Fig. 4

Kudzu blocks HIV-1 attachment. a Kudzu specifically affect the virus and not the cells. Kudzu (1:400) was either preincubated for 6 h with the HeLa-CD4-LTR-LacZ cells then washed and NL4-3 virus was added to the cells (“preincubation with compound/washed”) or simultaneously added with the virus to the cells (“virus + compound together”). β-Gal activity was measured 72 h later. Ralt.: Raltegravir, 100 nM. Results represent the mean ± SD of 3 independent experiments. b Absence of activity of Kudzu on infected HeLa-CD4-LTR-LacZ cells with VSV-G-NL4-3 pseudovirus. Efavirenz (Efav., 200 nM), Raltegravir (Ralt., 200 nM), AMD3100 (10 nM) and Enfuvirtide (1 μg/ml) were used as controls. Kudzu: 1:400 dilution. Viral supernatants recovered 72 h postinfection from cells were assayed for their p24 antigen content. The mean ± SD of 4 experiments is represented. c Activity of Kudzu on infected HeLa-CD4-LTR-LacZ cells with HXB2 gp160 pseudotyped NL4-3 env-. Same controls and Kudzu dilution as in b. Viral supernatants recovered 72 h post-infection from cells were assayed for their p24 antigen content. The mean ± SD of 4 independent experiments is presented. d No impact of Kudzu on interaction of monomeric YU2 gp120 and CD4-Ig receptor in ELISA. s: soluble. Emtricitabine (Emt., 500 nM) and soluble gp120 or CD4 were used as controls. The mean ± SD of 4 independent experiments is represented. e Kudzu does not affect fusion of HeLa-CD4-LTR-LacZ and HL2/3 cells. Emtricitabine (Emt., 100 nM), Enfuvirtide (1 μg/ml), cells only and untreated cells were used as controls. β-Gal activity was measured 48 h later. Data is the mean ± SD of 3 independent experiments. f No activity of Kudzu on shedding of transfected JRCSF gp160 in HEK293T cells, revealed by western blot. Data is representative of 3 independent experiments. CD4-Ig (45 μg) was used as a control. g No activity of Kudzu on shedding of NL4-3 gp120 from NL4-3 virus. HIV-1 directly loaded on gel (HIV-1+) was used as positive control of the gp120 protein. The other samples were filtered through a column and a fraction of the flow-through was analyzed by western blot. CD4 (30 μg) was used as control of the shedding.  Data is representative of 2 independent experiments and quantification of the 2 independent experiments is shown below the blot. h–j Kudzu significantly reduces the attachment of HIV-1 and HIV-2 to TZM-bl cells. h Schematic of the attachment assay. The attachment assay is performed by adding virus to cells in presence of the different compounds for 3 h at 4 °C. After this period of incubation at 4 °C, the cells are washed with cold PBS and then allowed to incubate at 37 °C for 72 h before luciferase is quantified. i Kudzu inhibits HIV-1 attachment. Enfuvirtide (a fusion inhibitor, 1 μg/ml) and Heparin (competes with Heparan sulfate proteoglycans binding to gp120, 1 mg/ml) were used as controls. The data is the mean ± SD of 3 independent experiments. j Kudzu inhibits HIV-2 CBL-20 attachment. AMD3100 (10 nM) and Heparin were used as controls. The data is the mean ± SD of 4 independent experiments. The two-tailed paired t test was used for statistical comparisons. **: p value < 0.005, ***: p value < 0.0005