Solution conformations of a linked construct of the Zika virus NS2B-NS3 protease

Mahawaththa, Mithun C.; Pearce, Benjamin J G; Szabó, Mónika; Graham, Bim; Klein, Christian D.; Nitsche, Christoph; Otting, Gottfried

doi:10.1016/j.antiviral.2017.03.011

Cited by 47 publications

(67 citation statements)

References 36 publications

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“…In agreement with previous studies , we also observed cleavage of linked WT ZIKV NS2B CF ‐NS3 pro . We therefore set out to identify the cleavage site, whether cleavage occurs in cis , that is, an individual protease cleaves its own linker or in trans , that is, a protease linker is cleaved by another ZIKV NS2B CF ‐NS3 pro protease in the sample and to compare the melting behaviors of cleaved and uncleaved protease constructs.…”

Section: Resultssupporting

confidence: 93%

“…The sizes of the autocatalytic cleavage products led us and others to conclude that there are two possible cleavage sites in ZIKV NS2B CF ‐NS3 pro , one at the NS2B CF C‐terminal R95 NS2B and one at R29 NS3 in the NS3 pro domain. The double mutation simultaneously removing both arginine residues successfully abrogated autocatalytic cleavage . Here, we mutated these sites individually and thus identified the relevant autocatalytic cleavage site to be at position R95 NS2B in the C‐terminus of the NS2B CF .…”

Section: Discussionmentioning

confidence: 99%

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Cis autocatalytic cleavage of glycine‐linked Zika virus NS2B‐NS3 protease constructs

et al. 2019

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The flaviviral heterodimeric serine protease NS2B‐NS3, consisting of the NS3 protease domain and the NS2B co‐factor, is essential for ZIKA virus maturation and replication in cells. For in vitro studies a ‘linked’ construct, where a polyglycine linker connects NS2BCF and NS3pro, is often used. This construct undergoes autocatalytic cleavage. Here, we show that linked ZIKV NS2BCF‐NS3pro is cleaved in cis in the NS2BCF exclusively at position R95 and not at the previously proposed alternate cleavage site at residue R29 in the NS3pro. Cleavage neither affects protease stability nor activity, despite some observed differences in spectroscopic behavior. This minimally modified construct may thus be useful for future structural and functional studies of the flaviviral protease, for example when testing new inhibitors.

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Section: Resultssupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Cis autocatalytic cleavage of glycine‐linked Zika virus NS2B‐NS3 protease constructs

et al. 2019

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“…To evaluate whether the N2CN3N construct can monitor the NS2B conformational changes, we performed the SLC experiment with/without an active-site inhibitor, Nitrophenyl p-guanidinobenzoate (NPGB), which was known to induce conformational change of NS2B by NMR studies [36, 45]. As shown in Fig 2B , addition of NPGB (30 μM) significantly increased SLC (4.2-fold), compared to that of the DMSO control.…”

Section: Resultsmentioning

confidence: 99%

“…However, NMR studies also indicated that (1) the C-terminal portion of NS2B was still found to be flexible; (2) conformational changes were also observed and could be induced upon buffer manipulations; and (3) active-site inhibitor further stabilized the “closed” conformation [36, 63–65]. In addition, recent NMR studies on the ZIKV NS2B-NS3 protease indicated that both closed and open conformations were observed even in the presence of different inhibitors [45, 66]. Overall these data indicated that although it is not clear what exact conformation of NS2B exists in vivo , the NS2B C-terminal region is quite flexible and may display multiple conformations, even though the “closed” conformation could be the main species.…”

Section: Discussionmentioning

confidence: 99%

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease

et al. 2017

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The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618.

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“…S1). A recent NMR study has revealed that the open conformation also exists in gZiPro in solution . In the present study, we compared NMR spectra of bZiPro and gZiPro.…”

mentioning

confidence: 91%

Structural characterization of the linked NS2B‐NS3 protease of Zika virus

Phoo

Loh

et al. 2017

FEBS Letters

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The Zika virus (ZIKV) NS2B-NS3 protease is an important drug target. The conventional flaviviral protease constructs used for structural studies contain the NS2B cofactor region linked to the NS3 protease domain via a glycine-rich flexible linker. Here, we examined the structural dynamics of this conventional Zika protease (gZiPro) using NMR spectroscopy. Although the glycine-rich linker in gZiPro does not alter the overall folding of the protease in solution, gZiPro is not homogenous in ion exchange chromatography. Compared to the unlinked protease construct, the artificial linker affects the chemical environment of many residues including H51 in the catalytic triad. Our study provides a direct comparison of ZIKV protease constructs with and without an artificial linker.

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Solution conformations of a linked construct of the Zika virus NS2B-NS3 protease

Cited by 47 publications

References 36 publications

Cis autocatalytic cleavage of glycine‐linked Zika virus NS2B‐NS3 protease constructs

Cis autocatalytic cleavage of glycine‐linked Zika virus NS2B‐NS3 protease constructs

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease

Structural characterization of the linked NS2B‐NS3 protease of Zika virus

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