An intramolecular bond at cluster of differentiation 81 ectodomain is important for hepatitis C virus entry

Abstract

Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver diseases; however, HCV vaccine remains unavailable to date. One main obstacle is the lack of an efficient small animal model. Cluster of differentiation 81 (CD81) is an essential entry coreceptor for HCV species specificity to humans, though the underlying mechanisms are yet to be fully elucidated. We performed structural, biophysical, and virologic studies on HCV nonpermissive CD81s from mice and African green monkeys [mouse cluster of differentiation 81 (mCD81) and African green monkey cluster of differentiation 81 (agmCD81)] and compared with human cluster of differentiation 81 (hCD81). We discovered an intramolecular hydrogen bond (Gln188-Nε2-H: Glu196-Oε2, 2 Å, 124°) within the large extracellular loop (LEL) of mCD81 and a salt bridge (Lys188-Nζ: Asp196-Oδ2, 2.4 Å) within agmCD81-LEL between residues 188 and 196. This structural feature is missing in hCD81. We demonstrated that the introduction of a single 188-196 bond to hCD81 impaired its binding affinity to HCV envelope glycoprotein 2 (HCV E2) and significantly decreased HCV pseudoviral particle (HCVpp) entry efficiency (4.92- to 8.42-fold) and cell culture-grown HCV (HCVcc) infectivity (4.55-fold), despite the availability of Phe186. For HCV nonpermissive CD81s, the introduction of Phe186 by Leu186F substitution alone was insufficient to confer HCV permissiveness. The disruption of the original 188-196 bond and Leu186F substitution were both required for potent binding to HCV E2 HCVpp entry efficiency and HCVcc infectivity. Our structural and biophysical analyses suggest that the intramolecular 188-196 bond restricts the intrinsic conformational dynamics of D-helix of CD81-LEL, which is essential for HCV entry, thus impairs HCV permissiveness. Our findings reveal a novel molecular determinant for HCV entry in addition to the well-characterized Phe186 and provide further guideline for selecting an HCV small animal model.

Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F

“…In both nanobody-bound structures, CD9EC2 adopts an arrangement that is globally similar to previously reported EC2-structures of CD81 (23,24,(40)(41)(42)(43). 4C8 and 4E8 bind nearly identical epitopes that span the C and D loops of CD9EC2 ( Fig.…”

“…The EC2-D loop displays a high amino-acid sequence variability among tetraspanins and has been proposed to mediate homo-and hetero-oligomeric interactions (5,(44)(45)(46). Previously reported structures of CD81 revealed the conformational plasticity of its EC2 D-loop, with both fully helical and partially unfolded arrangements (23,24,(40)(41)(42)(43). Our nanobody-bound CD9EC2 structures also display a minor conformational difference in D-loop with respect to each other (Fig.…”

“…Tetraspanin-mediated interactions that drive the clustering of single-pass membrane receptors into TEMs in plasma membranes have remained obscure. Structural insights into tetraspanins have so far encompassed the large extracellular EC2 loop of CD81 (23,(40)(41)(42)(43), full-length CD81 (24) and, more recently, full-length CD9 and a low-resolution cryo-EM map of CD9 in complex with EWI-2 (25).…”

Oligomerization of the Tetraspanin CD81 via the Flexibility of Its δ -Loop

“…Disulfide bridges form loops comprising the g and g/d sections, respectively. Although a, b, and ε show little conformational variation, the structures differ in their VD (g and d), which is poorly resolved, suggesting conformational fluctuations even in the packed crystal lattices (20)(21)(22). In addition, a solution NMR study of the LEL showed pronounced conformational flexibility in this region (23).…”

“…To date, conformational variability has been observed only experimentally in the isolated LEL (20)(21)(22)(23), without membrane anchoring and other putatively stabilizing regions such as the SEL. This raises the question as to whether the observed flexibility is maintained in the membraneembedded, full-length protein.…”