The ectodomain of BVDV1 E2 (residues 4–334, although residue 334 is not visible in the electron-density maps), bearing a mutation on the C-terminal glycosylation site (N298Q), was purified and crystallized as a covalently linked homodimer (Iourin et al., 2012
). E2 is an elongated molecule consisting of four domains, DA, DB, DC, DD, arranged linearly from N to C terminus (). All 17 cysteines are involved in disulfide bridges establishing one inter- and eight intramolecular bonds (A). As expected from the deglycosylation process, electron density for a single N-acetylglucosamine-linked asparagine is clearly defined for residues N117, N186, N230, and N298 in the structure obtained at pH 5 where N298 has not been mutated (C). Whereas for the pH 8 structure, as a result of the mutation N298Q, no glycan is seen at this position (A). The overall fold, despite being composed primarily of β strands, shows no similarity to class II fusion proteins of flaviviruses
that do not have the domains organized linearly along the polypeptide chain.
Amino Acid Conservation between Pestiviruses E2 Glycoprotein, Related to , , and
Domains DA and DB (residues 4–87 and 88–164, respectively) are the most distal from the viral membrane and are likely to be the most exposed on the virus surface, indeed the CSFV antigenic regions A and B/C map to exposed patches on the surface of these domains (). Both domains possess Ig-like folds (), consistent with a cell-receptor binding function, but no structure resembling a fusion loop or hydrophobic patch could be identified. CSFV E2 (65% sequence identity) binds to host cells, inhibiting infection by both CSFV and BVDV, implying a common cell receptor for pestiviruses
attaching through E2 (Hulst and Moormann, 1997
). Its role in cell binding is corroborated by the fact that E2 determines cell tropism of the virus (Liang et al., 2003
). A CSFV host cell binding peptide (Li et al., 2011
) maps to domain DB of BVDV1 E2 (B), more precisely to a solvent exposed β-hairpin extrusion. CD46 has been shown to be a cellular receptor of BVDV (Maurer et al., 2004
), and is a candidate for attachment to the β-hairpin.
Mapping of Epitopes of Classical Swine Fever Virus E2 onto the Structure of BVDV E2
Domains DA and DB Have Ig-fold, Related to
Domain DC (residues 165–271) could not be matched significantly to any known structure using the DALI server (Holm and Rosenström, 2010
); it is a highly extended disulfide-rich structure, composed of loops and antiparallel β strands, containing two glycosylation sites (N186 and N230). Among pestivirus E2 glycoproteins, there are two conserved glycosylation sites (N117 and N186) (A) that lie close together in the three-dimensional structure, and mutation of either of these leads to a marked reduction or loss of expression in baculovirus infected cells (Pande et al., 2005
). Homodimerization occurs through domain DD (residues 272–333), which is the most conserved domain among pestiviruses
(A and S1B). Dimers are covalently stabilized by a disulfide bridge and, surprisingly, by a domain swap between monomers (C), with DD, which is connected to the other domains via a long tether, being embedded in the adjacent subunit. Domain DD is composed of an extended loop that links domain DC to a β-hairpin. The hairpin loop folds back onto the β strands to form an hydrophobic pocket. Similarly to domain DC, no structural match could be found in the protein data bank. All disulfide bonds, apart from the C-terminal one, are involved in intramolecular bonds, so it is highly likely that the heterodimer E1-E2 is also formed via the cysteine involved in the homodimerization at the C terminus of E2 near the viral membrane. Moreover, the two swapped domains interact strongly with each other through hydrophobic patches rich in phenylalanines and tyrosines (C). During biogenesis, both glycoproteins E1 and E2 associate with the chaperone protein calnexin (Branza-Nichita et al., 2001
). The well-conserved hydrophobic patch of E2 is likely to be concealed temporarily through dimerization with chaperone proteins, and once released, used for homodimerization and heterodimerization, with both dimers being further stabilized by a disulfide bridge.
Interactions of BVDV1 E2 and Their Locations
The low pH structure of BVDV1 E2 (residues 1–339) is similar to the one at neutral pH apart from a rigid body movement of one of the monomers of some 12° and, intriguingly, by disordering of domain DA (C). In fusion proteins, histidine is believed to play a role in pH-induced conformational changes because its pKa is between the physiological and endosomal pH (Kampmann et al., 2006
). A drop in local pH on entry into the endosome would consequently change the protonation state of histidines and their local interactions. Pestivirus E2 glycoproteins possess only one strictly conserved histidine (H70) located in domain DA (A and A). H70 is surrounded by aliphatic chains from E70, R72, A73, and Y8 thus protonation of this conserved histidine is likely to destabilize domain DA whose stability is predicted to be sensitive to a drop of pH from 9 to 5 whereas the rest of the protein remains stable (). Remarkably, the distal domain of sindbis virus E2, which caps the fusion peptide of E1, also becomes disordered in low pH environment (), via a proposed histidine switch, to expose the fusion loop of E1 (Li et al., 2010
). HCV E2 likewise has a conserved histidine (H445) toward its N terminus acting as a pH sensor (Boo et al., 2012
), suggesting a similar mechanism.
Stability of BVDV1 E2 Domains over pH, Related to
Structural Comparison of Pestivirus and Alphavirus E2 Glycoproteins at High and Low pH, Related to
Because the membrane distal domains of BVDV1 E2 do not harbor a hydrophic peptide we propose that E2 is not the fusion protein. Furthermore BVDV1 E2 does not have a class II fusion protein fold and its shape instead resembles the alphavirus
E2 attachment glycoprotein that also contains Ig-like domains and partially unfolds at low pH but is not fusogenic (). We suggest instead that E1 is responsible for fusion. Consistent with this proposition, a fusion peptide-like motif located in the middle of the HCV E1 sequence (residues 264–290) has been proposed to play a role fusion (Drummer et al., 2007
; Li et al., 2009
). In pestivirus E1 proteins, a sequence similarly rich in hydrophobic amino acids can also be found between residues 57 and 85. However, E1 is about half the size of typical class II fusion proteins, so either it has a different fold, or, as suggested previously for HCV E1, a truncated class II fold (Garry and Dash, 2003
). In any event, E1 must span the distance to the host cell membrane, perhaps 100 Å to 150 Å and is therefore likely to be a thin, extended protein, possibly using E2 as a scaffold (the domain swap organization we observe would allow E2 to partly substitute for the E1 domain proximal to the viral membrane). This model is in line with the observation that the heterodimer is essential for membrane fusion, in contrast to alphaviruses
where E2 is not required once the fusion loop is exposed. Our model for the stages of pestivirus cell entry leading up to membrane fusion is shown in .
Proposed Model of the Fusion Mechanism of Pestiviruses
genus was moved from the Togaviridae
(containing the alphaviru
ses) to the Flaviviridae
because of similarity in genome organization (Collett et al., 1988
). The presence of heterodimers E1-E2 and our observation that BVDV1 E2 is reminiscent of alphavirus E2, suggest that the cell entry machineries of the pestivirus
and probably hepacivirus
genera are in fact closer to the Togaviridae
than to flaviviruses
Although BVDV has been used as a surrogate for HCV, there is no direct evidence that the glycoproteins are similar between these viruses. However, there is also no detectable sequence similarity between that the fusion proteins of the flaviviruses
although they share the same fold. Indeed a series of observations support the contention that HCV envelope glycoproteins are more similar to pestiviruses
than other viruses: (1) BVDV and HCV both require an extra step for membrane fusion beyond a simple pH drop, suggesting a similar entry mechanism (Krey et al., 2005
), (2) their genome organization is very similar: HCV has two glycoproteins E1 and E2 like alphaviruses
; however, the size of E1 is about half of E2, as seen in pestiviruses
, (3) BVDV and HCV E2 are both the immunodominant proteins that generate neutralizing antibodies, and (4) they both bind the cell receptors (Hulst and Moormann, 1997
; Pileri et al., 1998
). Together, these findings suggest that HCV will likely follow the unexpected pathway for cell attachment that we propose for BVDV1 and, more specifically that the BVDV1 E2 structures presented in this article should provide a useful model for HCV.