Sites of vulnerability in HCV E1E2 identified by comprehensive functional screening

Abstract

The E1 and E2 envelope proteins of hepatitis C virus (HCV) form a heterodimer that drives virus-host membrane fusion. Here, we analyze the role of each amino acid in E1E2 function, expressing 545 individual alanine mutants of E1E2 in human cells, incorporating them into infectious viral pseudoparticles, and testing them against 37 different monoclonal antibodies (MAbs) to ascertain full-length translation, folding, heterodimer assembly, CD81 binding, viral pseudoparticle incorporation, and infectivity. We propose a model describing the role of each critical residue in E1E2 functionality and use it to examine how MAbs neutralize infection by exploiting functionally critical sites of vulnerability on E1E2. Our results suggest that E1E2 is a surprisingly fragile protein complex where even a single alanine mutation at 92% of positions disrupts its function. The amino-acid-level targets identified are highly conserved and functionally critical and can be exploited for improved therapies and vaccines.

Keywords: CP: Microbiology; E1E2 mutation library; E1E2 structure-function; flaviviridae; hepatitis C virus; hepatitis C virus infectivity.

Figure 1.. Experimental and data analysis processes

Sequential analysis of E1E2 folding and function enabled amino acids involved in each step to be identified. This screening strategy also facilitates the exclusion of mutants that are locally misfolded or that have an expression defect (Davidson and Doranz, 2014; Paes et al., 2009). Residues identified as contributing directly to MAb epitopes were excluded from the corresponding structure-function analysis using said MAbs, as they impart no structural or functional information about E1E2.

Figure 2.. Identification of residues critical for E1E2 folding and assembly

The 545 mutants in the HCV E1E2 mutation library were tested for reactivity to MAbs to identify residues important for folding and assembly. The average MAb reactivity data (in relative fluorescence units [RFUs], normalized to reactivity with WT E1E2, mean of at least two measurements) were plotted for each clone. In all graphs, the horizontal dashed line indicates the threshold for identifying critical residues (≤30% of WT, representing ≥2 SD below WT). Critical residues for each folding step are shown as colored circles and noncritical residues as gray circles. Critical residues are plotted on an E1E2 composite structure (described below) using the same colors as in the graph. (A) Composite structure of the HCV E1E2 heterodimer. A composite structure of the E1E2 heterodimer was constructed. Dark ribbons represent crystal structures (dark blue for E1, red for E2; E1, PDB: 4UOI (Y192-P294) and 2KNU (T314-Q342); red - E2, PDB: 6MEI; R410-G647). Light-colored ribbons represent the predictive model (light blue for E1, pink for E2). Orange spheres represent cysteines, cyan represents glycosylation sequences, and green represents all other critical residues. Structural regions are (1) E1 N-terminal region (NTR) E384-Q409, (2) hypervariable region 1 (HVR1) E384-I411, (3) E1 fusion peptide-like region C272-L286, (4) E1 stem region (G315-A349), (5) E2 stem region (R648-W716), and (6) E2 NTR Y192-D206. (a) CD81 binding loop (BL) T519-D535, (b) β-sandwich R492-A566, and (c) back layer C597-N645. (B) Critical residues for E1 folding. E1 folding was tested using anti-E1 conformational MAbs HEPC-112 (shown here) and HCVE1-C1 (data in Table S3). Critical residues were defined as clones for which either MAb bound with reactivity ≤30% of WT level. Colored circles just above the threshold line correspond to clones showing >30% reactivity to HEPC112 but ≤30% reactivity to HCVE1-C1. (C) Critical residues for E2 folding. E2 folding was tested using 32 anti-E2 conformational anti-AR3 MAbs (shown here) or anti-AR1 MAbs (data in Table S3). Residues were considered critical if the corresponding clone showed mean reactivity ≤30% to the 10 AR1-or 23 AR3-type MAbs. Colored circles above the threshold correspond to clones with >30% reactivity to AR3-type MAbs but ≤30% reactivity to AR1-type MAbs. (D) Critical residues for E1E2 heterodimer assembly. E1E2 assembly was tested using 5 E1E2 heterodimer-specific MAbs. Gray circles below the 30% threshold correspond to clones showing mean reactivity <70% WT levels of reactivity in previous functional steps. (E) Critical residues for binding of the CD81-LEL peptide. The E1E2 mutation library was tested for binding to CD81-LEL. Gray circles below the 30% threshold correspond to clones showing mean reactivity <70% WT levels of reactivity in one or more previous functional steps.

Figure 3.. Identification of E1E2 amino acids critical for infectivity

(A) Infectivity values (mean of 5 experiments) for E1E2 clones with mutations critical for E1 or E2 folding, E1E2 heterodimer assembly, and CD81 binding. (B) Infectivity values (mean of 5 experiments) for clones not critical for functional steps (i.e., excluding clones colored in A). Green circles are critical residues for infectivity (clones with mean infectivity ≤15% and with ≥70% for E1E2 folding, E1E2 heterodimer assembly, and CD81-LEL binding). Gray circles below the threshold line represent clones with mean reactivity <70% of WT in previous functional steps. Important structural regions are marked, including HVR1, VR2, and VR3. (C) Critical residues for infectivity are shown as green spheres on the E1E2 composite structure. Residues in the stem and TM regions of E1 and E2 that lack structures are shown as cartoons. Annotations are as in Figure 1A. (D) Structural representation of E1E2 critical residues for infectivity, characterized by their conservation across the 7 HCV genotypes. Residues fully conserved across all 7 genotypes are shown as dark blue spheres. Residues with conserved properties are shown as green spheres. Non-conserved residues are shown as yellow spheres.

Figure 4.. Model for the roles of individual amino acids in E1E2 function

Proposed functional model describing the mechanism by which individual amino acids contribute to E1E2 function. (A) Critical residues for E1 and E2 folding and heterodimerization are shown on simplified representations of the protein structure. (B) Critical residues for HCV infectivity are assigned to their likely stage in the HCV entry pathway, based on available information of the pathway.

Figure 5.. Epitope locations for bNAbs, NAbs, and non-neutralizing MAbs (non-NAbs)

(A–D) MAb epitope residues are plotted on the composite E1E2 structure, categorized by (A) MAb neutralization status, (B) residues that contribute to MAb epitopes that are conformational, linear, or both, (C) conservation of the epitope residue, or (D) number of MAbs binding to that residue. Numbers and orientation correspond to Figure 1A.

Figure 6.. Co-localization of NAb epitopes with residues critical for E1E2 folding or function

NAb epitope residues that are also critical for functional steps are shown. The stem and TM regions of E1 and E2 are shown as cartoons.

Figure 7.. HCV E1E2 is remarkably fragile

(A) HCV E1E2 residues whose mutation to alanine had little effect on infectivity (>70% of WT levels) are shown as green spheres. Residues in the stem and TM regions of E1 and E2 are shown as cartoons. (B) E1E2 mutation library clones arranged in order of decreasing infectivity. Mutations in 309/545 clones (57%) eliminated infectivity (≤15% of WT level infectivity). Only 44 clones (8%) retained infectivity after mutation (>70% of WT levels).