Overall structure of ZIKV NS5

We were able to trace nearly the entire sequence of ZIKV NS5 (Fig. 1a), except for the first five N-terminal residues, residues 747–748 in the RdRp domain, and sixteen residues at the C terminus. The structure reveals an N-terminal classic S-adenosyl-L-methionine-dependent MTase domain situated on top of a C-terminal RdRp domain. The MTase domain is dominated by a Rossmann fold, with a seven-stranded β-sheet sandwiched by two α-helices from one side and another α-helix from the other side. Near to the catalytic site, the SAH molecule is surrounded by a set of conserved residues (Supplementary Fig. 2). In addition, the N-terminal extension sequence (α1–α4 in Supplementary Fig. 1) of the MTase domain pairs with its C-terminal extension sequence (α8–β9 in Supplementary Fig. 1) to form a helix bundle and an antiparallel two-stranded β-sheet, adding another structural layer onto the Rossmann fold (Fig. 1b). As with other viral RdRps20, the ZIKV NS5 RdRp adopts a capped right-hand structure with the Palm, Fingers and Thumb subdomains and a priming sequence poised to receive RNA substrates (Fig. 1b). The RdRp domain also harbours two zinc ions, as observed for the NS5 proteins of JEV and DENV3 (refs 18, 19).The associations of the MTase domain with the RdRp domain does not involve extensive interdomain contacts, leading to a modest buried surface area of ∼1,400 Å2. In fact, structural superposition of the MTase domain in full-length NS5 and the recently reported domain alone21 gives a root-mean-square deviation (RMSD) of 0.42 Å over 242 Cα atoms, indicating that the MTase–RdRp association does not lead to considerable conformational change of the MTase domain.

Figure 1: Structural overview of ZIKV NS5. (a) Colour-coded domain architecture of ZIKV NS5. (b) Orthogonal views of ribbon (left) and electrostatic surface (right) representations of ZIKV NS5. The MTase domain, the N-terminal extension, palm, fingers, priming loop and thumb of the RdRp domain, and the interdomain linker are coloured in slate, orange, pink, green, red, light blue and magenta, respectively. Zinc ions (purple) and SAH are shown in sphere representation. Full size image

Structural comparison with other flavivirus NS5 proteins

ZIKV NS5 shares ∼68% and ∼66% sequence identity, respectively, with its JEV and DENV3 counterparts (Supplementary Fig. 1). However, these NS5 homologues appear to antagonize the IFN signalling through different mechanisms: JEV NS5 suppresses IFN signalling likely through blocking phosphorylation of the IFN signalling components13, whereas DENV3 NS5 and ZIKV NS5 inhibit the IFN signalling through promoting protein degradation of signal transducer and activator of transcription 2 in an E3 ubiquitin ligase UBR4-dependent or -independent manner10,16. Along the line, we compared the structure of ZIKV NS5 with those of JEV NS5 and DENV3 NS5 (Fig. 2a,b). Remarkably, ZIKV NS5 superimposes well with JEV NS5, with an RMSD of 0.63 Å over 872 Cα atoms (Fig. 2a). In particular, the MTase–RdRp associations of ZIKV NS5 and JEV NS5 are both mediated by the same set of van de Waals contacts, involving the C-terminal extension of the MTase domain (P113, L115, Q117 and W121), and the Index, Ring and Middle fingers of the RdRp domain (Y350, R354, F466 and P584 in ZIKV NS5) (Fig. 2c,d). Subtle structural divergence between ZIKV NS5 and JEV NS5 was mainly observed for the N- and C-terminal extension of the MTase domain, the MTase-RdRp domain linker, and a segment in the Palm subdomain (residues E632-G653 in ZIKV NS5) (Supplementary Fig. 3a). Note that these regions have previously been linked to NS5-mediated immunosuppression13,14. Therefore, such structural divergence may underlie the distinct mechanisms of ZIKV NS5 and JEV NS5 in IFN antagonism. By contrast, structural superposition of ZIKV NS5 and DENV3 NS5 gives a RMSD of 6.06 Å over 844 Cα atoms, attributed in large part to the difference in the relative orientation between the MTase and RdRp domains (Fig. 2b). Unlike the structure of ZIKV NS5 in which the MTase domain sits on the back of the RdRp domain, the MTase domain of DENV3 NS5 approaches towards the front of the RdRp domain, resulting in a more compact conformation (Fig. 2b). Distinct from those of ZIKV NS5 (Fig. 2c,d) and JEV NS5, the MTase–RdRp association of DENV3 is mediated by a set of hydrogen bonding, cation-π and electrostatic interactions between the N- (Q63, E67 and R68) and C-terminal extensions (E252 and D254) of the MTase domain and the Index finger (F348, R352, E356 and K357) of the RdRp domain (Fig. 2e). These interactions of DENV3 NS5 appear to draw the MTase domain towards the NTP entrance of the RdRp domain, resulting in a ∼100° rotation of the MTase domain in relation to the RdRp domain (Fig. 2b). Another prominent structural difference between ZIKV/JEV NS5 and DENV3 NS5 arises from the substrate binding motifs of RdRp, including motif F in the Ring finger and motif G in the Pinky finger22,23 (Fig. 2b). The conformations of these two motifs appear to be stabilized by the MTase–Ring finger association of ZIKV/JEV NS5, but become disordered in DENV3 NS5 due to the loss of the corresponding interactions (Supplementary Fig. 3b).

Figure 2: Structural comparison of NS5 proteins from ZIKV and two other flaviviruses. Structural superposition of ZIKV NS5 with (a) JEV NS5 (PDB 4K6M) and (b) DENV3 NS5 (PDB 4V0Q). Alignment of the RdRp domains of ZIKV NS5 and DENV3 NS5 leads to a ∼100° change in orientation between the MTase and RdRp domains. The NTP entrance and RNA exit sites are labelled. (c–e) The MTase–RdRp domain interactions of (c) ZIKV NS5, (d) JEV NS5 and (e) DENV3 NS5. Full size image

The fact that the structure of ZIKV NS5 exhibits an extended domain conformation similar to that of JEV NS5, but differently from that of DENV3 NS5, raises a question on the functional implication of these two conformational states of NS5 proteins. On one hand, it is likely that the structures of ZIKV/JEV NS5 diverge from that of DENV3 NS5 through adaptive mutations of specific regions (for example, domain linker) during evolution, as proposed previously24. On the other hand, the high sequence conservation of both domain interfaces (Supplementary Fig. 1) strongly argues that the structures of ZIKV/JEV NS5 and DENV3 NS5 represent two alternative conformations of NS5 that may coexist in solution. Consistently, previous small-angle X-ray scattering analysis suggested the presence of a heterogeneous conformational ensemble of DENV3 NS5 in solution24, and mutations at the two alternative domain interfaces lead to compromised methyltransferase activity or viral replication function of DENV3 NS5 (ref. 19). Additional biochemical and cellular analyses are required to reveal the functional implication of these two alternative conformations of flavivirus NS5 proteins.

De novo RdRp assay of ZIKV NS5

To confirm that the ZIKV NS5 protein used for our structural study represents an active enzyme, we performed a de novo RdRp assay for ZIKV NS5 on a subgenomic ZIKV RNA template (Fig. 3a), using the recombinant ZIKV NS3 helicase domain (NS3-Hel) (Fig. 3b and Supplementary Fig. 4) as a negative control. We observed that the presence of ZIKV NS5 led to a time-dependent increase in the replication of the subgenomic ZIKV RNA at 33 °C (Fig. 3c and Supplementary Fig. 5). However, the reaction product became dominated by a shorter RNA at 23 °C, possibly due to early termination of the replication (Fig. 3c). On the other hand, the presence of ZIKV NS3-Hel failed to yield any RNA product (Fig. 3c). Together, these data not only confirm that the ZIKV NS5 protein used for the structural study is enzymatically active but also provide a basis for further functional characterization of ZIKV NS5.

Figure 3: De novo RNA synthesis by ZIKV NS5 protein. (a) The subgenomic ZIKV RNA contains an internal deletion from nucleotides 171 to 10,343 (GenBank accession no. KU963573.2). (b) SDS–polyacrylamide gel electrophoresis analysis of purified ZIKV NS5 and ZIKV NS3-Hel. (c) ZIKV de novo RNA replication assay. The subgenomic ZIKV RNA was incubated with recombinant ZIKV NS5 protein, ZIKV NS3-Hel or alone (mock). The relative amount of 32P-labelled RNA product is displayed in the autoradiograph of the PAGE gel. The reactions containing recombinant proteins were divided into four groups. Group1 was incubated at 23 °C for 30 min. Groups 2, 3 and 4 were incubated at 33 °C for 30, 60 or 120 min, respectively (Supplementary Fig. 6). Full size image

Identification of potential inhibitor-binding sites

Finally, we asked whether the structure of ZIKV NS5 permits us to identify potential inhibitor-binding sites for its enzymatic inhibition. A previous study, through fragment-based crystallography method, identified a pocket near to the active site of the DENV3 RdRp domain, termed ‘N pocket’, which binds to a small-molecule that inhibits DENV3 NS5-mediated RNA initiation and elongation (Fig. 4a)25. Detailed analysis of this inhibitor-binding site revealed that the critical residues for the inhibitor binding are also conserved in ZIKV NS5, arranged in a similar structural environment (Fig. 4b); therefore, suggesting that the same compound may also be inhibitory to the enzymatic activity of ZIKV NS5. Further enzymatic analysis is needed to test the possibility of applying this DENV3 inhibitor to suppress the activity of ZIKV NS5.