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. 2012 Feb;86(4):2337-46.
doi: 10.1128/JVI.06072-11. Epub 2011 Dec 7.

Crystal structure of the Japanese encephalitis virus envelope protein

Vincent C Luca  1 , Jad AbiMansour, Christopher A Nelson, Daved H Fremont
Affiliations

Crystal structure of the Japanese encephalitis virus envelope protein

Vincent C Luca et al. J Virol. 2012 Feb.

Abstract

Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.

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Figures

Fig 1
Fig 1
Crystal structure of JEV E ectodomain. JEV E possesses the three domains characteristic of flavivirus E with symmetry operators that allow for generation of the canonical E dimer. A JEV E diagram representation crystal structure is shown, with domain I highlighted in red, domain II highlighted in yellow, domain III highlighted in blue, and the crystallographic dimer generated from orthorhombic symmetry highlighted in gray. The structure is also shown rotated 90° into the page. The fusion loop is colored green, and the “k-l” loop and glycosylation site are indicated in both structures.
Fig 2
Fig 2
Relative buried surface area of dimeric flavivirus E protein structures. JEV E has a small dimer interface relative to other E crystal structures. Surface representations of known dimeric E protein crystal structures are displayed arranged in ascending order of buried surface area. Note that JEV E and TBEV E have visible solvent channels between subunits at the dimer interface, features that are absent in DV E dimers that have greater contact interfaces.
Fig 3
Fig 3
Comparison of E protein DI-DII hinge angles. The DI-DII hinge angle of JEV E is most similar to that of TBEV E. Various crystal structures of E were superimposed onto DI of JEV E, and the relative angle between DI and DII was determined using Dyndom. Proteins are colored according to the virus of origin, and the numbers on the left indicate the difference in angle between DI and DII of each E protein and JEV E. The DV3 E protein was omitted for clarity and because it varies by <1° from that of DV2 E.
Fig 4
Fig 4
Dimeric contact residues in E proteins from DV, TBEV, and JEV serocomplexes. Multiple loops of DI and DII have dimer contacts in TBEV and DV E that are lacking in JEV E. Loops colored green contribute to dimer contacts in the DV (A) and TBEV (B) E proteins but not in JEV E protein. (C) The equivalent loops are colored red in the JEV E structure. (D) The sequences corresponding to the numbered loops are aligned for all known dimeric E protein structures, with dimer contact residues highlighted in green. The parent virus of the E protein and its PDB ID are shown to the left of each sequence.
Fig 5
Fig 5
Multi-angle light scattering evaluation of E protein solution oligomeric state. E proteins from viruses of the JEV E serocomplex favor a monomeric solution state. Multi-angle light scattering was utilized to calculate the solution molecular mass (MM) of JEV E (A), SLEV E (B), WNV E (C) and DV2 E (D) over their elution profile on a S200 sizing column. JEV E, SLEV E, and WNV E had molecular masses corresponding to that of monomers, whereas DV2 E was that of a dimer. The UV absorbance trace is colored black, molar mass calculation in blue and fitted molar mass in red.
Fig 6
Fig 6
Comparison of E protein crystal structures to DV2 cryo-EM model. (A) Superimposition of the unmodified JEV E crystal structure onto the DV2 cryo-EM coordinates generates an effective model for the JEV virion. JEV E monomers were superimposed onto E proteins from the DV2 cryo-EM reconstruction. The enlarged window of E proteins at the 2-fold axis shows the only clashing main-chain loops, “b-c” and “h-i”, in cyan and magenta, respectively. (B) JEV E and DV2 E crystal structure backbones (green) are overlaid onto artificially generated dimers created by superimposing monomers from the crystal structure onto Dengue E dimers of the cryo-EM model (gray). (C) The table describes the buried surface areas from the crystal structures, the cryo-EM model dimers and the RMSD obtained by aligning the crystal structure dimers onto the cryo-EM models.
Fig 7
Fig 7
Conservation and localization of histidines of E proteins from the JEV and DV serocomplexes. Histidines on the lateral edge of DII and DIII are poorly represented in flaviviruses but conserved in the JEV serocomplex. Histidines of the DV2 E and JEV E proteins are shown in stick representation on one dimer subunit and labeled with their residue number for the given virus. Those colored green represent those conserved in all flaviviruses, those colored orange are conserved in only the DV2 (top) or JEV serocomplex (bottom), and those colored gray are not broadly conserved. Histidines fully conserved in the JEV serocomplex but not in other flaviviruses are found on the outer edge of the dimer and marked with an asterisk.
Fig 8
Fig 8
Mapping of neutralizing epitopes onto the JEV E protein and reconstructed virion. JEV E neutralizing epitopes are found at the DI-DII hinge, DI lateral ridge, DIII lateral ridge, and fusion loop. (A) Side chains of residues critical for binding by previously identified JEV neutralizing antibodies are colored green and in spherical representation. (B) 4G2 (G104, G106, and L107) maps to the fusion loop. (C) B2 (I126), NARMA3 (Q52), and 503 (Q52, I126, K136, and S275) map to the DI-DII hinge. (D) A3 (K179) maps to the DI lateral ridge. (E) E3 (G302) and E3.3 (I337, F360, and R387) map to the DIII lateral ridge. The regions described above have also been mapped onto the model of the JEV virion to reveal their arrangement and accessibility in the icosahedral assembly.
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