Structure of NS3 helicase from Zika virus

The Zika virus (ZIKV) has emerged as a major health hazard over the past year, linked to microcephaly in newborn infants and the Guillan-Barré syndrome in adults ¹. ZIKV belongs to the same Flavivirus genus as other mosquito-borne human pathogens, such as dengue virus (DENV1-4), tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), West Nile virus (WNV), Murray Valley encephalitis virus (MVEV) and Kokobera virus (KOKV), among others. The public health emergency posed by ZIKV has invigorated efforts to develop a vaccine, as well as measures to eradicate the Aedes mosquito vectors. In addition to these measures, it is equally important to develop antivirals based on the targeting of enzymatic activities central to the life cycle and survival of ZIKV. One such enzymatic activity is encoded by the C-terminal region of the non structural protein NS3 in flaviviruses, namely an RNA helicase (NS3-Hel) involved in genome replication and RNA synthesis ^{2, 3}. NS3-Hel belongs to the superfamily 2 (SF2) of helicases ⁴ and its inactivation in DENV2 renders the virus incapable of replicating properly ⁵. To help guide the discovery of antiviral compounds against ZIKV, we present here a high-resolution (1.62Å) crystal structure of NS3-Hel from the French Polynesia strain of the virus.

We expressed and purified ZIKV NS3-Hel (residues 171–617) of the H/PF/2013 strain as a soluble protein from E. coli (Online Methods). The structure was determined by molecular replacement, and refined to a resolution of 1.62 Å with R_work and R_free values of 16.1 % and 19.3 %, respectively (Supplementary Table 1). The refined model consists of one NS3-Hel molecule (residues 175–617), one pyrophosphate and six acetate ions, and 552 solvent molecules.

ZIKV NS3-Hel is composed of three domains of roughly similar size (Fig. 1a). Domains 1 and 2 (residues 182:327 and 328:480 respectively) comprise the tandem α/β RecA-like folds characteristic of SF1 and SF2 helicases ⁴. Domain 1 contains the classical motifs I/P-loop (or Walker A), Ia, II (or Walker B), and III, whereas domain 2 contains the motifs IV, IVa, V, and VI (Supplementary Fig. 1). These helicase motifs are typically associated with ATP binding and/or hydrolysis (motifs I, II, and VI), interdomain communication, and RNA binding (motifs Ia, IV, and V) and line a cleft at the interface of domains 1 and 2 (Fig. 1a, Supplementary Fig. 1). As with the closely related DENV4 helicase (Fig. 1a), for which there are structures in different ligand bound states ⁶, ATP is expected to bind to the “bottom” side of the cleft between domain 1 and 2, while the RNA would be accommodated in a tunnel that separates domains 1 and 2 from domain 3. Curiously, we observe electron density about the size of a pyrophosphate near the P-loop in the ATP binding cleft (Fig. 2a). Guided by its shape and size, we modeled this extra density both as glycerol (present in the purification buffer and cryoprotectant) and pyrophosphate, but the best fit is obtained with a pyrophosphate (Supplementary Fig. 2a), which may have been acquired during expression of the protein in E. coli⁷. The putative pyrophosphate is in a similar position as the β- and γ-phosphates of AMPNP in the DENV4 NS3-Hel/AMPNP structure ⁶ (Supplementary Fig. 2b), with the capacity to make hydrogen bonds with the main chain and side chains atoms of the conserved P-loop (Fig. 2a). It is not uncommon for an NTPase to co-purify with a pyrophosphate, as for example, in structures of KOKV NS3-Hel⁸ and H.pylori GTP cyclohydrolase⁹. A β-hairpin extends from domain 2 and interacts with domain 3 and has been proposed to act as a “wedge” to help separate the RNA strands of dsRNA for the unwinding reaction (Fig. 1a)⁶. Interestingly, the β-hairpin in our structure (residues 431–444) is slightly smaller than in the DENV4 helicase (430–445) (Fig. 2b) and may alter the kinetics of the RNA unwinding reaction. Domain 3 is predominantly α-helical. In addition to contacts with RNA it has also been implicated in interactions with the RNA-dependent RNA polymerase NS5 in other flaviviruses ¹⁰.

Overall, all three domains superimpose very well on equivalent domains in other flaviviruses (Supplementary Fig. 3a). ZIKV domains 1, 2, and 3 superimpose with r.m.s. deviations of 0.60 Å, 0.31 Å and 0.40 Å, respectively on the equivalent domains of DENV4. However, there are some qualitative differences in the electrostatic surface of ZIKV NS3-Hel when compared to other flaviviruses, including an RNA binding groove that is somewhat less basic (Supplementary Fig. 4). Additional comparison between ZIKV and the other flavivirus NS3-Hel structures highlights flexibility in the relative orientation of domains 1 and 2 (Fig. 2b), and in the conformation of loops implicated in ATP and RNA binding (Supplementary Figs. 3b and 3c). Compared to the apo DENV4 helicase, domain 2 of ZIKV helicase is rotated away from domain 1 by ~13°, leading to a wider ATP binding cleft (Fig. 2b). Interestingly, apo WNV, YFV, KOKV, and JEV also adopt the ZIKV-like partially open conformation ^{11, 12}, whereas DENV2 and MVEV assume the DENV4-like closed conformation ^{13, 14}. The P-loop (residues 196–203) and the RNA binding loop (residues 244–255) in our structure adopt conformations similar to that when DENV4 NS3-Hel binds to ATP and ssRNA - rather than the apoenzyme ⁶. The P-loops in other flavivirus apo structures also assume conformations similar to that in ZIKV (Supplementary Fig. 3b), whereas their RNA binding loops are partially disordered (Supplementary Fig. 3c). Altogether, the P-loop and the RNA-binding loop are the most flexible segments in NS3-Hel structures and intermittently sample conformations of the ligand bound state ¹⁵.

Is the ZIKV NS3-Hel a druggable target? We used the program FTMap to derive druggable “hotspots” on the ZIKV helicase surface ¹⁶. FTMap maps clusters of small organic molecules on a protein surface as putative drug/ligand binding sites. The two most prominent sites on the ZIKV NS3-Hel are between domains 1 and 2 (site 1) and at the confluence of domains 1 and 3 (site 2) (Fig. 3). Site 1 is close to the extra “pyrophosphate” density in our structure, while site 2 is within the RNA binding groove, close to the putative 3’ end of bound RNA. Both pockets possess polar and hydrophobic characteristics and appear well-suited for in-silico high throughput screening with drug-like molecules and/or fragment based screening ^{17, 18}. Also, the close proximity of the two pockets may lend to the design of inhibitors that can span both sites. Several inhibitors have been reported for DENV helicase ¹⁹. It will be interesting to see if they bind the ZIKV helicase and mitigate the ability of the virus to replicate.

In conclusion, we report here a high resolution crystal structure of ZIKV RNA helicase that will aid in the discovery of antiviral compounds against this emerging, pernicious pathogen.

Note added in proof: While this paper was under review, the structure of ZIKV NS3-Hel was published at 1.8 Å resolution²⁰, showing similar structure to that described here but without a pyrophosphate.