Non-nucleoside Inhibitors of Zika Virus RNA-Dependent RNA Polymerase.

Zika virus (ZIKV) remains a potentially significant public health concern because it can cause teratogenic effects, such as microcephaly in newborns and neurological disease, like Guillain-Barré syndrome. Together with efforts to develop a vaccine, the discovery of antiviral molecules is important to control ZIKV infections and to prevent its most severe symptoms. Here, we report the development of small nonnucleoside inhibitors (NNIs) of ZIKV RNA-dependent RNA polymerase (RdRp) activity. These NNIs target an allosteric pocket (N pocket) located next to a putative hinge region between the thumb and the palm subdomains that was originally described for dengue virus (DENV) RdRp. We first tested the activity of DENV RdRp N-pocket inhibitors against ZIKV RdRp, introduced chemical modifications into these molecules, and assessed their potency using both enzymatic and cell-based assays. The most potent compound had a 50% inhibitory concentration value of 7.3 µM and inhibited ZIKV replication in a cell-based assay with a 50% effective concentration value of 24.3 µM. Importantly, we report four high-resolution crystal structures detailing how these NNIs insert into the N pocket of ZIKV RdRp. Our observations point to subtle differences in the size, shape, chemical environment, and hydration of the N pocket from ZIKV RdRp from those of the N pocket from DENV RdRp that are crucial for the design of improved antiviral inhibitors with activity against ZIKV.IMPORTANCE Zika virus belongs to the Flavivirus genus, which comprises several important human pathogens. There is currently neither an approved vaccine nor antiviral drugs available to prevent infection by ZIKV. The nonstructural protein 5 (NS5) polymerase, which is responsible for replicating the viral RNA genome, represents one of the most promising targets for antiviral drug development. Starting from compounds recently developed against dengue virus NS5, we designed and synthesized inhibitors targeting Zika virus NS5. We show that these novel compounds inhibit viral replication by targeting the polymerase activity. High-resolution X-ray crystallographic structures of protein-inhibitor complexes demonstrated specific binding to an allosteric site within the polymerase, called the N pocket. This work paves the way for the future structure-based design of potent compounds specifically targeting ZIKV RNA polymerase activity.

NS5 from Dengue Virus Serotype 2 Can Adopt a Conformation Analogous to That of Its Zika Virus and Japanese Encephalitis Virus Homologues.

Flavivirus nonstructural protein 5 (NS5) contains an N-terminal methyltransferase (MTase) domain and a C-terminal polymerase (RNA-dependent RNA polymerase [RdRp]) domain fused through a 9-amino-acid linker. While the individual NS5 domains are structurally conserved, in the full-length protein, their relative orientations fall into two classes: the NS5 proteins from Japanese encephalitis virus (JEV) and Zika virus (ZIKV) adopt one conformation, while the NS5 protein from dengue virus serotype 3 (DENV3) adopts another. Here, we report a crystallographic structure of NS5 from DENV2 in a conformation similar to the extended one seen in JEV and ZIKV NS5 crystal structures. Replacement of the DENV2 NS5 linker with DENV1, DENV3, DENV4, JEV, and ZIKV NS5 linkers had modest or minimal effects on in vitro DENV2 MTase and RdRp activities. Heterotypic DENV NS5 linkers attenuated DENV2 replicon growth in cells, while the JEV and ZIKV NS5 linkers abolished replication. Thus, the JEV and ZIKV linkers likely hindered essential DENV2 NS5 interactions with other viral or host proteins within the virus replicative complex. Overall, this work sheds light on the dynamics of the multifunctional flavivirus NS5 protein and its interdomain linker. Targeting the NS5 linker is a possible strategy for producing attenuated flavivirus strains for vaccine design.IMPORTANCE Flaviviruses include important human pathogens, such as dengue virus and Zika virus. NS5 is a nonstructural protein essential for flavivirus RNA replication with dual MTase and RdRp enzyme activities and thus constitutes a major drug target. Insights into NS5 structure, dynamics, and evolution should inform the development of antiviral inhibitors and vaccine design. We found that NS5 from DENV2 can adopt a conformation resembling that of NS5 from JEV and ZIKV. Replacement of the DENV2 NS5 linker with the JEV and ZIKV NS5 linkers abolished DENV2 replication in cells, without significantly impacting in vitro DENV2 NS5 enzymatic activities. We propose that heterotypic flavivirus NS5 linkers impede DENV2 NS5 protein-protein interactions that are essential for virus replication.

Purification of Dengue and Zika Virus Non-structural Protein 5 for Crystallization and Screening of Antivirals.

Dengue Virus (DENV) and ZIKA Virus (ZIKV) are two important human pathogens that belong to the Flavivirus genus of positive strand RNA viruses. Symptoms of DENV infections range from asymptomatic or mild fever to life-threatening forms, while ZIKV can lead to teratogenic effects such as microcephaly in newborns and neurological disease like the Guillain-Barré syndrome.Non-Structural Protein 5 (NS5) is the largest and most conserved enzyme across flaviviruses and hence constitutes a prime target for developing pan-flavivirus antiviral inhibitors. NS5 results from the gene fusion between a methyltransferase at the N-terminus of the protein and an RNA-dependent RNA polymerase (RdRp) at the C-terminal end. The NS5 protein plays key roles in replication and modification of viral RNA and its inhibition by potent antiviral drugs could prevent severe symptoms associated with infections.We have optimized purification and crystallization protocols to obtain active recombinant proteins suitable for structure-based drug discovery for both the full-length NS5 protein and the polymerase domain of NS5 from DENV and ZIKV .

Ensa controls S-phase length by modulating Treslin levels.

The Greatwall/Ensa/PP2A-B55 pathway is essential for controlling mitotic substrate phosphorylation and mitotic entry. Here, we investigate the effect of the knockdown of the Gwl substrate, Ensa, in human cells. Unexpectedly, Ensa knockdown promotes a dramatic extension of S phase associated with a lowered density of replication forks. Notably, Ensa depletion results in a decrease of Treslin levels, a pivotal protein for the firing of replication origins. Accordingly, the extended S phase in Ensa-depleted cells is completely rescued by the overexpression of Treslin. Our data herein reveal a new mechanism by which normal cells regulate S-phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.The Greatwall/Ensa/PP2A-B55 pathway controls mitotic substrate phosphorylation and mitotic entry. Here the authors show that cells regulate S phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.

Characterization of the mechanisms controlling Greatwall activity.

Here we investigate the mechanisms regulating Greatwall (Gwl), a serine/threonine kinase essential for promoting the correct timing of mitosis. We identify Gwl as a unique AGC kinase that, unlike most AGC members, appears to be devoid of a hydrophobic motif despite the presence of a functional hydrophobic pocket. Our results suggest that Gwl activation could be mediated by the binding of its hydrophobic pocket to the hydrophobic motif of another AGC kinase. Our molecular modeling and mutagenic analysis also indicate that Gwl displays a conserved tail/linker site whose phosphorylation mediates kinase activation by promoting the interaction of this phosphorylated residue with two lysines at the N terminus. This interaction could stabilize the αC-helix and maintain kinase activity. Finally, the different phosphorylation sites on Gwl are identified, and the role of each one in the regulation of Gwl kinase activity is determined. Our data suggest that only the phosphorylation of the tail/linker site, located outside the putative T loop, appears to be essential for Gwl activation. In summary, our results identify Gwl as a member of the AGC family of kinases that appears to be regulated by unique mechanisms and that differs from the other members of this family.

The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A.

Initiation and maintenance of mitosis require the activation of protein kinase cyclin B-Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.