Crystal structure of glycoprotein C from Rift Valley fever virus

Abstract

Rift Valley fever virus (RVFV), like many other Bunyaviridae family members, is an emerging human and animal pathogen. Bunyaviruses have an outer lipid envelope bearing two glycoproteins, G(N) and G(C), required for cell entry. Bunyaviruses deliver their genome into the host-cell cytoplasm by fusing their envelope with an endosomal membrane. The molecular mechanism of this key entry step is unknown. The crystal structure of RVFV G(C) reveals a class II fusion protein architecture found previously in flaviviruses and alphaviruses. The structure identifies G(C) as the effector of membrane fusion and provides a direct view of the membrane anchor that initiates fusion. A structure of nonglycosylated G(C) reveals an extended conformation that may represent a fusion intermediate. Unanticipated similarities between G(C) and flavivirus envelope proteins reveal an evolutionary link between the two virus families and provide insights into the organization of G(C) in the outer shell of RVFV.

Fig. 1.

Overall fold of RVFV G_C and comparison with other class II membrane fusion proteins. (A) RVFV G_C has the same three-domain architecture as other class II proteins. Domain I is in red, domain II is in yellow with the fusion loop in orange, and domain III is in blue. Residue numbers follow M-segment polyprotein numbering, with corresponding G_C residue numbers in parentheses. In the structure, (B), the stem region (light gray), transmembrane anchor (medium gray), and cytoplasmic tail (dark gray) are missing. Glycans are linked to N794, N1035 (light blue), and N1077 (gray, disordered in the structure). The β-barrel connecting domains I and II is marked with a bracket. Disulfide bonds are in green. (C) Structure of Sindbis virus glycoprotein E1 [PDB ID code 3MUU (33], the most similar alphavirus envelope protein, with a Z-score of 15.1 in Dali (42). (D) Structure of West Nile virus glycoprotein E [PDB ID code 2I69 (30)], the most similar flavivirus glycoprotein, with a Z-score of 13.3. (E) The putative fusion loop of RVFV G_C and the fusion loops of Sindbis E1 and West Nile E. The hydrophobic residues that anchor the protein to the cellular membrane and the disulfide bonds are shown in ball-and-stick representation.

Fig. 2.

Nonglycosylated RVFV G_C has an extended conformation and glycosylated G_C forms a head-to-tail dimer in the crystal. (A) The structure of nonglycosylated G_C, colored as in Fig. 1, with glycosylated G_C (gray) superimposed using domain I as the reference. Arrows show the hinge motions of domains II and III relating the two structures. See also Fig. S3. (B, Left) RVFV G_C dimer. The overall arrangement closely resembles that of E proteins in mature flaviviruses and in crystal structures. (Right) E dimer from dengue type 2 virus [PDB ID code 1OAN (21)].

Fig. 3.

Dimer contacts involving the fusion loops of RVFV G_C and dengue type 2 E. (A) In the RVFV G_C dimer interface, Trp821 in the fusion loop forms a π-stacking interaction with Arg1047 in domain III of the dimer partner (DIII′). The other side of the Trp821 side chain is packed against Phe826. (B) In the dengue type 2 E dimer interface, the Trp101 side chain forms a hydrogen bond with the main chain carbonyl oxygen of Leu107. (Inset) The hydrophobic interaction of Trp101 with Lys310 in domain III of the dimer partner (DIII′).

Fig. 4.

Possible organization of the G_C dimer in the outer protein shell of RVFV. Six glycosylated G_C dimers fit into an asymmetric unit of the EM structure of RVFV (EMDataBank code EMD-1550) without major steric clashes and within the icosahedral symmetry constraints. In this configuration, 360 dimers (colored as in Fig. 2) are centered between capsomers, with the dimer dyad axes tangent to the viral surface. The glycans (light blue spheres) are either near the outer surface of the EM density or form contacts with another dimer. The G_C lattice occupies the inner half of the glycoprotein shell. A set of two-, three-, and fivefold icosahedral symmetry axes is labeled with standard symbols. The capsomer not centered on a symmetry axis is labeled “1.” Lower, density distribution from an electron microscopy image reconstruction on a virus-shaped surface with an average radius of 47 nm. Reproduced with permission from American Society for Microbiology (12).