Figure 7

Gelsolin knockdown restricts HIV-1 Env-mediated membrane fusion and infection in permissive cells, regardless viral tropism. (A) Luciferase-based assay of viral entry and infection by non-replicative X4- and R5-tropic HIV-1 viral strains in siRNA1-GSN- or siRNA2-GSN-silenced permissive lymphocytes (viral entry and infection was considered 100% in cells transfected with scrambled siRNA, i.e., controls). All the data were corrected using the values of non-productive infection (baseline) obtained in the presence of a neutralizing anti-CD4 mAb (5 μg/mL) under the same experimental conditions. A luciferase-based assay with equal inputs of pseudotyped VSV-G virions was performed to control the specificity of gelsolin knockdown-mediated effects on HIV-1 viral entry and infection, and by using specific siRNA1-GSN or siRNA2-GSN oligos. These data were corrected with non-productive baseline values obtained using non-infective virus-like particles lacking VSV-G Env under the same experimental conditions. Data are mean ± s.e.m. of three independent experiments carried out in triplicate. Bottom, Western blot of endogenous gelsolin knockdown prior to infection (top, infection histograms), showing representative data for each experimental condition. Cell-surface expression of viral receptors was analyzed under each experimental condition as shown in Figure 6B. (B) Effect of specific gelsolin silencing (siRNA(1 + 2)-GSN) in HeLa P5 cells on HIV-1 Env-mediated membrane fusion measured by β-Galactosidase production, and referred to the scrambled control condition (100% of membrane fusion). Treated HeLa P5 cells were fused with HeLa cells expressing X4-tropic (243) or R5-tropic (ADA) Env, in the presence or absence of an anti-CD4 mAb. Western blot of endogenous gelsolin knockdown prior to cell-to-cell fusion, showing representative data for each experimental condition. Cell-surface expression of viral receptors was analyzed under each experimental condition. Data are mean ± s.e.m. of four independent experiments carried out in triplicate * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test.