Crystal structure of the ZIKV NS5

We expressed the full-length NS5 from ZIKV strain MR766 that was originally isolated from Uganda Africa and determined its crystal structure at 3.0 Å resolution (Table 1, Supplementary Fig. 1). The polypeptide chains are well defined except for the N-terminal four residues and the C-terminal 16 residues (Fig. 1a, Supplementary Fig. 2). The MT is complexed with S-adenosyl-L-homocysteine (SAH), and the RdRp adopts a classic ‘right-hand’ structure consisting of three subdomains: fingers, palm and thumb (Fig. 1a,b). Two zinc ions are found in the fingers subdomain and at the junction of the palm and thumb subdomains of the RdRp.

Table 1 Statistics of crystallographic analyses. Full size table

Figure 1: Structure of full-length ZIKV NS5. (a) Ribbon representation showing the arrangement of the MT and the RdRp domains of ZIKV NS5. A top view look into the active site of the RdRp is shown on the left and a side view is shown on the right. The MT domain and structural motifs of the RdRp domain are coloured according b. The active site residues of the MT and the RdRp are shown by the pink and purple stick representations, respectively. The S-adenosyl-L-homocysteine (SAH) molecule that binds to the MT is shown by the magenta stick model. (b) Schematic representation of ZIKV NS5 showing the locations of key residues and structural motifs. Full size image

The overall structure of the ZIKV NS5 has striking similarities to that of the JEV NS5, with the RMS deviation of 0.55 Å for 751 Cα atoms (Fig. 2a). The MT of both proteins are also located at an acute angle to the RdRps. The ZIKV MT is in a distinct orientation relative to the DENV MT. Due to a short 3 10 -helix in the linker, the DEN MT is rotated toward the RdRp (Fig. 2b,c). Residues Arg363, Gln598 and Asn576 in the fingers subdomain of the ZIKV RdRp interact with the linker to prevent it from being more flexible (Fig. 2d).

Figure 2: Comparison of the NS5 structure of ZIKV to those of JEV and DENV. (a) Superposition of the structures of ZIKV NS5 with JEV NS5 (PDB, 4K6M). (b) Superposition of the structures of ZIKV NS5 with DENV NS5 (PDB, 4V0Q). (c) Distinct conformations of the linkers in ZIKV, JEV and DENV NS5 that are responsible for the altered orientations of the MT and RdRp domains in these proteins. The linkers are shown as stick models. The backbones of the MTs and RdRps that flank the linkers are shown as thin lines. Note that several residues of the JEV NS5 linker were not resolved. (d) Interactions between ZIKV NS5 linker and the fingers subdomain. The linker is shown as stick models (magenta). The extension (blue), portions of the MT (cyan) and the fingers subdomain (green) are shown as ribbons. Key residues R363, Q598 and N576 from the fingers subdomain that interact the linker are shown as sticks. Dashed lines indicate distance of <3.5 Å. Full size image

Additional interactions between the MT and the RdRp contribute to the orientation of the MT in the ZIKV NS5. In the MT, residues 112–128 that forms Loop 9, α6 and β4 interacts with the α14, Loop 32 and Loop 40 in the RdRp domain (Supplementary Fig. 3). Notably, residues in these structures are highly similar in the NS5 proteins of the ZIKV and JEV, providing an explanation for the similar orientations of the MT and RdRp in these two proteins (Supplementary Fig. 3a). In addition, Loop 32 of the DENV NS5 was disordered, likely contributing to the altered orientation of the MT in the DENV NS5.

The NS5 MTase domain

The MT of ZIKV NS5 adopts a classic α/β/α sandwich structure13. The ZIKV MT can be superimposed with the MTs from other flaviviruses with RMS deviations of <0.73 Å (Fig. 3a). The highly conserved structure allows assignment of the residues that function in RNA cap addition in the ZIKV MT. Residues that bind GTP, catalyse methyl transfer and bind the methyl donor S-adenosyl-methionine (SAM) are arranged in a line within a concave surface of the MT (Fig. 3b,c). The conserved catalytic tetrad of Lys61–Asp146–Lys182–Glu218 that forms the active site is positioned in the centre of the MT (Fig. 3c,d). The putative GTP binding pocket is located to the left of the active site. The SAM-binding pocket is located in a narrow crevice to the right of the catalytic pocket (Fig. 3a,c). SAH, the byproduct of methyl donation from SAM, has the adenylate embedded in a narrow portion of the active site channel, where the Oδ1 of Asp131 forms a H-bond with the N6 of adenine and the Nδ1 of His110 forms a H-bond with the ribose 2′ OH (ref. 7). The homocysteine portion of SAH interacts with loop residues 79–85 (Fig. 3d).

Figure 3: Structure of ZIKV NS5 methyltransferase domain (MT). (a) Comparison of the structures of the MT domains of ZIKV, DENV (PDB, 3P97), YFV (PDB, 3EVC), WNV (PDB, 2OY0) and JEV (PDB, 4K6M). SAH or SAM and GTP bound to the MT domains are shown by the stick models. (b) Surface of ZIKV NS5 MT involved in Cap-0 RNA binding coloured by electrostatic potential. Positively charged surface are coloured blue and negatively charged surface red. The Cap-0 RNA (5′-m7G 0ppp A 1 G 2 U 3 U 4 G 5 U 6 U 7 -3′) is modelled into the ZIKV NS5 MT by superposition of the DENV MT/Cap-0 RNA complex structure (PDB, 5DTO) onto ZIKV MT. (c) Surface representation of ZIKV NS5 MT showing the active site and the binding sites for GTP and SAM. (d) Key residues of ZIKV NS5 MT essential for GTP binding (orange), SAM binding (green) and catalysis (magenta). SAH is shown by the blue stick model. Full size image

The NS5 polymerase domain

Viral RdRps typically have extensive interactions between the fingers and thumb subdomains to encircle the active site of the polymerase14,15. Three channels are apparent in the ZIKV polymerase. Based on comparison with polymerases whose ternary structures have been determined and characterized16, the channels should bind the template RNA (template channel), guide the emergence of the template and nascent RNA (central channel) and enable the entry of the NTPs (NTP channel) (Fig. 4). Motifs A to G that are conserved in sequence and structure, line the active site cavity and contribute to nucleotide and template recognition and nucleotide polymerization.

Figure 4: Structure of ZIKV RNA-dependent RNA polymerase domain (RdRp). (a) Ribbon representation of the RdRp showing the locations of structural motifs that are critical for RNA synthesis. The extension (slate) is a unique structure of flaviviral NS5 connecting the RdRp with the MT via the linker. The priming loop (lime) extending from the thumb subdomain, forming a platform to coordinate with the NTP for polymerization. (b) Cut-away surface representation of ZIKV RdRp showing the locations of the template channel, the central channel and the NTP channel. Motifs G and F that form the encircled active site and also a constriction in the template channel are coloured cyan and orange. The priming loop is identified by ‘Pr’. Motif C and A that bind divalent metal ions are coloured blue and purple. (c) Electrostatic surface of ZIKV RdRp in two orientations. Positively charged surface is coloured blue and negatively charged surface red. (d) Locations of the key residues in the priming loop and active site of ZIKV RdRp. (e) Superposition of HCV RdRp (salmon) in complex with template RNA (slate sticks) and the initiation NTP (purple sticks, PDB, 4WTL) with ZIKV RdRp (green). Conserved residues of ZIKV RdRp are shown by the green sticks. Full size image

The active site of the ZIKV RdRp is enclosed due to the interaction between motifs F and G that project from the fingers to contact the thumb in the front of the RdRp (Fig. 4a,b). The back of the RdRp has a lattice of three loops (Fig. 4a,b). The template RNA will bend at a ca. 45° angle and emerge from the central channel. The NTP channel will merge at the confluence of the template and central channels (Fig. 4b,c). Here three aspartates from motifs C and A (Asp535, Asp665 and Asp666) that coordinate divalent metal ions for nucleotide polymerization are localized. The priming loop, which positions nucleotides for polymerization15, extends from the thumb subdomain is located at the confluence of the three channels (Fig. 4d). In the phage phi6 and HCV RdRp, a tyrosine in the priming element has been shown to form the priming platform by stacking interacting with the initiating nucleotide16,17. In the ZIKV NS5, Trp797 likely performs the role of stacking with first nucleotide to facilitate initiation of de novo RNA synthesis (Fig. 4d).

The RdRp of the hepatitis C virus (HCV), which belongs to the genus Hepacivirus of the Flaviviridae family has been extensively studied for the structures required for de novo initiation and elongation of RNA synthesis18. Residues in the ZIKV RdRp that should contact the RNA and NTPs are located at similar positions to their counterparts in the HCV RdRp ternary complex (Fig. 4e, Supplementary Fig. 4a), suggesting that ZIKV NS5 will have comparable recognition of the template, primer RNA and nucleotides for RNA synthesis. The priming loop of the ZIKV RdRp is larger than that of the HCV RdRp (Supplementary Fig. 4b,c), indicating that conformational changes from the current structure will take place to enable the elongation of the nascent RNA.

MTase interacts with the polymerase to affect RNA synthesis

The MT of the ZIKV NS5 connects to the fingers subdomain of the RdRp and overhangs the NTP channel of the RdRp (Fig. 5a). The MT interacts with the fingers subdomain of the RdRp primarily through a hydrophobic network that involves residues Pro113, Leu115 and Trp121 from the MT and Tyr350, Phe466 and Pro584 from the RdRp (Fig. 5b). The total buried surface area between the MT and the RdRp is ∼1,600 Å2. The close proximity of the MT to the RdRp suggests that the MT may impact RNA synthesis by the RdRp.

Figure 5: The MT affects RNA synthesis by the ZIKV RdRp. (a) Cut-away surface representation showing the locations of the MT and the RdRp in full-length ZIKV NS5. The MT overhangs the NTP channel and contacts the fingers subdomain of the RdRp. (b) Interactions between the MT domain (cyan) and the fingers subdomain (green). Dashed lines indicate distance <3.5 Å. (c) In vitro RNA synthesis catalysed by full-length ZIKV NS5 and Δ264 that lacks the MT. Each set of reactions were performed with 5, 20, 100 and 200 ng of NS5 protein or Δ264 (Supplementary Fig. 6). The PE of 46-nt denotes an elongated product RNA. DN denotes the 17-nt product RNA that initiated de novo with a NTP from the 3′-most template nucleotide. The templates used for RNA synthesis are shown in Supplementary Fig. 5. The relative amounts of the products made by Δ264 are normalized to those generated by the same concentration of the enzyme in the reaction with NS5. The results shown are reproducible in four independent assays. (d) Regions of ZIKV NS5 that contact the template RNA (PE46) for elongative RNA synthesis. Residues from peptides that are reversibly crosslinked to PE46 are shown in yellow. The structure shown is oriented to show the view at the back of the RdRp that connects to the MT. (e) Conformational changes of the RdRp in the absence of the MT. Comparison of eight Δ264 structures in one asymmetric unit reveals two distinct conformations in loop 312–323 and loop 742–750 located in the back of the RdRp and Motif G. Conformation 2 of Δ264 is similar to that in full-length NS5. (f) The different conformations of motif F in full-length NS5 (orange) and isolated RdRp (Δ264, green). (g) Surface representation showing the different conformations of motif F in full-length NS5 (orange) and isolated RdRp (Δ264, green). Full size image

To examine whether the interaction of the MT with the RdRp will affect RNA synthesis, we compared the RNA synthesis activity of NS5 to that of a truncated protein, Δ264, which lacks the MT (Supplementary Fig. 5a). The full-length ZIKV NS5 could initiate RNA synthesis de novo or elongate from a primed template in processes that will require distinct RdRp conformations (Fig. 5c, Supplementary Fig. 5b,c). NS5 that had the two aspartates in motif C replaced with alanines was unable to direct RNA synthesis either by de novo initiation or by elongation from a primed template (Supplementary Fig. 5b,c). Δ264 synthesized approximately half of the de novo-initiated RNA product as did full-length NS5 (Fig. 5c, Supplementary Fig. 6). However, with the template that directs elongative RNA synthesis, Δ264 synthesized sevenfold less product than did NS5. Our result demonstrates that the MT contributes to RNA synthesis by the RdRp, especially for elongative RNA synthesis.

The orientation of the MT relative to the template channel and the central channel suggests that it will affect RdRp interaction with the template RNA. A reversible crosslinking, mass spectrometric assay was used to map residues in NS5 that contact the template RNA19. The peptides from NS5 that contacted the RNA were mapped to the template channel, the fingers subdomain and all motifs in the RdRp except for motifs F and D (Fig. 5d, Supplementary Fig. 7a,b). Interestingly, the MT, especially the residues adjacent to the fingers subdomain of the RdRp, also had extensive contact with the template RNA.

Altered RdRp structure in the absence of MTase domain

To better understand the difference in RNA synthesis by NS5 and the RdRp, we determined the crystal structure of Δ264 at 3.0 Å resolution. The asymmetric unit contains eight RdRps that exist in two conformations (referred to as conformation 1 and 2) that vary in the locations of motif G, Loop 21 (residues 312–323) and Loop 51 (residues 742–750) that connect the thumb and fingers subdomains (Fig. 5e). The two conformations are affected by residue Arg483 that lies in the template channel. In conformation 1, the side chain of Arg483 inserts between motifs B and F and interacts with the carbonyl backbone of Gly604 and Trp476 (Fig. 5e, Supplementary Fig. 8a). In conformation 2, the locations of motifs B and F prevents the insertion of the side chain of Arg483. Instead, Arg483 interacts with Ala409 of motif G (Fig. 5e, Supplementary Fig. 8b). Full-length NS5 only has one conformation in the corresponding region and it resembles that of conformation 2 (Fig. 5e, Supplementary Fig. 8c). Notably, motif F in NS5 binds with the MT and has a different conformation relative to that in Δ264 (Fig. 5f). The net effect of the presence of the MT is that the RdRp has a reconfigured template channel and possesses a more open NTP channel (Fig. 5f,g). These changes will likely decrease RNA synthesis.

RNA synthesis by pandemic ZIKV NS5

The current pandemic ZIKV have been observed to be associated with serious human illness1. Strain Brazil/PE243/2015 that was identified from a patient from Recife in Brazil, has more than 35 amino acid substitutions in the NS5 when compared to MR766 (Fig. 6a,b). In terms of RNA synthesis in vitro, NS5 proteins from Brazilian/PE243/2015 and MR766 have comparable activities to direct de novo-initiated and elongative RNA synthesis (Fig. 6c). Mapping of residues changed in the NS5 protein of the Brazil/PE243/2015 onto the MR766 NS5 structure reveals that the substitutions are located on the surface of NS5 and are not involved in the core of the RdRp that can affect RNA synthesis (Fig. 6d).