CD81 Controls Beige Fat Progenitor Cell Growth and Energy Balance via FAK Signaling.

Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however, the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here, we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins, including PDGFRα, Sca1, and CD81. Notably, CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with αV/ß1 and αV/ß5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly, CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis.

Crystal Structure of a Full-Length Human Tetraspanin Reveals a Cholesterol-Binding Pocket.

Tetraspanins comprise a diverse family of four-pass transmembrane proteins that play critical roles in the immune, reproductive, genitourinary, and auditory systems. Despite their pervasive roles in human physiology, little is known about the structure of tetraspanins or the molecular mechanisms underlying their various functions. Here, we report the crystal structure of human CD81, a full-length tetraspanin. The transmembrane segments of CD81 pack as two largely separated pairs of helices, capped by the large extracellular loop (EC2) at the outer membrane leaflet. The two pairs of helices converge at the inner leaflet to create an intramembrane pocket with additional electron density corresponding to a bound cholesterol molecule within the cavity. Molecular dynamics simulations identify an additional conformation in which EC2 separates substantially from the transmembrane domain. Cholesterol binding appears to modulate CD81 activity in cells, suggesting a potential mechanism for regulation of tetraspanin function.

Structural insights into hepatitis C virus receptor binding and entry.

Hepatitis C virus (HCV) infection is a causal agent of chronic liver disease, cirrhosis and hepatocellular carcinoma in humans, and afflicts more than 70 million people worldwide. The HCV envelope glycoproteins E1 and E2 are responsible for the binding of the virus to the host cell, but the exact entry process remains undetermined1. The majority of broadly neutralizing antibodies block interaction between HCV E2 and the large extracellular loop (LEL) of the cellular receptor CD81 (CD81-LEL)2. Here we show that low pH enhances the binding of CD81-LEL to E2, and we determine the crystal structure of E2 in complex with an antigen-binding fragment (2A12) and CD81-LEL (E2-2A12-CD81-LEL); E2 in complex with 2A12 (E2-2A12); and CD81-LEL alone. After binding CD81, residues 418-422 in E2 are displaced, which allows for the extension of an internal loop consisting of residues 520-539. Docking of the E2-CD81-LEL complex onto a membrane-embedded, full-length CD81 places the residues Tyr529 and Trp531 of E2 proximal to the membrane. Liposome flotation assays show that low pH and CD81-LEL increase the interaction of E2 with membranes, whereas structure-based mutants of Tyr529, Trp531 and Ile422 in the amino terminus of E2 abolish membrane binding. These data support a model in which acidification and receptor binding result in a conformational change in E2 in preparation for membrane fusion.

The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity.

Hepatitis C virus (HCV) exploits the four entry factors CD81, scavenger receptor class B type I (SR-BI, also known as SCARB1), occludin, and claudin-1 as well as the co-factor epidermal growth factor receptor (EGFR) to infect human hepatocytes. Here, we report that the disintegrin and matrix metalloproteinase 10 (ADAM10) associates with CD81, SR-BI, and EGFR and acts as HCV host factor. Pharmacological inhibition, siRNA-mediated silencing and genetic ablation of ADAM10 reduced HCV infection. ADAM10 was dispensable for HCV replication but supported HCV entry and cell-to-cell spread. Substrates of the ADAM10 sheddase including epidermal growth factor (EGF) and E-cadherin, which activate EGFR family members, rescued HCV infection of ADAM10 knockout cells. ADAM10 did not influence infection with other enveloped RNA viruses such as alphaviruses and a common cold coronavirus. Collectively, our study reveals a critical role for the sheddase ADAM10 as a HCV host factor, contributing to EGFR family member transactivation and as a consequence to HCV uptake.

Transmembrane proteins tetraspanin 4 and CD9 sense membrane curvature.

Multiple membrane-shaping and remodeling processes are associated with tetraspanin proteins by yet unknown mechanisms. Tetraspanins constitute a family of proteins with four transmembrane domains present in every cell type. Prominent examples are tetraspanin4 and CD9, which are required for the fundamental cellular processes of migrasome formation and fertilization, respectively. These proteins are enriched in curved membrane structures, such as cellular retraction fibers and oocyte microvilli. The factors driving this enrichment are, however, unknown. Here, we revealed that tetraspanin4 and CD9 are curvature sensors with a preference for positive membrane curvature. To this end, we used a biomimetic system emulating membranes of cell retraction fibers and oocyte microvilli by membrane tubes pulled out of giant plasma membrane vesicles with controllable membrane tension and curvature. We developed a simple thermodynamic model for the partitioning of curvature sensors between flat and tubular membranes, which allowed us to estimate the individual intrinsic curvatures of the two proteins. Overall, our findings illuminate the process of migrasome formation and oocyte microvilli shaping and provide insight into the role of tetraspanin proteins in membrane remodeling processes.

CD81 costimulation skews CAR transduction toward naive T cells.

Adoptive cellular therapy using chimeric antigen receptors (CARs) has revolutionized our treatment of relapsed B cell malignancies and is currently being integrated into standard therapy. The impact of selecting specific T cell subsets for CAR transduction remains under investigation. Previous studies demonstrated that effector T cells derived from naive, rather than central memory T cells mediate more potent antitumor effects. Here, we investigate a method to skew CAR transduction toward naive T cells without physical cell sorting. Viral-mediated CAR transduction requires ex vivo T cell activation, traditionally achieved using antibody-mediated strategies. CD81 is a T cell costimulatory molecule that when combined with CD3 and CD28 enhances naive T cell activation. We interrogate the effect of CD81 costimulation on resultant CAR transduction. We identify that upon CD81-mediated activation, naive T cells lose their identifying surface phenotype and switch to a memory phenotype. By prelabeling naive T cells and tracking them through T cell activation and CAR transduction, we document that CD81 costimulation enhanced naive T cell activation and resultantly generated a CAR T cell product enriched with naive-derived CAR T cells.

Open conformation of tetraspanins shapes interaction partner networks on cell membranes.

Tetraspanins, including CD53 and CD81, regulate a multitude of cellular processes through organizing an interaction network on cell membranes. Here, we report the crystal structure of CD53 in an open conformation poised for partner interaction. The large extracellular domain (EC2) of CD53 protrudes away from the membrane surface and exposes a variable region, which is identified by hydrogen-deuterium exchange as the common interface for CD53 and CD81 to bind partners. The EC2 orientation in CD53 is supported by an extracellular loop (EC1). At the closed conformation of CD81, however, EC2 disengages from EC1 and rotates toward the membrane, thereby preventing partner interaction. Structural simulation shows that EC1-EC2 interaction also supports the open conformation of CD81. Disrupting this interaction in CD81 impairs the accurate glycosylation of its CD19 partner, the target for leukemia immunotherapies. Moreover, EC1 mutations in CD53 prevent the chemotaxis of pre-B cells toward a chemokine that supports B-cell trafficking and homing within the bone marrow, a major CD53 function identified here. Overall, an open conformation is required for tetraspanin-partner interactions to support myriad cellular processes.

Fluorescence Sensing of Eclampsia Biomarkers via the Immunosorbent Atom Transfer Radical Polymerization Assay.

Accurate and quantitative detection of pre-eclampsia markers is crucial in reducing pregnancy mortality rates. This study introduces a novel approach utilizing a fluorescent biosensor by the immunosorbent atom transfer radical polymerization (immuno-ATRP) assay to detect the pre-eclampsia protein marker CD81. The critical step used in this sensor is the novel signal amplification strategy of fluorescein polymerization mediated by ferritin-enhanced controlled radical polymerization, which combines with a traditional enzyme-linked immunosorbent assay (ELISA) to further reduce the detection limit of the CD81 protein concentration. The fluorescence intensity was linear versus logarithmic CD81 protein concentration from 0.1 to 10,000 pg mL-1, and the detection limit was 0.067 pg mL-1. Surprisingly, in 30% normal human serum (NHS), the sensor can also detect target protein over 0.1-10,000 pg mL-1, with 0.083 pg mL-1 for the detection limit. Moreover, the proposed biosensor is designed to be cost-effective, making it accessible, particularly in resource-limited settings where expensive detection techniques may not be available. The affordability of this method enables widespread screening and monitoring of preeclampsia, ultimately benefiting many pregnant women by improving their healthcare outcomes. In short, developing of a low-cost and susceptible direct detection method for preeclampsia protein markers, such as CD81, through the use of the immuno-ATRP assay, has significant implications for reducing pregnancy mortality. This method holds promise for early detection, precise treatment, and improved management of preeclampsia, thereby contributing to better maternal and fetal health.

Characterization and verification of CD81 as a potential target in lung squamous cell carcinoma.

CD81 is a cell surface transmembrane protein of the tetraspanin family, which critically regulates signal transduction and immune response. Growing evidence has shown that CD81 plays important roles in tumorigenesis and influences immunotherapy response. Here, combining bio-informatics and functional analysis, we find that CD81 is a risk factor in lung squamous cell carcinoma (LUSC), whereas a protective factor in lung adenocarcinoma. In LUSC with high expression of CD81, the autophagy and JAK-STAT signaling pathway are activated. Meanwhile, the expression level of CD81 is negatively correlated with tumor mutational load (TMB), microsatellite instability (MSI), and neoantigen (NEO). Furthermore, patients with LUSC and high expression of CD81 do not respond to immunotherapy drugs, but can respond to chemotherapy drugs. Importantly, depletion of CD81 suppresses the proliferation of LUSC cell, and enhances the sensitivity to cisplatin. Our findings suggest that CD81 represents a potential target for cisplatin-based chemotherapy in patients with LUSC.

Renal CD81 interacts with sodium potassium 2 chloride cotransporter and sodium chloride cotransporter in rats with lipopolysaccharide-induced preeclampsia.

The kidney regulates blood pressure through salt/water reabsorption affected by tubular sodium transporters. Expanding our prior research on placental cluster of differentiation 81 (CD81), this study explores the interaction of renal CD81 with sodium transporters in preeclampsia (PE). Effects of renal CD81 with sodium transporters were determined in lipopolysaccharide (LPS)-induced PE rats and immortalized mouse renal distal convoluted tubule cells. Urinary exosomal CD81, sodium potassium 2 chloride cotransporter (NKCC2), and sodium chloride cotransporter (NCC) were measured in PE patients. LPS-PE rats had hypertension from gestational days (GD) 6 to 18 and proteinuria from GD9 to GD18. Urinary CD81 in both groups tented to rise during pregnancy. Renal CD81, not sodium transporters, was higher in LPS-PE than controls on GD14. On GD18, LPS-PE rats exhibited higher CD81 in kidneys and urine exosomes, higher renal total and phosphorylated renal NKCC2 and NCC with elevated mRNAs, and lower ubiquitinated NCC than controls. CD81 was co-immunoprecipitated with NKCC2 or NCC in kidney homogenates and co-immunostained with NKCC2 or NCC in apical membranes of renal tubules. In plasma membrane fractions, LPS-PE rats had greater amounts of CD81, NKCC2, and NCC than controls with enhanced co-immunoprecipitations of CD81 with NKCC2 or NCC. In renal distal convoluted tubule cells, silencing CD81 with siRNA inhibited NCC and prevented LPS-induced NCC elevation. Further, PE patients had higher CD81 in original urines, urine exosomes and higher NKCC2 and NCC in urine exosomes than controls. Thus, the upregulation of renal CD81 on NKCC2 and NCC may contribute to the sustained hypertension observed in LPS-PE model. Urine CD81 with NKCC2 and NCC may be used as biomarkers for PE.

A conserved epitope III on hepatitis C virus E2 protein has alternate conformations facilitating cell binding or virus neutralization.

Epitope III, a highly conserved amino acid motif of 524APTYSW529 on the hepatitis C virus (HCV) E2 glycoprotein, resides in the critical loop that binds to the host receptor CD81, thus making it one of the most important antibody targets for blocking HCV infections. Here, we have determined the X-ray crystal structure of epitope III at a 2.0-Å resolution when it was captured by a site-specific neutralizing antibody, monoclonal antibody 1H8 (mAb1H8). The snapshot of this complex revealed that epitope III has a relatively rigid structure when confined in the binding grooves of mAb1H8, which confers the residue specificity at both ends of the epitope. Such a high shape complementarity is reminiscent of the "lock and key" mode of action, which is reinforced by the incompatibility of an antibody binding with an epitope bearing specific mutations. By subtly positioning the side chains on the three residues of Tyr527, Ser528, and Trp529 while preserving the spatial rigidity of the rest, epitope III in this cocrystal complex adopts a unique conformation that is different from previously described E2 structures. With further analyses of molecular docking and phage display-based peptide interactions, we recognized that it is the arrangements of two separate sets of residues within epitope III that create these discrete conformations for the epitope to interact selectively with either mAb1H8 or CD81. These observations thus raise the possibility that local epitope III conformational dynamics, in conjunction with sequence variations, may act as a regulatory mechanism to coordinate "mAb1H8-like" antibody-mediated immune defenses with CD81-initiated HCV infections.

The iron chaperone and nucleic acid-binding activities of poly(rC)-binding protein 1 are separable and independently essential.

Poly(rC)-binding protein (PCBP1) is a multifunctional adaptor protein that can coordinate single-stranded nucleic acids and iron-glutathione complexes, altering the processing and transfer of these ligands through interactions with other proteins. Multiple phenotypes are ascribed to cells lacking PCBP1, but the relative contribution of RNA, DNA, or iron chaperone activity is not consistently clear. Here, we report the identification of amino acid residues required for iron coordination on each structural domain of PCBP1 and confirm the requirement of iron coordination for binding target proteins BolA2 and ferritin. We further construct PCBP1 variants that lack either nucleic acid- or iron-binding activity and examine their functions in human cells and mouse tissues depleted of endogenous PCBP1. We find that these activities are separable and independently confer essential functions. While iron chaperone activity controls cell cycle progression and suppression of DNA damage, RNA/DNA-binding activity maintains cell viability in both cultured cell and mouse models. The coevolution of RNA/DNA binding and iron chaperone activities on a single protein may prove advantageous for nucleic acid processing that depends on enzymes with iron cofactors.

Targeting the tetraspanin CD81 reduces cancer invasion and metastasis.

Tetraspanins are an evolutionary conserved family of proteins involved in multiple aspects of cell physiology, including proliferation, migration and invasion, protein trafficking, and signal transduction; yet their detailed mechanism of action is unknown. Tetraspanins have no known natural ligands, but their engagement by antibodies has begun to reveal their role in cell biology. Studies of tetraspanin knockout mice and of germline mutations in humans have highlighted their role under normal and pathological conditions. Previously, we have shown that mice deficient in the tetraspanin CD81 developed fewer breast cancer metastases compared to their wild-type (WT) counterparts. Here, we show that a unique anti-human CD81 antibody (5A6) effectively halts invasion of triple-negative breast cancer (TNBC) cell lines. We demonstrate that 5A6 induces CD81 clustering at the cell membrane and we implicate JAM-A protein in the ability of this antibody to inhibit tumor cell invasion and migration. Furthermore, in a series of in vivo studies we demonstrate that this antibody inhibits metastases in xenograft models, as well as in syngeneic mice bearing a mouse tumor into which we knocked in the human CD81 epitope recognized by the 5A6 antibody.

Synthetic Peptides with Genetic-Codon-Tailored Affinity for Assembling Tetraspanin CD81 at Cell Interfaces and Inhibiting Cancer Metastasis.

Probing biomolecular interactions at cellular interfaces is crucial for understanding and interfering with life processes. Although affinity binders with site specificity for membrane proteins are unparalleled molecular tools, a high demand remains for novel multi-functional ligands. In this study, a synthetic peptide (APQQ) with tight and specific binding to the untargeted extracellular loop of CD81 evolved from a genetically encoded peptide pool. With tailored affinity, APQQ flexibly accesses, site-specifically binds, and forms a complex with CD81, enabling in-situ tracking of the dynamics and activity of this protein in living cells, which has rarely been explored because of the lack of ligands. Furthermore, APQQ triggers the relocalization of CD81 from diffuse to densely clustered at cell junctions and modulates the interplay of membrane proteins at cellular interfaces. Motivated by these, efficient suppression of cancer cell migration, and inhibition of breast cancer metastasis were achieved in vivo.

Modified E2 Glycoprotein of Hepatitis C Virus Enhances Proinflammatory Cytokines and Protective Immune Response.

Hepatitis C virus (HCV) is characterized by a high number of chronic cases owing to an impairment of innate and adaptive immune responses. CD81 on the cell surface facilitates HCV entry by interacting with the E2 envelope glycoprotein. In addition, CD81/E2 binding on immunity-related cells may also influence host response outcome to HCV infection. Here, we performed site-specific amino acid substitution in the front layer of E2 sequence to reduce CD81 binding and evaluate the potential of the resulting immunogen as an HCV vaccine candidate. The modified sE2 protein (F442NYT), unlike unmodified sE2, exhibited a significant reduction in CD81 binding, induced higher levels of proinflammatory cytokines, repressed anti-inflammatory response in primary monocyte-derived macrophages as antigen-presenting cells, and stimulated CD4+ T cell proliferation. Immunization of BALB/c mice with an E1/sE2F442NYT nucleoside-modified mRNA-lipid nanoparticle (mRNA-LNP) vaccine resulted in improved IgG1-to-IgG2a isotype switching, an increase in neutralizing antibodies against HCV pseudotype virus, a B and T cell proliferative response to antigens, and improved protection against infection with a surrogate recombinant vaccinia virus-expressing HCV E1-E2-NS2aa134-966 challenge model compared to E1/unmodified sE2 mRNA-LNP vaccine. Further investigation of the modified E2 antigen may provide helpful information for HCV vaccine development. IMPORTANCE Hepatitis C virus (HCV) E2-CD81 binding dampens protective immune response. We have identified that an alteration of amino acids in the front layer of soluble E2 (sE2) disrupts CD81 interaction and alters the cytokine response. Immunization with modified sE2F442NYT (includes an added potential N-linked glycosylation site and reduces CD81 binding activity)-mRNA-LNP candidate vaccine generates improved proinflammatory response and protective efficacy against a surrogate HCV vaccinia challenge model in mice. The results clearly suggested that HCV E2 exhibits immunoregulatory activity that inhibits induction of robust protective immune responses. Selection of engineered E2 antigen in an mRNA-LNP platform amenable to nucleic acid sequence alterations may open a novel approach for multigenotype HCV vaccine development.

Natural flavonoids effectively block the CD81 receptor of hepatocytes and inhibit HCV infection: a computational drug development approach.

Hepatitis C virus (HCV) infection is a major public health concern, and almost two million people are infected per year globally. This is occurred by the diverse spectrum of viral genotypes, which are directly associated with chronic liver disease (fibrosis, and cirrhosis). Indeed, the viral genome encodes three principal proteins as sequentially core, E1, and E2. Both E1 and E2 proteins play a crucial role in the attachment of the host system, but E2 plays a more fundamental role in attachment. The researchers have found the "E2-CD81 complex" at the entry site, and therefore, CD81 is the key receptor for HCV entrance in both humans, and chimpanzees. So, the researchers are trying to block the host CD81 receptor and halt the virus entry within the cellular system via plant-derived compounds. Perhaps that is why the current research protocol is designed to perform an in silico analysis of the flavonoid compounds for targeting the tetraspanin CD81 receptor of hepatocytes. To find out the best flavonoid compounds from our library, web-based tools (Swiss ADME, pKCSM), as well as computerized tools like the PyRx, PyMOL, BIOVIA Discovery Studio Visualizer, Ligplot+ V2.2, and YASARA were employed. For molecular docking studies, the flavonoid compounds docked with the targeted CD81 protein, and herein, the best-outperformed compounds are Taxifolin, Myricetin, Puerarin, Quercetin, and (-)-Epicatechin, and outstanding binding affinities are sequentially - 7.5, - 7.9, - 8.2, - 8.4, and - 8.5 kcal/mol, respectively. These compounds have possessed more interactions with the targeted protein. To validate the post docking data, we analyzed both 100 ns molecular dynamic simulation, and MM-PBSA via the YASARA simulator, and finally finds the more significant outcomes. It is concluded that in the future, these compounds may become one of the most important alternative antiviral agents in the fight against HCV infection. It is suggested that further in vivo, and in vitro research studies should be done to support the conclusions of this in silico research workflow.

Transcript CD81-215 may be a long noncoding RNA of stromal origin with tumor-promoting role in colon cancer.

The role of tetraspanin CD81 in malignant transformation is best studied in colorectal cancer, and it appears that other transcripts beside the fully coding mRNA may also be dysregulated in malignant cells. Recent data from a comprehensive pan-cancer transcriptome analysis demonstrated differential activity of two alternative CD81 gene promoters in malignant versus nonmalignant gut mucosa. The promoter active in gut mucosa gives rise to transcripts CD81-203 and CD81-213, while the promoter active in colon and rectal cancer gives rise to transcripts CD81-205 and CD81-215. Our study aimed to explore the biomarker potential of the transcripts from the alternative CD81 gene promoters in colon cancer, as well as to investigate their structure and potential function using in silico tools. The analysis of the transcripts' expression in several colon cell lines cultivated in 2D and 3D and a set of colon cancer and healthy gut mucosa samples by qPCR and RNA sequencing suggested their low expression and stromal origin. Expression patterns in tumor and nontumor tissue along with in silico data suppose that the transcript CD81-215 may be a noncoding RNA of stromal origin with possible involvement in signaling related to malignant transformation.

Tetraspanin heterogeneity of small extracellular vesicles in human biofluids and brain tissue.

Extracellular vesicles (EVs) are particles released from most cell types delimited by a lipid bilayer. Small EVs (sEVs) are nanosized (<200 nm) and include exosomes. Brain-derived sEVs may provide a source for new biomarkers of brain status. CD9, CD63, and CD81 are major members of the tetraspanin family frequently used as sEV markers. However, according to a recent report, tetraspanins were not equally expressed in all sEVs, but rather show heterogeneity that reflects the expression levels in their secretory cells. We therefore investigated tetraspanin heterogeneity of sEVs in biofluids commonly used for clinical laboratory tests, and those in the brain. Expression levels and distributions of CD9, CD63 and CD81 on sEVs were determined in serum, plasma, and cerebrospinal fluid (CSF) samples collected from each healthy donor, and in post-mortem brain tissue samples. We found heterogeneous mixes of sEVs with various tetraspanin combinations among sEVs, and the predominant types and heterogeneous patterns of tetraspanins were specific to sample type. Hierarchical clustering revealed that brain sEVs were similar to those in the CSF, but different from those in peripheral blood. Our findings both provide basic information and contribute to the development of biomarkers for neurological and psychiatric disorders.

The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling.

A key role is emerging for the cytoskeleton in coordinating receptor signaling, although the underlying molecular requirements remain unclear. Here we show that cytoskeleton disruption triggered signaling requiring not only the B cell receptor (BCR), but also the coreceptor CD19 and tetraspanin CD81, thus providing a mechanism for signal amplification upon surface-bound antigen stimulation. By using superresolution microscopy, we demonstrated that endogenous IgM, IgD, and CD19 exhibited distinct nanoscale organization within the plasma membrane of primary B cells. Upon stimulation, we detect a local convergence of receptors, although their global organization was not dramatically altered. Thus, we postulate that cytoskeleton reorganization releases BCR nanoclusters, which can interact with CD19 held in place by the tetraspanin network. These results not only suggest that receptor compartmentalization regulates antigen-induced activation but also imply a potential role for CD19 in mediating ligand-independent "tonic" BCR signaling necessary for B cell survival.

Cholesterol sensing by CD81 is important for hepatitis C virus entry.

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in both health and disease.