Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: The DENV2 NS2B/NS3 SLC assays. (A) The DENV2 NS2B/NS3 SLC constructs. NLuc aa 1-416-NS2B (named as NLuc-NS2B), NS2B-NLuc aa 1-416 (NS2B-NLuc), NLuc416-NS2B aa 49-66 (NLuc-E66stop), NS2B (aa 49-66)-NLuc416 (E66stop-NLuc), GST-NS3-CLuc aa 398-550 (GNC), and GST-CLuc (aa 398-550)-NS3 (GCN). (B) The positions of NLuc and CLuc are important. Equal concentrations (100 nM) of each pair of NS2B/NS3 constructs were mixed (or alone) and incubated with luciferin substrate. ***P < 0.001. In all bar graphs, means and SD from triplicate experimental data were shown, unless otherwise specified. (C) Effects of detergent at 0.05% concentration. **P < 0.01; ***P < 0.001. (D) Dose-response of NLuc-E66stop/GCN pair. Nluc-E66stop at 20 nM was included in each experiment. Concentration of GCN was varied as indicated. ***P < 0.001. (E) The DENV2 MBP-NS3 fusion protein specifically inhibited the SLC by NLuc-E66stop and GCN (80 nM each). MBP-NS3 or MBP were at 3.25 μM each. ***P < 0.001. (F) Dose-response inhibition of the SLC signals from NLuc-E66stop and GCN by “cold” MBP-NS3. Experimental data were fitted using the sigmoidal function with the Origin6.0 software. (G) NS2B mutations greatly reduced SLC. GCN was paired with equal molar of NLuc-E66stop or NLuc-E66stop mutants (L51A, L53A, and V59A). ***P < 0.001.

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: Construct, Incubation, Concentration Assay, Inhibition, Software

Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: HTS assay identified potent orthosteric protease inhibitors. (A) Dose-response inhibition of the SLC signals from NLuc-E66stop and GCN by SK-12. Experimental data were fitted using the sigmoidal function with the Origin6.0 software. (B) HTS parameters using purified NLuc-E66stop and GCN. NLuc-E66stop and GCN at 100 nM were used with DMSO or SK-12 (40 μM). n = 8. ***P < 0.001. (C) NS3 pockets were accessible to small molecule inhibitor when NS2B and NS3 were co-expressed. Plasmids of NLuc-E66stop and GCN were co-transformed into Escherichia coli BL21 (DE3). Cells were grown to OD600 of 0.6 and were induced by IPTG. Cells were continuously grown for 2 h, collected and resuspended in luciferase assay buffer. About 100 μl of cells was dispensed into a 96-well plate, incubated with 1% DMSO or SK-12 (40 μM) for 2 h, then mixed with substrate luciferin n = 8. ***P < 0.001. (D) Summary of HT screening of the NCGC Pharmaceutical Collection in all plates. Statistics were generated by averaging those of 20 plates that were calculated from 32 wells in each plate. (E) SDS-PAGE analysis of purified His-MBP-NS3 (lane 2) and His-NS2B (Lane 3). Lane 1, Bio-Rad broad range molecular weight (MW) standard. (F) CD spectrum of purified His-MBP-NS3. (G) Sigmoidal curve fittings of dose-response inhibitions of the His-NS2B/His-MBP-NS3 protease activities by drugs. Inset: schematic representations of identified drugs.

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: HTS Assay, Inhibition, Software, Purification, Transformation Assay, Luciferase, Incubation, Generated, SDS Page, Molecular Weight

Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: Drugs directly bind to the NS3 protease domain and disrupt interactions between NS2B and NS3. (A) GST pull-down assay. GST-NS3 or the GST-tag (10 μg) was immobilized on the Glutathione sepharose-4B affinity beads (GE HealthCare). The FLAG-tagged NS2B (10 μg) was incubated with the beads for 2 h, and subjected to western blots (WB), using anti-FLAG (Genscript) and anti-GST antibodies (GE HealthCare). (B) Dose-dependent inhibition of NS2B-NS3 interactions by drugs, using the GST pull-down assay. The assay was performed the same as in A, except that two-fold dilution series of drugs were incubated with the GST-NS3 beads overnight prior to incubation with the FLAG-NS2B. Bottom panels showed normalized binding of FLAG-tag NS2B to GST-NS3. The binding of NS2B to NS3 in the absence of each drug (DMSO control) was set as 100%. The relative binding of NS2B to NS3 in the presence of each drug was normalized to the DMSO control. n = 3. (C) PTSA for binding of drugs to the MBP-NS3 protein. ΔTm was defined as Tm−drug−TmDMSO. (D) SPR sensorgrams of kinetic data for the binding of drugs to refolded NS3. The refolded His-NS3 was coupled to a ProteOn GLH sensor chip (∼15 000 RU). Each drug with three-fold dilutions was injected. Global fitting of data to a 1:1 binding model is shown in dark black.

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: Pull Down Assay, Incubation, Western Blot, Inhibition, Binding Assay, FLAG-tag, Injection

Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: Drugs inhibit viral polyprotein precursor (PP) processing. (A) Lineweaver-Burk plot of kinetics experimental data for inhibition of the His-NS2B/His-MBP-NS3 protease complex by drugs. The DENV2 MBP-NS3 (100 nM) was mixed with temoporfin (3, 1.5, and 0.75 μM), niclosamide (30, 15, and 7.5 μM), or nitazoxanide (30, 15, and 7.5 μM) for 30 min. The DENV2 His-NS2B (1 μM) was added together with the Abz substrate at various concentrations (800-25 μM in two-fold dilutions). (B-D) Western blots (WB) analysis of dose-dependent inhibition of ZIKV NS3 expression by temoporfin (B), niclosamide (C), and nitazoxanide (D) using the GTX133309 ZIKV α-NS3 antibody (GeneTex) (left panel), respectively. The experiment was performed at the 48 h time point. Middle panel, NS3 expression (lower bands) normalized to the GAPDH loading control. Right panel, accumulated PP normalized to the DMSO control. **P < 0.01; ***P < 0.001. (E) MS/MS spectra obtained from the fragmentation of the precursor ion at m/z corresponding to representative ZIKV peptides. Fragment ions corresponding to y- and b-ions were observed (red lines).

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: Inhibition, Western Blot, Expressing, Tandem Mass Spectroscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tupaia MAVS Is a Dual Target during Hepatitis C Virus Infection for Innate Immune Evasion and Viral Replication via NF-κB.

doi: 10.4049/jimmunol.2000376

Figure Lengend Snippet: FIGURE 1. HCV cleaves tMAVS and jeopardizes the host antiviral response. (A) Cleavage of endogenous tMAVS protein during HCV infection. PTHs were harvested after HCV (JFH-1; MOI = 10) infection at the indicated times or without HCV infection (mock). Cell lysates were immu- noblotted using the anti-VISA Ab and anti-HCV NS3 Ab, respectively. The ACTB was used as the loading control. (B) Expression of endogenous tMAVS (red) and HCV protein (HCV-GFP, green) in PTHs with (MOI = 10) or without HCV infection (mock) for 72 h. tMAVS was immunostained by using anti-VISA Ab to show the colocalization with HCV-GFP. Nuclei were stained with DAPI. (C) Change of endogenous tMAVS protein in the presence of HCV NS3/4A overexpression. TSPRCs were transfected with NS3/4A-HA vector or empty vector together with pDsRed-Mito for 48 h (concentration ratio 10:1). Endogenous tMAVS was immunostained to show the colocalization with mitochondria by using anti-VISA Ab. Nuclei were stained with DAPI. (D) Alignment of the MAVS transmembrane (TM) sequences of the Chinese tree shrew and other mammals retrieved from GenBank. The TM region predicted by the TMpred server (http://www.ch.embnet.org/software/TMPRED_form.html) is shown in box. Residues at the 506th and 508th sites are marked with different colors. (E) Activation of the IFN-b–Luc reporter in TSPRCs with overexpression of tMAVS and NS3/4A (+) or without NS3/4A (2). A change of Cys at the 508th position in the WT hMAVS to Arg (C508R) was used as the control for being unable to be cleaved by NS3/4A. TSPRCs were transfected with the IFN-b–Luc reporter vector (100 ng), TK (10 ng, as an inner control), and hMAVS-Flag or tMAVS-Flag expression vector (WT) or C508R (200 ng) for 24 h, then were transfected with empty vector (2NS3/4A; 200 ng) or NS3/4A-HA (+NS3/4A; 200 ng) for 12 h before the harvest for luciferase assay. Firefly luciferase activity was normalized as being 100% for overexpression of MAVS WT or C508R in the absence of NS3/4A. Immunoblot analyses showing MAVS, NS3/4A, and ACTB expression were listed in the below panel. Data shown are representative of three independent experiments. Bars represent means 6 SD of three biological repeats in this representative. *p , 0.05, 2NS3/4A versus +NS3/4A, Student t test. (F) Localization of tMAVS in the presence of NS3/4A. Expression vectors for Flag-NS3/4A and tMAVS-EGFP or empty vector were transfected into TSPRCs; Flag-NS3/4A was immunostained using Flag Ab. All data in (A)–(C) and (F) are representative of three independent experiments with similar results.

Article Snippet: The other Abs used in this study included rabbit monoclonal anti-IRF3 (4302; Cell Signaling Technology), mouse monoclonal anti-HCV NS3 (ab13830; Abcam), mouse monoclonal anti-HCV core (MA1-080; Thermo Fisher Scientific), rabbit monoclonal anti-histone H3 (D1H2) (4499; Cell Signaling Technology), mouse monoclonal anti–b-actin (ACTB) (E1C602-2; EnoGene), mouse monoclonal anti-tubulin (Shanjin, ZA-0435), peroxidase-conjugated anti-mouse Ab (474-1806; KPL), and peroxidase-conjugated anti-rabbit Ab (074-1506; KPL).

Techniques: Infection, Control, Expressing, Staining, Over Expression, Transfection, Plasmid Preparation, Concentration Assay, Software, Activation Assay, Luciferase, Activity Assay, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tupaia MAVS Is a Dual Target during Hepatitis C Virus Infection for Innate Immune Evasion and Viral Replication via NF-κB.

doi: 10.4049/jimmunol.2000376

Figure Lengend Snippet: FIGURE 2. Residue Glu506 in tMAVS cannot protect it from being cleaved by HCV NS3/4A and has different effects on activating NF-kB– Luc reporters. The residues Glu506 (E506) and Val506 (V506) in MAVS had different effects on the activation of IFN-b–Luc reporter (A) and NF-kB– Luc reporter (B), but not ISRE-Luc reporter (C), in the presence of HCV NS3/4A protease, WT, hMAVS, or tMAVS. The experimental procedure and normalization of luciferase reporter activities are the same as Fig. 1E. Data are representative of three independent experiments with similar re- sults. Bars represent means 6 SD of three biological repeats in this rep- resentative. *p , 0.05, **p , 0.01, Student t test.

Article Snippet: The other Abs used in this study included rabbit monoclonal anti-IRF3 (4302; Cell Signaling Technology), mouse monoclonal anti-HCV NS3 (ab13830; Abcam), mouse monoclonal anti-HCV core (MA1-080; Thermo Fisher Scientific), rabbit monoclonal anti-histone H3 (D1H2) (4499; Cell Signaling Technology), mouse monoclonal anti–b-actin (ACTB) (E1C602-2; EnoGene), mouse monoclonal anti-tubulin (Shanjin, ZA-0435), peroxidase-conjugated anti-mouse Ab (474-1806; KPL), and peroxidase-conjugated anti-rabbit Ab (074-1506; KPL).

Techniques: Residue, Activation Assay, Luciferase

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tupaia MAVS Is a Dual Target during Hepatitis C Virus Infection for Innate Immune Evasion and Viral Replication via NF-κB.

doi: 10.4049/jimmunol.2000376

Figure Lengend Snippet: FIGURE 4. HCV NS3/4A inhibits tMAVS-induced IRF3 signaling, but not NF-kB pathway. TSPRCs were transfected with the indicated luciferase vector (100 ng), expression vector of tTRAF3 (A), tTRAF6 (B), tTBK1 (C), or tIKKb (D) (100 ng), together with tMAVS WT (upper) or tMAVS E506V (lower) vector (100 ng) for 24 h, then were transfected with empty vector (2NS3/4A; 200 ng) or with increased amount of NS3/4A-HA (+NS3/4A; 100 or 200 ng, with empty vector to reach a total amount of 200 ng) for 24 h before the luciferase analysis. Overexpression of tMAVS WT or E506V, together with each indicated effector (tTRAF3, tTRAF6, tTBK1, or tIKKb), was used as the control for evaluating the inhibitory effect by NS3/4A. All data are representative of three inde- pendent experiments with similar results. Bars repre- sent means 6 SD of three biological repeats in the representative. *p , 0.05, **p , 0.01, Student t test.

Article Snippet: The other Abs used in this study included rabbit monoclonal anti-IRF3 (4302; Cell Signaling Technology), mouse monoclonal anti-HCV NS3 (ab13830; Abcam), mouse monoclonal anti-HCV core (MA1-080; Thermo Fisher Scientific), rabbit monoclonal anti-histone H3 (D1H2) (4499; Cell Signaling Technology), mouse monoclonal anti–b-actin (ACTB) (E1C602-2; EnoGene), mouse monoclonal anti-tubulin (Shanjin, ZA-0435), peroxidase-conjugated anti-mouse Ab (474-1806; KPL), and peroxidase-conjugated anti-rabbit Ab (074-1506; KPL).

Techniques: Transfection, Luciferase, Plasmid Preparation, Expressing, Over Expression, Control