LAUSR

Dear Editor, In 2015, a Zika virus (ZIKV) outbreak began in South and Central America and in the Caribbean, and has since spread to both North America and Asia. It has been revealed that ZIKV is the primary cause of severe neurological pathologies, such as neonatal microcephaly and Guillain–Barré syndrome. ZIKV infection can also damage mouse testes, posing a potential threat to the mammalian reproductive system. Similar to the dengue virus (DENV) and the West Nile virus (WNV), ZIKV is a mosquito-borne flavivirus containing a single-stranded positive-sense RNA genome, which encodes three structural proteins (C, prM/M, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The mature form of the flavivirus C protein is ~12 kDa, and it plays a critical role in encapsulation of the RNA genome. In addition, the C protein interacts with intracellular lipid droplets for viral particle formation and inhibits host RNA silencing to suppress the immune response. The multiple functions of the flavivirus C protein in the viral life cycle make it an attractive target for drug development. We set out to conduct structural studies of the ZIKV C protein in order to understand the role of the flavivirus C protein during encapsulation of the viral RNA genome. The flavivirus C protein is a highly basic macromolecule with a disordered N-terminus, which has caused problems in structural studies. Only an NMR structure of the DENV2 C protein (residues 21–100) and a 3.2-Å crystal structure of the WNV C protein (residues 24–98) have been previously reported. To overcome the poor yield and solubility problems of the ZIKV C protein, the core domain (residues 24–98) and a longer fragment (residues 2–104) (Supplementary information, Figure S1) from the ZIKV C protein were fused to the maltosebinding protein (MBP, residues 1–370), resulting in MBP-ZIKVCC (ZIKVCC) and MBP-ZIKVCL (ZIKVCL), respectively. Both fusion proteins are very soluble, and eluted as monodispersed peaks from a size exclusion column (Supplementary information, Figure S2) with the elution volumes matching those expected for the dimers. ZIKVCC successfully produced two different crystal forms. Both crystals belong to the P21 space group, but the unit cell parameters are different. We were able to solve the two crystal structures of ZIKVCC at 2.0 and 2.9 Å, respectively. In the highresolution structure, there are two ZIKVCC molecules in an asymmetric unit (ASU), forming a dimer with twofold noncrystallographic symmetry. These two fusion proteins dimerize solely via the capsid while the MBPs are on the outside of the dimer interface, participating in crystal packing (Supplementary information, Figure S3). In contrast, the low-resolution structure consists of two identical dimers in an ASU; however, superimposition of the dimers from these two structures individually shows that although the orientation of the MBP molecules is different, the central ZIKVCC molecules are identical (Supplementary information, Figure S4). These data indicate that ZIKVCC exists as a biological dimer in structure, and its oligomerization state is similar to that of the DENV C protein. For clarity, only the dimer of the ZIKVCC at high resolution is discussed in this study. Within the ZIKVCC dimer, each protomer adopts the all-alpha helical conformation, from Nto C-termini, designated α1, α2, α3, and α4 (Fig. 1a and Supplementary information, Figure S5). These helices are connected by the loops between them. The rootmean-square deviations between the ZIKV C protomer and those of the WNV and DENV are 1.6 and 1.8 Å, respectively (for Cα; PDB IDs: 1SFK and 1R6R). The helical parts (α2, α3, and α4) aligned well, and the most distinct feature between these flavivirus C protomers lies in their N-termini (Supplementary information, Figure S6). The N-terminus of the ZIKVCC protomer is composed of a long, structured loop and a short helix (α1), whereas the Ntermini of the WNV and DENV C protomers only consist of a much longer α1. An overlay of these structures shows that the Ntermini of the ZIKV and WNV C protomers share the same orientation, but the N-terminus of the DENV C protomer adopts a completely different orientation (Supplementary information, Figure S6A and B). The diverse N-terminal structures of the flavivirus C proteins indicate the inherently flexible nature of this region, which might be able to adopt different conformations for various physiological processes. The two ZIKV C protomers associate into a dimer with a buried area of 2270 Å, which is much larger than those for DENV C protein (1650 Å) and WNV C protein (1530 Å). The dimer contact interface can be roughly divided into three layers: Nterminus-N-terminus (layer I), α2–α2 (layer II), and α4–α4 (layer III). In layer I, it is notable that the structured N-terminal loop connected to α1 contributes to strong interactions between the two protomers, which are also stabilized by a core of hydrophobic residues (L30, L33, L37, L38, I50, L51, and L54 of both protomers; Fig. 1b). This feature explains why the ZIKV C dimer has a larger contact interface than the DENV and WNV C proteins (Fig. 1b and Supplementary information, Figure S6C). In layer II, α2 and α2 are aligned in an antiparallel style. Along the helix–helix interface, the hydrophobic residues M46, I50, and F53 (Fig. 1c), which are well conserved among flaviviruses, play a major role in the helix–helix interaction. In layer III, α4 and α4 also form an antiparallel helix pair. The conserved hydrophobic residues (I81, F84, L88, M91, L92, and I95) are responsible for the hydrophobic interactions (Fig. 1d). The conserved residues in the dimerization interface have been shown to be critical for its physiological role in flaviviruses. Mutation of these residues (F56K and F84K) could disrupt the protein folding, resulting in a soluble aggregate (Supplementary information, Figure S7A).