ARTICLE
Received 15 Oct 2016 | Accepted 30 Jan 2017 | Published 27 Mar 2017
The recent outbreak of Zika virus (ZIKV) has imposed a serious threat to public health. Here we report the crystal structure of the ZIKV NS5 protein in complex with S-adenosyl-L-homocysteine, in which the tandem methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains stack into one of the two alternative conformations of avivirus NS5 proteins. The activity of this NS5 protein is veried through a de novo RdRp assay on a subgenomic ZIKV RNA template. Importantly, our structural analysis leads to the identication of a potential drug-binding site of ZIKV NS5, which might facilitate the development of novel antivirals for ZIKV.
DOI: 10.1038/ncomms14763 OPEN
The structure of Zika virus NS5 reveals a conserved domain conformation
Boxiao Wang1,*, Xiao-Feng Tan1,*, Stephanie Thurmond2, Zhi-Min Zhang1, Asher Lin1, Rong Hai2 & Jikui Song1
1 Department of Biochemistry, University of California, Riverside, Riverside, California 92521, USA. 2 Department of Plant Pathology and Microbiology, University of California, Riverside, Riverside, California 92521, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to R.H. (email: mailto:[email protected]
Web End [email protected] ) or to J.S. (email: mailto:[email protected]
Web End [email protected] ).
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14763
The outbreak of Zika virus (ZIKV) in the Americas and West Pacic islands in the past year has become a worldwide health concern, affecting more than 60
countries to date1,2. Increasing evidence has linked ZIKV infection to microcephaly in newborn infants3, and to GuillainBarr syndrome in adults4. The fact that no vaccines or therapeutics for prevention or treatment of ZIKV infection are currently available further deepens the concern. To develop effective antivirals against ZIKV infection, it is urgent to gain a comprehensive structural and mechanistic understanding of the molecular machineries underpinning the life cycle of ZIKV.
ZIKV belongs to the family of avivirus that includes a variety of mosquito-borne human pathogens, such as dengue virus (DENV14), yellow fever virus, West Nile virus, Spondweni virus and Japanese encephalitis virus (JEV)5. The genome of ZIKV is organized into the form of a single positive strand RNA, encoding in total three structural proteins (C, prM/M and E) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5)6. Among these, NS5 is the largest NS protein, containing an N-terminal methyltransferase (MTase) domain responsible for viral RNA capping and a C-terminal RNA-dependent RNA polymerase (RdRp) domain for viral RNA synthesis, with evidence indicating that the MTase and RdRp domains cooperate in RNA synthesis initiation and elongation7. In addition, NS5 proteins have been shown to inhibit the type I interferon (IFN) signalling to evade antiviral defence in the host816. The essential role of NS5 in viral replication and immunosuppression makes it an ideal target for antivirals17. However, the molecular mechanism underlying the enzymatic action of NS5 remains poorly understood. Thus far, crystal structures of full-length NS5 proteins have only been reported for JEV and DENV3 (refs 18,19). Intriguingly, despite with B65%
sequence identity (Supplementary Fig. 1), JEV NS5 and DENV3 NS5 adopt different orientations between the MTase and RdRp domains18,19, raising the question of how the MTase and RdRp domains of NS5 cooperate during RNA replication and capping.
To illuminate the structure and mechanism of NS5 proteins, and, more importantly, to explore potential druggable sites for ZIKV, we determined the crystal structure of full-length ZIKV NS5 in complex with S-adenosyl-L-homocysteine (SAH), by-product of cofactor S-adenosyl-L-methionine at 3.3 resolution. Our structural analysis reveals that ZIKV NS5 folds into one of the two alternative conformations of avivirus NS5 proteins, providing functional implication for the conformational dynamics of avivirus NS5 proteins. We further verify the enzymatic activity of ZIKV NS5 through a de novo RdRp assay using a subgenomic ZIKV RNA as template. Finally, we show that the structure of ZIKV NS5 provides a framework for future development of novel antivirals against ZIKV infection.
ResultsOverall structure of ZIKV NS5. We were able to trace nearly the entire sequence of ZIKV NS5 (Fig. 1a), except for the rst ve N-terminal residues, residues 747748 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 b-sheet sandwiched by two a-helices from one side and another a-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 (a1a4 in Supplementary Fig. 1) of the MTase domain pairs with its C-terminal extension sequence (a8b9 in Supplementary Fig. 1) to form a helix bundle and an antiparallel two-stranded b-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
a
MTase RdRp
Palm FingersPalm
Priming loop
Fingers
MTase NE Palm Thumb
Fingers
1 266 275 304 369 397 495 576 597 720 888 903
Thumb 786 810
b
N
MTase
MTase
90
SAH
SAH
Domain linker
NE
Fingers Palm
Thumb
RdRp
C
RdRp
Priming loop
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, ngers, 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.
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14763 ARTICLE
a b
~100
ZIKV NS5 JEV NS5
ZIKV NS5 DENV3 NS5
MTase
MTase
RdRp RdRp
NTP entrance
NTP entrance
RNA exit RNA exit
c
Q117
Q117
L115
P113
P113
W121
W121
F351
e
E252 D254
Y350
P584
F466
d
R354
R68
Q63
W
D256
Y119
W
L115
F348 E67 R352 E356 K357
E299
P585
F467
R355
Figure 2 | Structural comparison of NS5 proteins from ZIKV and two other aviviruses. 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 B100 change in orientation between the MTase and RdRp domains. The NTP entrance and RNA exit sites are labelled. (ce) The MTaseRdRp domain interactions of (c) ZIKV NS5, (d) JEV NS5 and (e) DENV3 NS5.
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 B1,400 2. 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 over 242 Ca atoms, indicating that the MTaseRdRp association does not lead to considerable conformational change of the MTase domain.
Structural comparison with other avivirus NS5 proteins. ZIKV NS5 shares B68% and B66% 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 over 872 Ca atoms (Fig. 2a). In particular, the
MTaseRdRp 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 ngers 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 of6.06 over 844 Ca 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 MTaseRdRp association of DENV3 is mediated by a set of hydrogen bonding, cation-p and electrostatic interactions between the N- (Q63, E67 and R68) and C-terminal extensions (E252 and D254) of the MTase domain and the Index nger (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 B100 rotation of the MTase
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14763
a
5-Noncoding region 3-Noncoding region
10,343
Coding region
171
b c
ZIKV NS5
Mock
ZIKV NS3-Hel
2 3 4 1 2 3 4
Marker
ZIKV NS3-Hel
ZIKV NS5
1
100 kDa
75 kDa
50 kDa
Subgenomic ZIKV RNA
37 kDa
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) SDSpolyacrylamide gel electrophoresis analysis of puried 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).
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 nger and motif G in the Pinky nger22,23 (Fig. 2b). The conformations of these two motifs appear to be stabilized by the MTaseRing nger association of ZIKV/JEV NS5, but become disordered in DENV3 NS5 due to the loss of the corresponding interactions (Supplementary Fig. 3b).
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 specic 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 avivirus NS5 proteins.
De novo RdRp assay of ZIKV NS5. To conrm 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 conrm 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.
Identication 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, identied 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.
DiscussionOur structural study of ZIKV NS5 sheds light onto the conformational dynamics and functional regulation of ZIKV NS5. The observation that ZIKV NS5 adopts one of the two dened conformations of avivirus NS5 proteins provides rst evidence on the conservation of domain conformation within NS5 proteins, which may be required for their functional regulation. To conrm the enzymatic activity of the recombinant ZIKV NS5 protein, we developed a de novo RdRp assay of ZIKV NS5 on a subgenomic ZIKV RNA template, which provides basis for future mechanistic characterization of ZIKV NS5. Furthermore, we identied that the small molecular inhibitor-binding site of DENV3 NS5 is structurally conserved in ZIKV NS5, thereby revealing a potential mechanism for functional inhibition of ZIKV NS5. This study provides a foundation for
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14763 ARTICLE
a b
R737
M761
W803
T794
Q802
Y766 M765
S796
R739
T796
M763 W805
E804
Y768
S798
R729
S710
H711
R731
S712
G801
A799
H512 H800
H713
K802
Q514
L513
L511
H800
H798
L514
C711
L516
C709
future dissection of the functional coupling between the MTase and RdRp domains and a framework for the design of novel inhibitors against ZIKV infection.
Methods
Expression and purication of ZIKV NS5 and NS3 helicase. The DNA sequence encoding full-length ZIKV NS5 or ZIKV NS3-Hel (residues 171617) was amplied from the cDNA of ZIKV/Macaca mulatta/UGA/MR-766/1947 and inserted into a modied pRSFDuet-1 vector (Novagen) (see Supplementary Table 1 for primer sequences), in which the NS5 or NS3-Hel gene was preceded by an N-terminal His6-
SUMO tag and ULP1 (ubiquitin-like protease 1) cleavage site. The obtained plasmids were then transformed into BL21 (DE3) RIL cell strain (Agilent Technologies) for expression. The cells were rst grown at 37 C and then shifted to room temperature when A600 reached 1.0, followed by the addition of 0.4 mM isopropyl b-D-galactoside for induction. After another 18 h of cell growth, the cells were collected and the His6-
SUMO-tagged ZIKV NS5 or ZIKV NS3-Hel was puried using a Ni-NTA afnity column. ZIKV NS5 was further puried on a Phenyl Sepharose column (GE Healthcare) for separation from degraded protein products, followed by removal of the His6-SUMO tag through ULP1 cleavage and size-exclusion chromatography on a
Superdex 200 16/600 column (GE Healthcare) pre-equilibrated in buffer containing 25 mM Tris, pH 7.5, 500 mM NaCl, 5 mM DTT (dithiothreitol) and 5% glycerol. The ZIKV NS3-Hel fusion protein was rst subject to ULP1 cleavage, and subsequently puried on a Phenyl Sepherase column and a Superdex 200 16/600 column preequilibrated in buffer containing 25 mM Tris, pH 7.5, 250 mM NaCl, 5 mM DTT and 5% glycerol. SDSpolyacrylamide gel electrophoresis analysis indicated that the purities of NS5 and NS3-Hel proteins were 495% and 490%, respectively. Protein solution of puried NS5 and NS3-Hel, with concentrations of B20 andB70 mg ml 1, respectively, were stored at 80 C.
Crystallization and X-ray data collection. Full-length ZIKV NS5 was mixed with SAH and GTP in a 1:3:3 molar ratio for complex formation. Initial crystallization conditions were identied through sparse-matrix screens (Hampton Research Inc.). The crystals were subsequently reproduced by hanging-drop vapour diffusion method at 4 C, from drops mixed from 1 ml of ZIKV NS5 and 1 ml of precipitant solution (0.70.9 M lithium sulfate, 0.1 M MES, pH 67). Crystals were soaked for 1 min in a cryoprotectant solution, comprising of crystallization buffer and 20% glycerol, before ash frozen in liquid nitrogen.
The X-ray diffraction data for ZIKV NS5 were collected on the BL 5.0.3 beamline at the Advanced Light Source, Lawrence Berkeley National Laboratory. The diffraction data were indexed, integrated and scaled using the HKL2000 program26. The structure was solved using the molecular replacement method in PHASER27, with the structure of Japanese encephalitis virus NS5 (PDB ID: 4K6M) as search model. The resulting electron density revealed that there are two molecules of ZIKV NS5 in each asymmetric unit. Despite being present in the crystallization mixture, GTP molecules were not modelled, presumably due to low occupancy under the crystallization condition. The structure of ZIKV NS5 was improved by iterative model building and renement with Coot28 and PHENIX29 software packages. The same R-free test set was used throughout the renement. The statistics for data collection and structural renement of ZIKV NS5 is summarized in Table 1.
De novo RdRp assay. For ZIKV, the de novo RdRp reaction (20 ml) contained 50 mM Tris (pH 8.0), 10 mM NaCl, 5 mM MgCl2, 2 mM MnCl2, 10 mM DTT,0.5 mM ATP, 0.5 mM UTP, 0.5 mM GTP, 5 mM CTP, 15 mCi of [a-32P]
(10 mCi ml 1, 3,000 Ci mmol 1; Perkin-Elmer), 1 mg of RNA template and 2 mg of ZIKV NS5 protein or ZIKV NS3-Hel. The RNA template was in vitro transcribed from a PCR product using T7 polymerase (New England BioLabs). The PCR product contained a T7 promoter, followed by a cDNA fragment representing a ZIKV subgenome with deletion of nucleotides 17110,343 (GenBank accession no. KU963573.2). The de novo RdRp reaction mixtures were incubated at 23 C for 30 min, or 33 C for 30, 60 and 120 min. The nal reactions were further extracted with phenolchloroform and precipitated with isopropanol. The RNA pellet was dissolved in 20 ml of 1 denaturing gel loading dye, and loaded onto a 10%
denaturing polyacrylamide gel with 7 M urea. 32P-labelled RNA results were detected via the autoradiograph of the PAGE gel.
Data availability. Coordinates and structure factors for ZIKV NS5SAH complexes have been deposited in the Protein Data Bank under accession code 5TMH. The PDB accession codes 5TMH, 5JJR, 4K6M and 4V0Q and GenBank entry KU963573.2 were used in this study. All other data are available from the corresponding authors on reasonable request.
Figure 4 | Identication of a potential inhibitor-binding site in ZIKV NS5. (a) Binding of a small-molecule inhibitor of DENV3 NS5 at its N pocket. The residues of DENV3 NS5 and small-molecule inhibitor are shown in blue and yellow sticks, respectively. The hydrogen bonding interactions are depicted as dashed lines. (b) The residues of ZIKV NS5 corresponding to the inhibitor binding site of DENV3 NS5 are shown in pink sticks.
Table 1 | Data collection and renement statistics.
ZIKV NS5
Data collection
Space group P2 21 21
Cell dimensionsa, b, c () 95.1, 136.5, 197.0 a, b, g () 90, 90, 90
Wavelength 0.9764 Resolution () 50.003.30 (3.423.30)* Rsym or Rmerge 31.3 (83.5)
I/sI 5.3 (1.7)
Completeness (%) 99.4 (97.1)
Redundancy 6.2 (5.7)
CC1/2 0.979 (0.748)
Renement
Resolution () 48.33.28 (3.363.28)
No. of reections 39,405 (3,511)Rwork/Rfree 0.262/0.293 (0.344/0.405)
No. of atoms
Protein 13,391 Ligand 38B factors
Protein 54.12 Ligand 68.85 r.m.s.d.
Bond lengths () 0.002 Bond angles () 0.55
*Values within parentheses are for highest-resolution shell.
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Acknowledgements
This work was supported by March of Dimes Foundation (1-FY15-345),Kimmel Scholar Award from Sidney Kimmel Foundation for Cancer Research and NIH (1R35GM119721) (to J.S.). The Berkeley Center for Structural Biology is supported,in part, by the National Institutes of Health, National Institute of General Medical Sciences and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Ofce of Science, Ofce of Basic Energy Sciences and of the US Department of Energy under Contract No DE-AC02-05CH11231.
Author contributions
B.W., S.T. and R.H. cloned ZIKV NS5. B.W., A.L. and J.S. puried the protein sample. B.W. and X.-F.T. crystallized ZIKV NS5 and collected the X-ray data set. X.-F.T. andZ.-M.Z. determined the crystal structure. R.H. and S.T. performed enzymatic assay. R.H. and J.S. organized the study. J.S. prepared the manuscript with input fromall the authors.
Additional information
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How to cite this article: Wang, B. et al. The structure of Zika virus NS5 reveals a conserved domain conformation. Nat. Commun. 8, 14763 doi: 10.1038/ncomms14763 (2017).
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r The Author(s) 2017
6 NATURE COMMUNICATIONS | 8:14763 | DOI: 10.1038/ncomms14763 | http://www.nature.com/naturecommunications
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Copyright Nature Publishing Group Mar 2017
Abstract
The recent outbreak of Zika virus (ZIKV) has imposed a serious threat to public health. Here we report the crystal structure of the ZIKV NS5 protein in complex with S-adenosyl-L-homocysteine, in which the tandem methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains stack into one of the two alternative conformations of flavivirus NS5 proteins. The activity of this NS5 protein is verified through a de novo RdRp assay on a subgenomic ZIKV RNA template. Importantly, our structural analysis leads to the identification of a potential drug-binding site of ZIKV NS5, which might facilitate the development of novel antivirals for ZIKV.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer