Region of hepatitis C virus E2 required for membrane binding

Here, we describe regions of HCV E2 that are required to regulate the CD81-binding loop for CD81 binding and membrane binding, yielding unique mechanistic insights into HCV entry. The CD81-binding loop of E2 is involved in membrane binding, but this loop is necessary but deficient for membrane binding. The N-terminal region flanking the unbound CD81-binding loop is also required for membrane binding. This N-terminal region containing AS412 and the front layer forms the CD81-binding site, the primary site of antibody-mediated neutralization affecting Ile422-mediated membrane binding. So I would guess that this mechanism is fine-tuned rather than just the sum of its parts.

Here we show the structure of the E2core+stem fragment bound to non-neutralizing Fab 2A12 determined at 2.5 Å resolution. The structures of the E2core+stem, E2core, eE2, and ΔHVR1 eE2/tCD81-LEL complexes offer unique mechanistic insights during the early stages of infection and model the changes that occur during receptor binding and endosomal acidification. produce (Fig. 6). The E2core region contains a folded globular domain. AS412 and the front layer are key determinants for proper alignment of the CD81 binding loop and CD81 interactions. In the absence of CD81, Ile422 in the anterior layer holds her CD81-binding loop in a retracted position. Both E2core and E2core+ stems failed to bind hCD81 in vitro, indicating the importance of her N-terminal region (384–459) in receptor binding. HCV, like avian sarcoma leukemia virus (ASLV), uses a hybrid method of membrane attachment that requires cellular receptors and low pH.^{twenty four}Under these conditions, E2 undergoes two conformational changes. The front layer interacts with her CD81 and folds the AS412 region around her CD81, with an internal loop (CD81-binding loop) extending away from the core (Fig. 7a). Her two invariant hydrophobic residues (Tyr529 and Trp531) at the tip of the CD81-binding loop extend towards the host and insert into the membrane. Deletion of the E2 front layer abolished the CD81 interaction and freed the CD81-binding loop (Fig. 7b). However, the E2core+ stem is incapable of membrane binding, suggesting that the CD81-binding loop alone is insufficient for membrane binding and requires a front layer for proper orientation (Fig. 6). Together, these four structures provide a mechanistic picture of the flexibility of the CD81-binding loop, the regulatory function of the N-terminal region, and a detailed understanding of the viral entry process.

a When CD81 is present and the pH is low, the frontal layer wraps around CD81 and the CD81-binding loop extends toward the host membrane. b Deletion of the prelayer of E2 releases the CD81-binding loop, making it incapable of binding CD81 even at low pH and abolishing its ability to interact with the membrane.

All enveloped viruses interact with and fuse with the cell membrane to deliver the viral genome into the cell. The E2/tCD81-LEL complex is modeled on the full-length structure of CD81 (PDB ID: 5TCX^{twenty five}) Tyr529 and Trp531 are located at the tip of the CD81 binding loop that extends toward the bilayer (Fig. Five).These residues are invariant across all major genotypes and are required for CD81 binding and viral entry^26,27Superposition of the E2core+stem structure to the eE2/full-length CD81 complex places Tyr529 and Trp531 at distances of more than 35 Å and 31 Å from the idealized phosphatidylcarbonyl layer, respectively, and away from the outer leaflet of the membrane. (Fig. Five). The N-terminal region of E2 may help direct the CD81-binding loop to the membrane. Figure model. Five Use an idealized bilayer. However, the flexibility of the CD81-binding loop may be compromised by the curvature of the host membrane within the endosome and the presence of other factors (i.e., E1, OCLN, and/or CLDN) that require loop flexibility for membrane insertion. may be advantageous during potential HCV infection. .

Viral membrane fusion proteins are divided into three classes (I, II, and III) based on structural criteria with four mechanisms of fusion triggering.^{twenty four}Currently, HCV fusion proteins have not been conclusively identified and classified. The HCV E1 glycoprotein is proposed to be the fusion subunit of the E1E2 heterodimer because it contains a putative fusion peptide^28,29However, the CD81-binding loop of E2 has membrane-binding properties and E2 contains an IgG-like beta-sandwich fold similar to type II fusion proteins.^{twenty four,30}A more complex taxonomy, E1 and E2, are required for efficient virus/host fusion and are functions that do not belong to any class. In most cases, the CD81-binding loop is embedded in the target membrane and aids in the fusion process. All fusogenic viral proteins are trimers, although TM helices and E1 may influence oligomerization, but no evidence for E2 trimers has been found. The cryoEM structure of the E1E2 glycoprotein in complex with three neutralizing Fabs was recently published^{twenty one}, which contains a single copy of the E1E2 heterodimer. Similarly, inclusion of the stem did not produce trimers in the eE2+ stem structure, which may be due to the 2A12 Fab interacting at the carboxyl terminus of the E2 protein. Further studies are needed to gain insight into the HCV fusion process.

SR-BI and CD81 have been shown to directly interact with E2 after initial attachment to susceptible cells. SR-BI binds through her HVR1 at the N-terminus, whereas CD81 binds to the E2 protein through a discontinuous region containing AS412, AS432, the CD81-binding loop, and the back layer.^6,16However, the molecular mechanism of the interaction between SR-BI and E2 remains unclear. CD81 with a low pH trigger may be sufficient to induce conformational changes. SR-BI may be effective in inducing conformational changes in an HCV isolate-dependent manner.^31,32.

Using a secondary receptor, CD81, for fusion is advantageous for the virus, consistent with previous observations.¹³Although E2 and CD81-LEL form a complex at neutral pH and may form a complex at the cell surface, the stability and homogeneity of the complex increases at low pH and endosomal acidification. matches tCD81-LEL adopts an open conformation with the D-helix unwound, whereas human CD81 is most frequently observed in the closed conformation, and intermediate and open conformations have also been described.³³The E2/tCD81-LEL structure is only a snapshot of the complex after acidification and before the fusion-induced conformational change. Similar to the description of other viral fusion proteins, additional structural and mechanistic studies are required to adequately describe the fusion mechanism of HCV.

The E2 HVRs contain a significant proportion of HCV sequence variants. These regions are one mode of neutralizing antibody evasion. In patients, HVR1 is rapidly altered by antibody-induced antigenic drift. Broadly neutralizing antibodies are directed against conserved conformational epitopes of HCV E2 that compete for the CD81 binding site. The CD81 binding loop is surface exposed and protected from antibody-mediated neutralization by conformational obfuscation. Identification of the CD81-binding loop in membrane insertion provides a target for HCV vaccine design.Relatively high conservation of CD81-binding loop sequences among different HCV genotypes and observation of membrane-binding loops from other viruses, including HIV³⁴Ebora^35,36,37impact³⁸and dengue³⁹ is a major target for vaccines, and neutralizing antibodies suggest that the CD81-binding loop of HCV may be a potential therapeutic target. Information obtained from these atomic crystal structures may aid in the development of preventive HCV vaccine designs.