18,32 . While in the presence of PMPC, the observed values for this angle are above 155 ∘ .…”

“…We compute the free energy landscape from the 1.5-microsecond trajectory ( Figure 3a ); the barrier for binding/unbinding is very low ∼ 4 kcal/mol. Interestingly, the unbinding of PMPC from the EC2 induces an allosteric movement of the transmembrane helix 4 (TM4) ( Figure 3b , d ), which has only been described recently 32 in the absence of cholesterol in the binding site. The observed opening reported enabling the export of cholesterol 18 involves the detachment of helix B and involves the recruitment of a partner, CD19 18, 33 .…”

Single-nucleotide variants in human CD81 influence hepatitis C virus infection of hepatoma cells

“…Further studies, including pseudoparticles expressing glycoproteins from other HCV genotypes, will help to determine if hCD81 variants affect HCV binding in a genotypedependent manner. Grove et al showed an excellent agreement between their J6 HCVpp and J6/JFH HCVcc assays when comparing single-and double-amino acid exchange mutants, which affect the conformation of CD81 [43]. In our HCVpp assay, the entry step was not affected by the different hCD81 variants, which is in contrast to the results from our HCVcc assay.…”

“…Banse et al, demonstrated using chimeric CD81 that the backbone domains play a role during the post-binding steps, being critical for productive HCV entry [26]. The specific residues in the backbone that are important for HCV uptake remain poorly understood although a cholesterol-coordinating residue (E219) seems important [26,43]. In this study, we aimed to investigate the effect of five non-synonymous SNVs, specifically R36L, A54V, V211M, A213T and M220I, on HCV infectivity.…”