Hepatitis C virus nonstructural protein NS3 unfolds viral G-quadruplex RNA structures.

Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the nonstructural proteins of HCV, NS3 (nonstructural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)-forming sequences in the HCV-positive and HCV-negative strands. We performed fluorescence anisotropy binding, G4 reporter duplex unwinding, and G4RNA trapping assays to determine the binding and G4 unfolding activity of NS3. Our data suggest that NS3 can unfold the conserved G4 structures present within the genome and the negative strand of HCV. Additionally, we found the activity of NS3 on a G4RNA was reduced significantly in the presence of a G4 ligand. The ability of NS3 to unfold HCV G4RNA could imply a novel biological role of the viral helicase in replication.

Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft.

The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well-understood. Previous molecular dynamics simulations conducted by our group have identified Motif V as a potential "communication hub" for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. These results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

Zika virus NS4A N-Terminal region (1-48) acts as a cofactor for inducing NTPase activity of NS3 helicase but not NS3 protease.

Among Flaviviridae, in West Nile virus (WNV) and Hepatitis C virus (HCV), the non-structural protein NS4A modulates the NTPase activity of viral helicases during nucleic acid unwinding through its N-terminal disordered residues (1-50). In HCV, the acidic NS4A also serves as a cofactor for regulating the NS3 protease activity. However, in case of Zika virus (ZIKV), the role of NS4A and its impact on activities of NS3 helicase and protease is not known. In order to elucidate the role of NS4A, we checked the NTPase activity of NS3 helicase and protease activity of NS3 protease in presence of NS4A N-terminal region (residues 1-48) peptide. Our enzyme kinetics results together with binding experiment clearly demonstrate that NS3 helicase in presence of NS4A peptide increased the rate of ATP hydrolysis whereas the protease activity of NS3 protease was not affected. Therefore, like WNV and HCV, our results establish a role of ZIKV NS4A being a cofactor for modulating the NTPase activity of ZIKV NS3 helicase.

The Transactions of NS3 and NS5 in Flaviviral RNA Replication.

Dengue virus (DENV) replication occurs in virus-induced vesicles that contain the replication complex (RC) where viral RNA, viral proteins and host proteins participate in RNA-RNA, RNA-protein and protein-protein interactions to ensure viral genome synthesis. However, the details of the multitude of interactions involved in the biogenesis of the infectious virion are not fully understood. In this review, we will focus on the interaction between non-structural (NS) proteins NS3 and NS5, as well as their interactions with viral RNA and briefly also the interaction of NS5 with the host nuclear transport receptor protein importin-α. The multifunctional NS3 protease/helicase and NS5 methyltransferase (MTase)/RNA-dependent RNA polymerase (RdRp) contain all the enzymatic activities required to synthesize the viral RNA genome. The success stories of drug discovery and development with Hepatitis C virus (HCV), a member of the Flaviviridae family, has led to the view that DENV NS3 and NS5 may be attractive antiviral drug targets. However, more than 10 years of intensive research effort by Novatis has revealed that they are not "low hanging fruits" and therefore, the search for potent directly acting antivirals (DAAs) remains a pipeline goal for several medium to large drug discovery enterprises. The effort to discover DAAs for DENV has been boosted by the epidemic outbreak of the closely related flavivirus member - Zika virus (ZIKV). Because the viral RNA replication occurs within a molecular machine that is composed several viral and host proteins, much interest has turned to characterising functionally essential protein-protein interactions in order to identify potential allosteric inhibitor binding sites within the RC.

Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening.

The flavivirus family contains several important human pathogens, such as Zika virus (ZIKV), dengue, West Nile, and Yellow Fever viruses, that collectively lead to a large, global disease burden. Currently, there are no approved medicines that can target these viruses. The sudden outbreak of ZIKV infections in 2015⁻2016 posed a serious threat to global public health. While the epidemic has receded, persistent reservoirs of ZIKV infection can cause reemergence. Here, we have used X-ray crystallography-based screening to discover two novel sites on ZIKV NS3 helicase that can bind drug-like fragments. Both sites are structurally conserved in other flaviviruses, and mechanistically significant. The binding poses of four fragments, two for each of the binding sites, were characterized at atomic precision. Site A is a surface pocket on the NS3 helicase that is vital to its interaction with NS5 polymerase and formation of the flaviviral replication complex. Site B corresponds to a flexible, yet highly conserved, allosteric site at the intersection of the three NS3 helicase domains. Saturation transfer difference nuclear magnetic resonance (NMR) experiments were additionally used to evaluate the binding strength of the fragments, revealing dissociation constants (KD) in the lower mM range. We conclude that the NS3 helicase of flaviviruses is a viable drug target. The data obtained open opportunities towards structure-based design of first-in-class anti-ZIKV compounds, as well as pan-flaviviral therapeutics.

Molecular dynamics simulations of Zika virus NS3 helicase: Insights into RNA binding site activity.

America is still suffering with the outbreak of Zika virus (ZIKV) infection. Congenital ZIKV syndrome has already caused a public health emergency of international concern. However, there are still no vaccines to prevent or drugs to treat the infection caused by ZIKV. The ZIKV NS3 helicase (NS3h) protein is a promising target for drug discovery due to its essential role in viral genome replication. NS3h unwinds the viral RNA to enable the replication of the viral genome by the NS5 protein. NS3h contains two important binding sites: the NTPase binding site and the RNA binding site. Here, we used molecular dynamics (MD) simulations to study the molecular behavior of ZIKV NS3h in the presence and absence of ssRNA and the potential implications for NS3h activity and inhibition. Although there is conformational variability and poor electron densities of the RNA binding loop in various apo flaviviruses NS3h crystallographic structures, the MD trajectories of NS3h-ssRNA demonstrated that the RNA binding loop becomes more stable when NS3h is occupied by RNA. Our results suggest that the presence of RNA generates important interactions with the RNA binding loop, and these interactions stabilize the loop sufficiently that it remains in a closed conformation. This closed conformation likely keeps the ssRNA bound to the protein for a sufficient duration to enable the unwinding/replication activities of NS3h to occur. In addition, conformational changes of this RNA binding loop can change the nature and location of the optimal ligand binding site, according to ligand binding site prediction results. These are important findings to help guide the design and discovery of new inhibitors of NS3h as promising compounds to treat the ZIKV infection.

Four Aromatic Sulfates with an Inhibitory Effect against HCV NS3 Helicase from the Crinoid Alloeocomatella polycladia.

Bioassay-guided separation of a lipophilic extract of the crinoid Alloeocomatella polycladia, inhibiting the activity of HCV NS3 helicase, yielded two groups of molecules: cholesterol sulfate and four new aromatic sulfates 1-4. The structures of the aromatics were elucidated by spectroscopic analysis in addition to theoretical studies. The aromatic sulfates 1-4 showed moderate inhibition against NS3 helicase with IC50 values of 71, 95, 7, and 5 µM, respectively.

Single point mutations in the helicase domain of the NS3 protein enhance dengue virus replicative capacity in human monocyte-derived dendritic cells and circumvent the type I interferon response.

Dengue is the most prevalent arboviral disease worldwide. The outcome of the infection is determined by the interplay of viral and host factors. In the present study, we evaluated the cellular response of human monocyte-derived DCs (mdDCs) infected with recombinant dengue virus type 1 (DV1) strains carrying a single point mutation in the NS3hel protein (L435S or L480S). Both mutated viruses infect and replicate more efficiently and produce more viral progeny in infected mdDCs compared with the parental, non-mutated virus (vBACDV1). Additionally, global gene expression analysis using cDNA microarrays revealed that the mutated DVs induce the up-regulation of the interferon (IFN) signalling and pattern recognition receptor (PRR) canonical pathways in mdDCs. Pronounced production of type I IFN were detected specifically in mdDCs infected with DV1-NS3hel-mutated virus compared with mdDCs infected with the parental virus. In addition, we showed that the type I IFN produced by mdDCs is able to reduce DV1 infection rates, suggesting that cytokine function is effective but not sufficient to mediate viral clearance of DV1-NS3hel-mutated strains. Our results demonstrate that single point mutations in subdomain 2 have important implications for adenosine triphosphatase (ATPase) activity of DV1-NS3hel. Although a direct functional connection between the increased ATPase activity and viral replication still requires further studies, these mutations speed up viral RNA replication and are sufficient to enhance viral replicative capacity in human primary cell infection and circumvent type I IFN activity. This information may have particular relevance for attenuated vaccine protocols designed for DV.

Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase.

Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure-activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.

Kyasanur Forest disease virus NS3 helicase: Insights into structure, activity, and inhibitors.

Kyasanur Forest disease virus (KFDV), a tick-borne flavivirus prevalent in India, presents a serious threat to human health. KFDV NS3 helicase (NS3hel) is considered a potential drug target due to its involvement in the viral replication complex. Here, we resolved the crystal structures of KFDV NS3hel apo and its complex with three phosphate molecules, which indicates a conformational switch during ATP hydrolysis. Our data revealed that KFDV NS3hel has a higher binding affinity for dsRNA, and its intrinsic ATPase activity was enhanced by dsRNA while being inhibited by DNA. Through mutagenesis analysis, several residues within motifs I, Ia, III, V, and VI were identified to be crucial for NS3hel ATPase activity. Notably, the M419A mutation drastically reduced NS3hel ATPase activity. We propose that the methionine-aromatic interaction between residues M419 and W294, located on the surface of the RNA-binding channel, could be a target for the design of efficient inhibitor probes. Moreover, epigallocatechin gallate (EGCG), a tea-derived polyphenol, strongly inhibited NS3hel ATPase activity with an IC50 value of 0.8 µM. Our computational docking data show that EGCG binds at the predicted druggable hotspots of NS3hel. Overall, these findings contribute to the development and design of more effective and specific inhibitors.

Mutational analyses, pharmacophore-based inhibitor design and in silico validation for Zika virus NS3-helicase.

Zika virus is responsible for causing Zika infections and was declared as a public health emergency of international concern in February 2016. The Zika virus NS3-helicase is a viable drug target for the design of inhibitors due to its essential role in the replication of viral genome. The viral RNA is unwound by the NS3-helicase in order to enable the reproduction of viral genome by the NS5 protein. Zika virus infections in humans are being reported for the last 15 years. We have therefore carried out amino acid mutational analyses of NS3-helicase. NS3-helicase has two crucial binding sites: the ATP and RNA binding sites. The cofactor-ATP based pharmacophore was generated for virtual screening of ZINC database using Pharmit server, that is followed by molecular docking and molecular dynamics simulations of potential hits as probable Zika virus NS3-helicase inhibitors at the cofactor binding site. The drug-like properties of the molecules were analysed and, DFT calculations were performed on the five best molecules to reveal their stability in solvent phase compared to gas phase, the HOMO and LUMO and electrostatic potential maps to analyze the electronic and geometric characteristics. These are significant findings towards the discovery of new inhibitors of Zika virus NS3-helicase, a promising drug target to treat the Zika virus infection.Communicated by Ramaswamy H. Sarma.

Inhibition of hepatitis C virus replication and viral helicase by ethyl acetate extract of the marine feather star Alloeocomatella polycladia.

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC(50) of 11.7 µg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC(50) value of 23 to 44 µg/mL, which is similar to the IC(50) value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.

Molecular docking analysis of fluoroquinolones and other natural and synthetic compounds with the HCV NS3 helicase.

It is of an interest to document the molecular docking analysis of fluoroquinolones and other natural and synthetic compounds with the HCV NS3 helicase. Data shows that three fluoroquinolones interacted with the NS3 helicase in the catalytic region, targeting some of the amino acids known to play a crucial role in NS3 helicase activity. Similarly, binding energy shows that the fluoroquinolones were comparable to the thiazolpiperazinyl derivatives, while superior to several of the synthetic and natural derivatives. The results show three fluoroquinolones to be potent helicase inhibitors that can be repurposed as supplemental therapy against HCV especially in cases non-responsive to DAAAs.

Crystal structure of the Ilheus virus helicase: implications for enzyme function and drug design.

BACKGROUND: The Ilheus virus (ILHV) is an encephalitis associated arthropod-borne flavivirus. It was first identified in Ilheus City in the northeast Brazil before spreading to a wider geographic range. No specific vaccines or drugs are currently available for the treatment of ILHV infections. The ILHV helicase, like other flavivirus helicases, possesses 5'-triphosphatase activity. This allows it to perform ATP hydrolysis to generate energy as well as sustain double-stranded RNA's unwinding during ILHV genome replication. Thus, ILHV helicase is an ideal target for inhibitor design. RESULTS: We determined the crystal structure of the ILHV helicase at 1.75-Å resolution. We then conducted molecular docking of ATP-Mn2+ to the ILHV helicase. Comparisons with related flavivirus helicases indicated that both the NTP and the RNA-ILHV helicase binding sites were conserved across intra-genus species. This suggested that ILHV helicase adopts an identical mode in recognizing ATP/Mn2+. However, the P-loop in the active site showed a distinctive conformation; reflecting a different local structural rearrangement. ILHV helicase enzymatic activity was also characterized. This was found to be relatively lower than that of the DENV, ZIKV, MVE, and ALSV helicases. Our structure-guided mutagenesis revealed that R26A, E110A, and Q280A greatly reduced the ATPase activities. Moreover, we docked two small molecule inhibitors of DENV helicase (ST-610 and suramin) to the ILHV helicase and found that these two molecules had the potential to inhibit the activity of ILHV helicase as well. CONCLUSION: High-resolution ILHV helicase structural analysis demonstrates the key amino acids of ATPase activities and could be useful for the design of inhibitors targeting the helicase of ILHV.

Molecular mechanism of ATP and RNA binding to Zika virus NS3 helicase and identification of repurposed drugs using molecular dynamics simulations.

Congenital Zika virus syndrome has caused a public health emergency of international concern. So far, there are no drugs available to prevent or treat the infection caused by Zika virus. The Zika virus NS3 helicase is a potential protein target for drug discovery due to its vital role in viral genome replication. NS3 helicase unwinds the viral RNA to enable the reproduction of the viral genome by the NS5 protein. NS3 helicase has two crucial binding sites; the ATP binding site and the RNA binding site. We used molecular docking and molecular dynamics (MD) simulations to study the structural behavior of Zika virus NS3 helicase in its apo form and in the presence of ATP, single-stranded RNA, and both ATP-RNA to understand their potential implications in NS3 helicase activity. Further, we have carried out virtual screening of FDA approved drugs, followed by molecular docking to identify the ATP-competitive hit molecules as probable Zika virus NS3 helicase inhibitors. The MD simulations trajectories were analyzed using normal mode analysis and principal component analysis that reveals fluctuations in the R-loop. These findings aid in understanding the molecular mechanisms of the simultaneous binding of ATP and RNA, and guide the design and discovery of new inhibitors of the Zika virus NS3 helicase as a promising drug target to treat the Zika virus infection. Communicated by Ramaswamy H. Sarma.

In silico testing of flavonoids as potential inhibitors of protease and helicase domains of dengue and Zika viruses.

Background: Dengue and Zika are two major vector-borne diseases. Dengue causes up to 25,000 deaths and nearly a 100 million cases worldwide per year, while the incidence of Zika has increased in recent years. Although Zika has been associated to fetal microcephaly and Guillain-Barré syndrome both it and dengue have common clinical symptoms such as severe headache, retroocular pain, muscle and join pain, nausea, vomiting, and rash. Currently, vaccines have been designed and antivirals have been identified for these diseases but there still need for more options for treatment. Our group previously obtained some fractions from medicinal plants that blocked dengue virus (DENV) infection in vitro. In the present work, we explored the possible targets by molecular docking a group of molecules contained in the plant fractions against DENV and Zika virus (ZIKV) NS3-helicase (NS3-hel) and NS3-protease (NS3-pro) structures. Finally, the best ligands were evaluated by molecular dynamic simulations. Methods: To establish if these molecules could act as wide spectrum inhibitors, we used structures from four DENV serotypes and from ZIKV. ADFR 1.2 rc1 software was used for docking analysis; subsequently molecular dynamics analysis was carried out using AMBER20. Results: Docking suggested that 3,5-dicaffeoylquinic acid (DCA01), quercetin 3-rutinoside (QNR05) and quercetin 3,7-diglucoside (QND10) can tightly bind to both NS3-hel and NS3-pro. However, after a molecular dynamics analysis, tight binding was not maintained for NS3-hel. In contrast, NS3-pro from two dengue serotypes, DENV3 and DENV4, retained both QNR05 and QND10 which converged near the catalytic site. After the molecular dynamics analysis, both ligands presented a stable trajectory over time, in contrast to DCA01. These findings allowed us to work on the design of a molecule called MOD10, using the QND10 skeleton to improve the interaction in the active site of the NS3-pro domain, which was verified through molecular dynamics simulation, turning out to be better than QNR05 and QND10, both in interaction and in the trajectory. Discussion: Our results suggests that NS3-hel RNA empty binding site is not a good target for drug design as the binding site located through docking is too big. However, our results indicate that QNR05 and QND10 could block NS3-pro activity in DENV and ZIKV. In the interaction with these molecules, the sub-pocket-2 remained unoccupied in NS3-pro, leaving opportunity for improvement and drug design using the quercetin scaffold. The analysis of the NS3-pro in complex with MOD10 show a molecule that exerts contact with sub-pockets S1, S1', S2 and S3, increasing its affinity and apparent stability on NS3-pro.

Dengue Virus NS4b N-Terminus Disordered Region Interacts with NS3 Helicase C-Terminal Subdomain to Enhance Helicase Activity.

Dengue virus replicates its single-stranded RNA genome in membrane-bound complexes formed on the endoplasmic reticulum, where viral non-structural proteins (NS) and RNA co-localize. The NS proteins interact with one another and with the host proteins. The interaction of the viral helicase and protease, NS3, with the RNA-dependent RNA polymerase, NS5, and NS4b proteins is critical for replication. In vitro, NS3 helicase activity is enhanced by interaction with NS4b. We characterized the interaction between NS3 and NS4b and explained a possible mechanism for helicase activity modulation by NS4b. Our bacterial two-hybrid assay results showed that the N-terminal 57 residues region of NS4b is enough to interact with NS3. The molecular docking of the predicted NS4b structure onto the NS3 structure revealed that the N-terminal disordered region of NS4b wraps around the C-terminal subdomain (CTD) of the helicase. Further, NS3 helicase activity is enhanced upon interaction with NS4b. Molecular dynamics simulations on the NS4b-docked NS3 crystal structure and intrinsic tryptophan fluorescence studies suggest that the interaction results in NS3 CTD domain motions. Based on the interpretation of our results in light of the mechanism explained for NS3 helicase, NS4b-NS3 interaction modulating CTD dynamics is a plausible explanation for the helicase activity enhancement.

Novel insights into the function of an N-terminal region of DENV2 NS4B for the optimal helicase activity of NS3.

Dengue virus NS3 is a prototypical DEx(H/D) helicase that binds and hydrolyzes NTP to translocate along and unwind double-stranded nucleic acids. NS3 and NS4B are essential components of the flavivirus replication complex. Evidences showed that NS4B interacted with NS3 and modulated the helicase activity of NS3. Despite important insights into structural, mechanistic, and cellular aspects of the NS3 function, there is still a gap in understanding how it coordinates the helicase activities within the replicase complex for efficient replication. Here, using the DENV2 as a model, we redefined the critical region of NS4B required for NS3 function by pull-down and MST assays. The FRET-based unwinding assay showed that NS3 would accelerate unwinding duplex nucleic acids in the presence of NS4B (51-83). The simulated NS3-NS4B complex models based on the rigid-body docking delineated the potential interaction sites located in the conserved motif within the core domain of NS3. Mutations in motif I (I190A) and motif III (P319L) of NS3 interfered with the unwinding activity stimulated by NS4B. Upon binding to the NS3 helicase, NS4B assisted NS3 to dissociate from single-stranded nucleic acid and enabled NS3 helicase to keep high activity at high ATP concentrations. These results suggest that NS4B probably serves as an essential cofactor for NS3 to coordinate the ATP cycles and nucleic acid binding during viral genome replication.

Targeting the NTPase site of Zika virus NS3 helicase for inhibitor discovery.

The major threats linked to Zika virus (ZIKV) are microcephaly, Guillain-Barre syndrome, and the ability to transfer through sexual transmission. Despite these threats, Zika specific FDA approved drugs or vaccines are not available as of yet. Additionally, the involvement of pregnant women makes the drug screening process lengthy and complicated in terms of safety and minimum toxicity of the molecules. Since NS3 helicase of ZIKV performs the critical function of unwinding double-stranded RNA during replication, it is considered as a promising drug target to block ZIKV replication. In the present study, we have exploited the NTPase site of ZIKV NS3 helicase for screening potential inhibitor compounds by molecular docking, and molecular dynamics (MD) simulation approaches. NS3 helicase hydrolyzes the ATP to use its energy for unwinding RNA. We have chosen twenty natural compounds from ZINC library with known antiviral properties and a helicase focused library (HFL) of small molecules from Life Chemicals compounds. After going through docking, the top hit molecules from ZINC and HFL library were further analysed by MD simulations to find out stable binding poses. Finally, we have reported the molecules with potential of binding at NTPase pocket of ZIKV NS3 helicase, which could be further tested on virus through in vitro experiments to check their efficacy.Communicated by Ramaswamy H. Sarma.

Crystallographic Snapshots of the Zika Virus NS3 Helicase Help Visualize the Reactant Water Replenishment.

Zika virus (ZIKV), a positive-strand RNA virus belonging to the Flavivirus genus, has become an urgent public health concern since recent outbreaks worldwide. Its genome replication is facilitated by the viral NS3 protein bearing helicase function. The NS3 helicase uses energy derived from adenosine triphosphate (ATP) hydrolysis to unwind RNA duplexed regions. Structural studies of the flavivirus NS3 helicases have suggested a conserved mechanism of ATP hydrolysis. However, the process of the reactant water replenishment, a key part of the hydrolysis cycle, remains elusive. Here, we report two high-resolution crystal structures of ZIKV NS3 helicase in complex with adenosine diphosphate (ADP) and Mn2+, one with the reactant water already loaded as previously observed and the other with the water molecule still in a loading state. These data suggest that the reactant water replenishment can occur between the release of phosphate and the release of ADP and improves the structural basis of the NS3 ATP hydrolysis cycle.