Extended Data Fig. 2: vBcl-2 interaction with NM23-H2 is required for infectious virion production.
a, Experimental schematic to assess vBcl-2 knockout (KO) effects on KSHV reactivation. iSLK-BAC16.vBcl-2 WT and iSLK-BAC16-vBcl-2 KO cells were induced (Dox/NaB, 60-72 h), and supernatants infected naïve SLK cells for 24 h (results in b-d). b, Bright field (BF) and GFP images of SLK cells infected with virus from induced iSLK-BAC16 cells. Scale bars, 50 μm. c,d, Virus titres determined by two-fold serial dilution of supernatants collected 72 h post-reactivation (a) and quantification of GFP-positive SLK cells per well by flow cytometry at 24 h post-infection (c), expressed as infectious units/ml (d). e, Schematic for assessing vBcl-2 KO impact on KSHV de novo infection in HCT116 cells infected with vBcl-2 WT or vBcl-2 KO KSHV at MOI = 10, and supernatant collected were used to infect SLK cells for 24 h (results in f-h). f, GFP images of SLK cells infected in e. Scale bars, 430 μm. g, Flow cytometry analysis of GFP-positive SLK cell percentages and mean fluorescence intensity from e. h, qPCR analysis of viral genome copies in supernatants from e. (n = 3 independent experiments). i, Close-up view of site variations between NM23-H2 (yellow) and -H1 (cyan) in the structural model of NM23-H2 and vBcl-2. The corresponding residues of NM23-H2 and NM23-H1 are shown in stick. The ΔG for the hydrogen (H)-bonding interactions of NM23-H2 Q50 was noted65, which was lost when Q50 was replaced by E50. j, Co-IP of Flag-NM23-H2 (WT/mutant) with HA-vBcl-2 in HEK293T cells. k-m, iSLK-BAC16.vBcl-2 WT cells transduced with ctrl or NM23-H1 shRNA induced as above and supernatants infected SLK cells. GFP/BF images are shown in k, virus titer determined in l (n = 3), and protein expression shown in m. Data in j,m are representative of three independent experiments (see Source Data for uncropped data). Data in d,h,l are mean ± s.d. analyzed by two-tailed Student’s t test and one-way ANOVA with Tukey’s post hoc test. *, p < 0.05; **, p < 0.01; ****, p < 0.0001. ns, not significant. Exact P values are provided in Source Data Extended Data Fig. 2.
