Extended Data Fig. 9: VBNI-1 disrupts KSHV vBcl-2 interaction with NM23-H2 and impairs late stage KSHV lytic replication.
a, HTRF assay. His-NM23-H2 is bound by an anti-His-Tb HTRF donor fluorophore. GST-vBcl-2 is bound by an anti-GST-d2 HTRF acceptor. vBcl-2 binding to NM23-H2 produces FRET. The addition of small molecule inhibitor(s) reduces FRET. b, HTRF showing dose-dependent vBcl-2 binding to NM23-H2 (3 nM; red) or NM23-H1(25 nM; blue fitted by nonlinear regression. c, HTRF comparison of vBcl-2 binding to NM23-H2 vs. BID BH3 peptide in the presence of ABT-263; IC50 values indicated (n = 3). d-g, Co-IP of endogenous NM23-H2, Beclin1, and BAK with vBcl-2 in HEK293T cells expressing HA-vBcl-2 after VBNI-1 treatment at increasing doses for 36 h (d,e) or with VBNI-1 (2 μM) at increasing timepoints (f,g). Interactions with indicated binding partners were quantified in (e,g; n = 3). h, The effects of VBNI-1 on NM23-H1/H2 kinase activity measured by Transcreener ADP2-FI assay; inhibition curves with IC50 values shown. i, Docking model (left) shows the electrostatic surface of vBcl-2 harbouring VBNI-1 (sphere). Overlay (right) of vBcl-2-VBNI-1 with vBcl-2-NM23-H2 highlights potential steric clash between VBNI-1 and NM23-H2 interface residue K128. For clarity, the vBcl-2 molecule bound to VBNI-1 is not shown. j, SPR analysis of VBNI-1 interaction with vBcl-2 across concentrations. k, Cytotoxicity of VBNI-1 in SLK (blue) and BJAB (red) cells; median effective concentration (EC50) values indicated. l, Dose-dependent inhibition of KSHV production by VBNI-1 in reactivated iSLK-BAC16.vBcl-2 WT but not KO cells; EC50 values indicated. m-o, Virus production (m,n) and gene expression (o) following VBNI-1 treatment and indicated shRNA knockdowns post-reactivation. GFP/BF images shown in m; quantified in n (n = 3). Scale bars, 430 μm. Data in d,f,j,m are from one experiment that is representative of three independent experiments. See Source Data for uncropped data (d,f). Data in e,g,n,o represent mean ± s.d. analyzed by one-way ANOVA followed by Tukey’s post hoc test. **, p < 0.01; ****, p < 0.0001; ns, not significant. Exact P values are provided in Source Data Extended Data Fig. 9.
