The crystal structure of Zika virus NS 5 reveals conserved drug targets
2017; Springer Nature; Volume: 36; Issue: 7 Linguagem: Inglês
10.15252/embj.201696241
ISSN1460-2075
AutoresWenqian Duan, Hao Song, Haiyuan Wang, Yan Chai, Chao Su, Jianxun Qi, Yi Shi, George F. Gao,
Tópico(s)Insect symbiosis and bacterial influences
ResumoArticle2 March 2017free access Transparent process The crystal structure of Zika virus NS5 reveals conserved drug targets Wenqian Duan CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Hao Song Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Haiyuan Wang CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China College of Animal Sciences and Technology, Guangxi University, Nanning, China Search for more papers by this author Yan Chai CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chao Su CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China College of Veterinary Medicine, China Agricultural University, Beijing, China Search for more papers by this author Jianxun Qi CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yi Shi Corresponding Author [email protected] orcid.org/0000-0002-3053-2687 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China Center for Influenza Research and Early-warning (CASCIRE), Chinese Academy of Sciences, Beijing, China Shenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third People's Hospital, Shenzhen, China Search for more papers by this author George F Gao Corresponding Author [email protected] orcid.org/0000-0002-3869-615X CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China Center for Influenza Research and Early-warning (CASCIRE), Chinese Academy of Sciences, Beijing, China Shenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third People's Hospital, Shenzhen, China National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), Beijing, China Search for more papers by this author Wenqian Duan CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Hao Song Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Haiyuan Wang CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China College of Animal Sciences and Technology, Guangxi University, Nanning, China Search for more papers by this author Yan Chai CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chao Su CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China College of Veterinary Medicine, China Agricultural University, Beijing, China Search for more papers by this author Jianxun Qi CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yi Shi Corresponding Author [email protected] orcid.org/0000-0002-3053-2687 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China Center for Influenza Research and Early-warning (CASCIRE), Chinese Academy of Sciences, Beijing, China Shenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third People's Hospital, Shenzhen, China Search for more papers by this author George F Gao Corresponding Author [email protected] orcid.org/0000-0002-3869-615X CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China Center for Influenza Research and Early-warning (CASCIRE), Chinese Academy of Sciences, Beijing, China Shenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third People's Hospital, Shenzhen, China National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), Beijing, China Search for more papers by this author Author Information Wenqian Duan1,2,‡, Hao Song3,‡, Haiyuan Wang1,4, Yan Chai1, Chao Su1,5, Jianxun Qi1, Yi Shi *,1,2,3,6,7 and George F Gao *,1,2,3,6,7,8 1CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 2Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China 3Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China 4College of Animal Sciences and Technology, Guangxi University, Nanning, China 5College of Veterinary Medicine, China Agricultural University, Beijing, China 6Center for Influenza Research and Early-warning (CASCIRE), Chinese Academy of Sciences, Beijing, China 7Shenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third People's Hospital, Shenzhen, China 8National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), Beijing, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 10 64806050; E-mail: [email protected] *Corresponding author. Tel: +86 10 64807688; E-mail: [email protected] EMBO J (2017)36:919-933https://doi.org/10.15252/embj.201696241 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Zika virus (ZIKV) has emerged as major health concern, as ZIKV infection has been shown to be associated with microcephaly, severe neurological disease and possibly male sterility. As the largest protein component within the ZIKV replication complex, NS5 plays key roles in the life cycle and survival of the virus through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains. Here, we present the crystal structures of ZIKV NS5 MTase in complex with an RNA cap analogue (m7GpppA) and the free NS5 RdRp. We have identified the conserved features of ZIKV NS5 MTase and RdRp structures that could lead to development of current antiviral inhibitors being used against flaviviruses, including dengue virus and West Nile virus, to treat ZIKV infection. These results should inform and accelerate the structure-based design of antiviral compounds against ZIKV. Synopsis The crystal structures of ZIKV NS5 MTase in complex with an RNA cap analogue (m7GpppA) and the free NS5 RdRp reveal conserved features of ZIKV MTase and RdRp structures that will aid in the structure-based design of antiviral compounds against ZIKV. Crystal structure of Brazilian ZIKV NS5 MTase bound to S-adenosyl-L-methionine (SAM) reveals a conserved SAM-binding pocket. The tertiary complex structure of ZIKV MTase with SAM and RNA analogue (m7GpppA) reveals both conserved and specific features on the cap-binding site for antiviral inhibitor design. ZIKV NS5 RdRp structure reveals a tighter and more closed conformation compared to other flaviviruses. ZIKV NS5 RdRp possesses conserved drug binding sites including the RNA template entry tunnel and N-pocket, implicating current available drugs targeting Dengue virus RdRp should be tested for anti-ZIKV activity. Introduction Zika virus (ZIKV), a mosquito-borne flavivirus, has become a major public health concern over the past year (Calvet et al, 2016). ZIKV infection in pregnant women can cause congenital malformations including fetal and newborn microcephaly, and serious neurological complications, such as Guillain–Barré syndrome (Carteaux et al, 2016; Driggers et al, 2016; Mecharles et al, 2016; Mlakar et al, 2016; Zhang et al, 2016). In addition to transmission by mosquitoes, ZIKV has been shown to be able to establish long-term persistent infection (Mansuy et al, 2016a) and be transmitted by sexual activity (Davidson et al, 2016; Deckard et al, 2016; Prisant et al, 2016). More importantly, ZIKV was detected in human semen and spermatozoa (Mansuy et al, 2016b), and ZIKV infections of male adult mice can cause testicular and epididymal damage, resulting in cell death and destruction of the seminiferous tubules (Govero et al, 2016; Ma et al, 2016). These findings pose new challenges for controlling outbreaks caused by this virus. However, there is currently no available drug approved to treat or prevent ZIKV infections. Given this urgent situation, an important strategy to prevent the spread of ZIKV would be to develop antivirals to inhibit viral protein activities, which are central to survival of the virus. In particular, antivirals could also be used to avoid fetal neurological disorders or even male sterility induced by ZIKV infections. A good target for designing antiviral inhibitors is the viral replication machinery, inhibition of which could block viral replication. Like other flaviviruses, such as Dengue virus (DENV), Japanese encephalitis virus (JEV), Yellow fever virus (YFV), West Nile virus (WNV) and tickborne encephalitis virus (TBEV), ZIKV is an enveloped, single-stranded, positive-sense RNA virus carrying a cap-1 structure (m7G0pppAm2′-O-G-RNA) at its 5′ end. The RNA genome of ZIKV translates into a long polyprotein in the cytoplasm of the infected cells. The polyprotein is further cleaved and processed by either host or viral proteases into three structural and seven nonstructural (NS) proteins. The structural proteins are precursor membrane (prM) protein, envelope (E) protein, capsid (C) protein, which constitute the virus particle, and seven NS proteins include NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, which perform essential functions in genome replication, polyprotein processing and manipulation of cellular processes for virus infections. RNA replication occurs within a multi-protein replication complex (RC) comprising of both NS proteins and host cofactors, which assemble on endoplasmic reticulum (ER)-derived membranes (Welsch et al, 2009). Among them, NS5 is the largest enzyme and the most conserved protein of the RC. NS5 carries two essential enzymatic activities, methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp), which are strictly required for the viral replication, and therefore constitute promising targets for the development of antiviral compounds. NS5 MTase locates at the N-terminus of the NS5 protein, methylates guanosine N-7 and ribose 2′-O positions of the viral RNA cap, which is crucial for genome stability, efficient translation and evasion of the host immune response (Zhou et al, 2007). MTases from various flaviviruses perform the two cap methylations in a sequential manner: N-7 methylation followed by 2′-O-methylation, that is, G0pppAG-RNA–>m7G0pppAG-RNA–>m7G0pppAm2′-O-G-RNA, using S-adenosyl-L-methionine (SAM) as the methyl donor and generate S-adenosyl-L-homocysteine (SAH) as a by-product (Zhao et al, 2015). N-7-MTase activity has been shown to be essential for viral replication by biochemical studies and reverse genetic analysis, while 2′-O-MTase defective viruses can replicate but are highly attenuated (Dong et al, 2010a; Zust et al, 2011, 2013). Therefore, suppression of both N7- and 2′-O-MTase activities is important for the development of antivirals. The C-terminus of the NS5 protein is the RdRp that synthesizes the genome RNA in the absence of a primer strand, which is called a de novo initiation mechanism. Flavivirus RdRp contains functional nuclear localization sequence (NLS), which is the key region for interactions with other viral and host proteins. NS5 interacts with the NS3 protease-helicase and several host proteins, including β-importin (Yap et al, 2007; Tay et al, 2015). In addition to its enzymatic functions, NS5 acts as an antagonist of the host interferon response by interacting with and promoting the degradation of the signal transducer and activator of transcription 2 protein (Ashour et al, 2009), and the same mechanism has been proven in ZIKV recently (Grant et al, 2016; Kumar et al, 2016). The importance of NS5 in viral replication and host immune response modulation makes it an attractive target for developing broad-spectrum antiviral inhibitors. During the preparation of our manuscript, Javier Coloma et al have reported the high-resolution crystal structures of NS5 MTase from the H/PF/2013 strain (isolated from French Polynesia) in complex with SAM or SAM and 7-methyl guanosine diphosphate (m7Gpp) (Coloma et al, 2016). Here, we have studied the NS5 protein of the BeH819015 strain (isolated from Brazil in 2015, responsible for the ZIKV outbreak in South America), and determined the crystal structure of the NS5 MTase at a resolution of 2.5 Å in the presence of bound SAM and the complex structure of ZIKV MTase with both SAM and a bigger RNA analogue (m7GpppA) at a resolution of 2.6 Å, and the NS5 RdRp at a resolution of 1.8 Å. These studies provide important structural information to help guide the development of antiviral compounds against ZIKV NS5. Results Structure of ZIKV NS5 MTase bound to an RNA cap analogue m7GpppA The crystal structure of ZIKV NS5 MTase was solved by molecular replacement to a resolution of 2.5 Å, with Rwork and Rfree values of 23.5 and 25.7%, respectively (Table 1). The ZIKV MTase has an overall globular fold and can be subdivided into three subdomains (Fig 1A). The subdomain 1 (N-terminal extension) consists of a helix-turn-helix motif followed by a strand and a helix. The subdomain 2 forms the core structure, adopts the SAM-dependent methyltransferase fold and folds into a seven-stranded β-sheet surrounded by four α-helices. The subdomain 3 (C-terminal extension) comprises an α-helix and two β-stands (Fig 1A and B). Although no SAM or SAH was added during purification or crystallization, SAM, the methyl donor for the methylation reaction of MTase, is co-purified and clearly visible in the electron density in the active site (Fig 1A). This was also observed in other MTase structures (Mastrangelo et al, 2007; Bollati et al, 2009a,b; Jansson et al, 2009). The core domain contains a binding site for SAM, referred to here as the SAM-binding pocket (Fig 1C). The SAM molecule binds to the SAM-binding pocket mainly by hydrogen-bonding interactions and van der Waals interactions, contacting with residues S56, G86, W87, K105, H110, D131, V132 and D146 (Figs 1C and 2), which are conserved among different flavivirus MTases (Fig 1D). Table 1. Data collection and refinement statistics ZIKV NS5 MTase (PDB code 5WZ1) ZIKV NS5 MTase with RNA analogue (PDB code 5WZ2) ZIKV NS5 RdRp (PDB code 5WZ3) Data collection Wavelength (Å) 0.97853 0.97907 0.72929 Space group P 21 C 2 P 21 Cell dimensions a, b, c (Å) 107.55, 86.85, 107.56 75.13, 78.27, 136.38 63.53, 84.85, 69.20 α, β, γ (°) 90.00, 97.39, 90.00 90.00, 90.10, 90.00 90.00, 113.37, 90.00 Resolution (Å) 50.00–2.50 (2.59–2.50)aa Values in parentheses are for highest resolution shell. 50.00–2.60 (2.69–2.60) 50.00–1.80 (1.86–1.80) R merge bb Rmerge = ΣiΣhkl|Ii− |/ΣiΣhklIi, where Ii is the observed intensity and is the average intensity from multiple measurements. 0.195 (1.085) 0.161 (0. 815) 0.078 (0.612) I/σI 8.075 (1.409) 9.362 (1.953) 22.098 (3.064) CC 1/2 0.997 (0.695) 0.993 (0.640) 0.994 (0.823) Completeness (%) 98.6 (98.2) 99.8 (98.6) 98.5 (92.5) Redundancy 5.7 (5.9) 4.7 (4.5) 6.1 (5.2) Refinement Resolution (Å) 45.45–2.50 42.46–2.60 34.16–1.80 No. reflections 63,210 23,751 61,141 Rwork/Rfreecc Rwork = Σ | |Fo|−|Fc| |/Σ |Fo|, where Fo and Fc are the structure-factor amplitudes from the data and the model, respectively. Rfree is the R factor for a subset (5%) of reflections that was selected prior to refinement calculations and was not included in the refinement. 0.235/0.257 0.202/0.247 0.178/0.206 No. atoms Protein 16,168 6,297 4,641 Ligand/ion 216 234 2 Water 0 90 503 B-factors Protein 40.6 48.4 27.6 Ligand/ion 34.0 60.6 18.3 Water – 40.4 34.2 Rms. deviations Bond lengths (Å) 0.004 0.004 0.005 Bond angles (°) 0.719 0.913 0.958 Ramachandran plot Favored (%) 95.57 96.76 97.30 Allowed (%) 4.23 2.72 2.52 Outliers (%) 0.19 0.52 0.18 a Values in parentheses are for highest resolution shell. b Rmerge = ΣiΣhkl|Ii− |/ΣiΣhklIi, where Ii is the observed intensity and is the average intensity from multiple measurements. c Rwork = Σ | |Fo|−|Fc| |/Σ |Fo|, where Fo and Fc are the structure-factor amplitudes from the data and the model, respectively. Rfree is the R factor for a subset (5%) of reflections that was selected prior to refinement calculations and was not included in the refinement. Figure 1. Structure of ZIKV MTase in complex with the cofactor SAM Cartoon representation of ZIKV methyltransferase colored by three subdomains: subdomain 1 (magenta), subdomain 2 (green) and subdomain 3 (orange). The cofactor SAM is shown as sticks. The secondary structure element nomenclature corresponds to the DENV2 MTase (Egloff et al, 2002). Topology diagram for ZIKV MTase. The colors are coherent with the above representation of the structure. The electrostatic surface potential of ZIKV MTase. Red and blue colors indicate negative potential and positive potential, respectively. The cofactor SAM is shown as sticks colored in yellow. Surface representation of the ZIKV MTase colored according to sequence conservation from the most conserved (dark magenta) to the most divergent (dark cyan) based on an alignment of MTase sequences from 73 flaviviruses using the ConSurf server (Ashkenazy et al, 2016; Xu et al, 2016). Download figure Download PowerPoint Figure 2. Detailed interaction of ZIKV MTase bound to the cofactor SAM and the RNA analogue m7GpppAOverall structure showing ZIKV MTase in complex with the cofactor SAM and the RNA analogue (m7GpppA). The 2Fo−Fc electron density map for SAM or RNA analogue contoured at 1.0 sigma is represented, respectively, in gray. The hydrogen bond interactions of ZIKV MTase bound to SAM and m7GpppA are shown in dashed lines. Download figure Download PowerPoint The overall structure of the ZIKV MTase is highly like other flaviviruses MTase structures, with root-mean-square differences (rmsd) of 0.41–0.83 Å (Fig EV1). Of note, among these structures, three regions, residues 48–52, 173–l77 and 245–249, show the greatest structural differences. The differences are partially due to the insertion or deletion of amino acid residues in these loops. Besides the SAM pocket, the core domain contains a pocket that binds to the GTP cap of the viral RNA, referred to as the cap-binding site, and a positively charged groove between these two pockets referred to as the RNA-binding site (Fig 1C). We compared the ZIKV MTase structure with all the other available flavivirus MTases structures, including DENV2, YFV, DENV3, WNV, Murray Valley encephalitis virus (MVEV), Wesselsbron virus (WESSV), Modoc virus (MODV), Meaban virus (MEAV), Yokose virus (YOKV) and JEV (Egloff et al, 2002; Assenberg et al, 2007; Mastrangelo et al, 2007; Zhou et al, 2007; Bollati et al, 2009a,b; Geiss et al, 2009; Jansson et al, 2009; Lu & Gong, 2013; Coutard et al, 2014). The electrostatic surface potential maps of these proteins show very similar patterns, especially the positively charged surface in the RNA-binding site (Fig EV2). We then analyzed the conserved features based on the alignment of MTase sequences from 73 flaviviruses (Fig 1D). The most conserved regions are the three binding sites: the SAM-binding pocket, cap-binding pocket and RNA-binding site, indicating a conserved methylation mechanism for different flaviviruses. Click here to expand this figure. Figure EV1. Superposition of the ZIKV MTase structure with other flavivirus MTase structuresRibbon representation of each structure is colored separately (ZIKV, green; DENV2, PDB: 1L9K, light blue; WNV, PDB: 2OY0, cyan; YFV, PDB: 3EVC, magenta; DENV3, PDB: 4CTJ, blue; MVEV, PDB: 2PX2, pink; WESSV, PDB: 3ELU, orange; MODV, PDB: 2WA1, deep olive; MEAV, PDB: 2OXT, red; YOKV, PDB: 3GCZ, purple; JEV, PDB: 4K6M, yellow). The loops that show significant differences are labeled. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Comparison of the electrostatic surfaces of ZIKV MTase structure with other flavivirus MTase structuresRed and blue colors indicate negative potential and positive potential, respectively. SAM and SAH are shown as sticks and colored in yellow and cyan, respectively. PDB codes of structures used for analysis are described in the Fig EV1 legend. Download figure Download PowerPoint To elucidate the molecular mechanism of ZIKV MTase in recognizing capped RNA and to know the detailed mechanism of RNA analogue inhibition, we determined the crystal structure of ZIKV MTase in complex with a RNA analogue at 2.6 Å, with Rwork and Rfree values of 20.5 and 25.5%, respectively (Table 1). Little conformational changes of the protein were observed upon RNA analogue binding. The RNA analogue binds to the cap-binding site, formed by helices A1, A2 and a loop (amino acids 208–218), in a stacked conformation (Fig 2). The second nucleobase A1 stacks against the first nucleobase 7mG0, displaying a hairpin-like shape. The aromatic side chain of residue F24 arranges in parallel with the two base moieties of 7mG0 and A1, below the 7mG0, forming strong π–π interaction. The exocyclic 2′-amine group of 7mG0 interacts via hydrogen bonding with the main-chain carbonyl groups of residues L16, N17 and M19, while the 2′-hydroxyl group is hydrogen-bonded to the amino group of residue K13 side chain and the oxygen of residue N17 main-chain. The amino group K13 side chain also forms a hydrogen bond with the 3′-hydroxyl group of 7mG0 ribose. The 2′-hydroxyl group of the A1 ribose interacts via hydrogen bond with the hydroxyl group of residue S152. In addition, the first phosphate of triphosphate forms a hydrogen bond with residue S215 (Fig 2). Interestingly, we found that ZIKV MTase contains nonpolar hydrophobic residues A21 and L22 near the cap-binding site, while in other flaviviruses they are all polar residues (DENV2: K22/S23; DENV3: R22/K23; WESSV: K21/Q22) at the same sites (Fig 3). Moreover, the shorter side chain of A21 in ZIKV MTase compared to Arg or Lys in other flaviviruses makes the ZIKV MTase cap-binding pocket more shallow and "open". Figure 3. Comparison of the structures of ZIKV MTase with other flavivirus MTases in complex with SAM/SAH and the RNA molecule A–D. The ZIKV MTase in complex with the cofactor SAM and the RNA analogue m7GpppA (A), DENV2 MTase in complex with SAH and m7GpppA (PDB: 2P3O) (Egloff et al, 2007) (B), DENV3 MTase in complex with SAH and m7GpppAGUUGUU (PDB: 5DTO) (Zhao et al, 2015) (C) and WESSV MTase in complex with SAM and m7GpppG (PDB: 3EMB) (Bollati et al, 2009b) (D) are shown respectively. The RNA molecule, SAM, and SAH are shown as sticks and colored in white, yellow and cyan, respectively. The variable residues near the capping binding site are highlighted in dotted boxes and labeled. Download figure Download PowerPoint As the host also has RNA MTases for methylation, we compared our ZIKV MTase structure with human RNA guanine-7 methyltransferase (RNMT) structure in complex with its activating subunit, RNMT-activating miniprotein (RAM) (Varshney et al, 2016), and mRNA 2′-O-methylation MTase CMTr1 (Smietanski et al, 2014) (Fig 4). For SAM-binding pocket, ZIKV MTase has a conserved additional hydrophobic pocket located next to the binding site (Fig 4B). This pocket has been proven to be important both for viral replication and cap methylations and for specific inhibitor design targeting flavivirus MTase (Dong et al, 2010b). For the cap-binding site, cellular and viral MTase interact with the guanosine cap in very different ways. In the structure of the human CMTr1, m7G is bound in a deep pocket, and the orientation and binding mode is quite different from m7G binding to ZIKV MTase (Fig 4E and F). Therefore, both the SAM-binding pocket and cap-binding site have diverse properties between the cellular and viral enzymes, making the ZIKV MTase an attractive target for the development of inhibitors. Figure 4. Comparison of the structures of ZIKV MTase with human RNA MTases A–F. The ZIKV MTase in complex with the cofactor SAM and the RNA analogue m7GpppA shown in cartoon (A) and electrostatic surface (B). Human RNA guanine-7 methyltransferase RNMT-RAM in complex with SAH (PDB: 5E8J) (Varshney et al, 2016) shown in cartoon (C) and electrostatic surface (D). Human 2′-O-ribose MTase CMTr1 in complex with a methyl group donor SAM and a capped oligoribonucleotide (PDB: 4N48) (Smietanski et al, 2014) shown in cartoon (E) and electrostatic surface (F). The RNA molecule, SAM, and SAH are shown in sticks and colored in magenta, yellow and cyan, respectively. Download figure Download PowerPoint Structure of ZIKV NS5 RdRp The crystal structure of ZIKV NS5 RdRp was solved here by molecular replacement to a resolution of 1.8 Å, with Rwork and Rfree values of 17.8 and 20.6%, respectively (Table 1). The ZIKV RdRp displays an overall spherical fold, and has the canonical right-hand conformation consisting of fingers (α1-α9 from residues 275 to 498, and α12 to β3 from residues 544 to 607), palm (α10, α11 and 310 helix η1 from residues 499 to 543, and α14 to 310 helix η2 from residues 608 to 709) and thumb (β6 to α23 from residues 710 to 887) subdomains (Fig 5A). The ZIKV RdRp fold is analogous to that of other flaviviruses like DENV, WNV and JEV (Malet et al, 2007; Yap et al, 2007; Lu & Gong, 2013). One special characteristic of flavivirus RdRp is that it contains the NLS region, which is distributed between the fingers and thumb subdomains, consisting of βNLS and α/βNLS sequences. In ZIKV RdRp structure, the βNLS (residues 316–367) including a helix-turn-helix motif (α2-α3) lies on top of the thumb subdomain, and the α/βNLS (residues 368–415) consisting of helices α5 and α6 are located between the fingers and the palm subdomain (Fig 5A). It has been shown that the NLS region is necessary for DENV NS5 structure stabilization (Yap et al, 2007). The NLS could be recognized by cellular factors such as β-importin to transport NS5 to nucleus, which is helpful for viral replication (Uchil et al, 2006). This region is also thought to interact with NS3 helicase, which may modulate their respective enzymatic activities (Johansson et al, 2001). Figure 5. Structure of ZIKV NS5 RdRp A–C. Ribbon representation of the overall fold with the three subdomains: fingers (sky blue), palm (green) and thumb (light pink) (A). The putative NLS region is colored in yellow. The priming loop is highlighted by an arrow. The catalytic residues are indicated by sticks. The two zinc-binding sites are highlighted by dashed circles, and detailed interactions are shown as sticks (B and C). The zinc ions are displayed as gray spheres, and the water interacting with the zinc ion is displayed as pink sphere. The 2Fo−Fc electron density maps contoured at 1.0 sigma are represented respectively in gray. D. Surface representation of the ZIKV RdRp colored according to sequence conservation from the most conserved (dark magenta) to the most divergent (dark cyan) based on an alignment of RdRp sequences from 73 flaviviruses using the ConSurf server (Ashkenazy et al, 2016; Xu et al, 2016). E. The electrostatic surface potential of ZIKV RdRp. Red and blue colors indicate negative potential and positive potential, respectively. Download figure Download PowerPoint There are four flexible loops in the fingers subdomain (β1-α2 loop, α3-α4 loop, α6-α7 loop and α7-α8 loop),
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