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Langat flavivirus protease NS3 binds caspase-8 and induces apoptosis.

ABSTRACT: The flavivirus NS3 protein plays an important role in the cleavage and processing of the viral polyprotein and in the synthesis of the viral RNA. NS3 recruits NS2B and NS5 proteins to form complexes possessing protease and replicase activities through protease and nucleoside triphosphatase/helicase domains. We have found that NS3 also induces apoptosis. Expression of the Langat (LGT) virus NS3 protein resulted in a cleavage of cellular DNA and reduced the viability of cells. Coexpression of NS3 with apoptotic inhibitors (CrmA and P35) and addition of caspase peptide substrates (Z-VAD-FMK and Z-IETD-FMK) to NS3-transfected cells blocked NS3-induced apoptosis. In cotransfection experiments, NS3 bound to caspase-8 and enhanced caspase-8-mediated apoptosis. NS3 and caspase-8 colocalized in the cytoplasm of transfected cells. Deletion analysis demonstrated that at least two regions of NS3 contribute to its apoptotic activities. The protease and helicase domains are each able to bind to caspase-8, while the protease domain alone induces apoptosis. The protease domain and tetrahelix region of the helicase domain are required for NS3 to augment caspase-8-mediated apoptosis. Thus, the LGT virus NS3 protein is a multifunctional protein that binds to caspase-8 and induces apoptosis.

SUBMITTER: Prikhod'ko GG

PROVIDER: S-EPMC137024 | biostudies-literature | 2002 Jun

REPOSITORIES: biostudies-literature

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Langat flavivirus protease NS3 binds caspase-8 and induces apoptosis.

Prikhod'ko Grigori G GG Prikhod'ko Elena A EA Pletnev Alexander G AG Cohen Jeffrey I JI

Journal of virology 20020601 11

The flavivirus NS3 protein plays an important role in the cleavage and processing of the viral polyprotein and in the synthesis of the viral RNA. NS3 recruits NS2B and NS5 proteins to form complexes possessing protease and replicase activities through protease and nucleoside triphosphatase/helicase domains. We have found that NS3 also induces apoptosis. Expression of the Langat (LGT) virus NS3 protein resulted in a cleavage of cellular DNA and reduced the viability of cells. Coexpression of NS3 ...[more]

PMID: 11991998

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Project description: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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Langat flavivirus protease NS3 binds caspase-8 and induces apoptosis.

Publications

Langat flavivirus protease NS3 binds caspase-8 and induces apoptosis.

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