Crystal structure of glycoprotein E2 from bovine viral diarrhea virus

Abstract

Pestiviruses, including bovine viral diarrhea virus, are important animal pathogens and are closely related to hepatitis C virus, which remains a major global health threat. They have an outer lipid envelope bearing two glycoproteins, E1 and E2, required for cell entry. They deliver their genome into the host cell cytoplasm by fusion of their envelope with a cellular membrane. The crystal structure of bovine viral diarrhea virus E2 reveals a unique protein architecture consisting of two Ig-like domains followed by an elongated β-stranded domain with a new fold. E2 forms end-to-end homodimers with a conserved C-terminal motif rich in aromatic residues at the contact. A disulfide bond across the interface explains the acid resistance of pestiviruses and their requirement for a redox activation step to initiate fusion. From the structure of E2, we propose alternative possible membrane fusion mechanisms. We expect the pestivirus fusion apparatus to be conserved in hepatitis C virus.

Fig. 1.

Topology and overall protein fold of BVDV E2. (A) The three-domain topology of E2. Domain I is in red, domain II is in yellow, and domain III is in shades of blue: light blue for module IIIa, medium blue for module IIIb, and dark blue for module IIIc. Residue numbers follow BVDV polyprotein numbering. The transmembrane domain (gray) is missing in the structure. (B and C) E2 has a unique architecture consisting of two Ig-like domains (I and II) followed by an elongated β-stranded domain with a new fold (III). The only histidine conserved in pestivirus E2 is His762 (magenta). A host cell binding sequence forms a protruding β hairpin and is a candidate for CD46 binding. Glycans (cyan) are linked to N809, N878, N922, and N990. Disulfide bonds are in green. See also Fig. S1.

Fig. 2.

BVDV E2 forms covalently linked dimers. (A) The structure of the BVDV E2 dimer, colored as in Fig. 1. The two subunits in the end-to-end dimer are related by a dyad axis, except for domain I, whose orientation relative to domain II differs by 42° in the two subunits. The viral lipid envelope is represented by a gray rectangle (not to scale). (B) The dimer interface consists predominantly of a large cluster of aromatic residues in module IIIc. The aromatic side chains are tightly clustered in a planar array at the membrane-proximal end of E2. (C and D) Disulfide bond links each monomer across the dyad axis via Cys987. The aromatic side chains interact mostly via π stacking and hydrophobic interactions. Hydrogen bonds across the dimer interface are shown as black dashed lines.

Fig. 3.

Potential domain swap at the conserved dimer interface. A loop in module IIIc (residues 979–984) is disordered in the structure of E2 (dashed lines). Because the ends connecting to the loop are close to the dyad, residues on the C-terminal end of the loop (985–1023) may cross the dyad into the dimer partner. (A) The nonswapped structure, as deposited. (B) The domain-swapped structure, which doubles the buried surface area within the dimer interface. (C) Module IIIc is the most conserved region of E2 in pestiviruses. The sequence of module IIIc has similarity to the C-terminal region of HCV E2 proteins. Residue numbers (Left) follow polyprotein numbering. The secondary structure elements of BVDV E2 (blue arrows) and predicted secondary structure elements of HCV2b E2 (green arrows, cylinders) are shown.

Fig. 4.

Mapping of antigenic regions. (A) Location of the four antigenic domains (A–D) of CSFV E2 in the structure of BVDV E2 viewed in the same orientation as in Fig. 1C in space-filling representation. Antigenic domains B (green) and C (blue) are in domain I. Domains A (pink and salmon) and D (salmon) map to domain II. Most of the neutralization-escape mutations (magenta) and glycans (cyan) are exposed on the opposite face of E2 (B), suggesting that this face is exposed on the viral surface. Residues forming the E2 dimer interface are in yellow.

Fig. 5.

Possible pestivirus membrane fusion mechanisms. Three alternative mechanisms are proposed for insertion of a fusion motif into the target host cell membrane. (A) E2 forms a disulfide-linked dimer and is associated with E1 on the surface of the infectious virion. The black pentagon represents the unprotonated side chain of His762. (B) Activation of E2 by the reduced pH of the endosome, disulfide isomerase activity and proteolytic cleavage of E2 near the C-terminal transmembrane domain causes E2 dimers to dissociate. The cluster of aromatic residues in domain IIIc functions as a fusion motif. E2 remains associated to the viral membrane via E1. Both glycoproteins contribute to the subsequent fusogenic conformational change. (C) E1 contains the fusion motif. E2 functions as a coeffector of fusion providing structural integrity to the fusion complex. Protonation of His762 controls exposure of the fusion motif in E1 by destabilizing an interaction between E2 and the fusion motif. (D) Domain I contains an as yet unidentified fusion motif, which becomes exposed under specific conditions, for example in the presence of a lipid bilayer. This topology would place the fusion motif of the opposite end of E2 from the viral membrane, as would be expected in a fusion protein. (C and D) If domain IIIc does not function as the fusion motif, the array of aromatic side chains in domain IIIc could insert into cellular membrane, positioning the fusion motif toward the target membrane. See also Fig. S5.