Figure 2

Effects of rottlerin on viral entry. Macrophages (5 × 10⁵/ml) were untreated or treated with rottlerin (5 μM) (A) for 30 minutes or (B) 2hours. (A) Cells were then stained for CD4 or CCR5 and analyzed by FACS. Dashed lines correspond to unstained cells, solid lines to specific staining. (B) Cells were then transduced with equal MOI of VSV-G- or JR-FL-pseudotyped vectors carrying a GFP reporter plasmid. Two days later, GFP-positive cells were counted using microscopy. Results are represented as relative levels of infection, with infected control cells set to 100%. Error bars are from triplicates of a single experiment. Results are representative of 3 independent experiments. (C) Cell fusion was measured by syncytia formation in a co-culture of cells expressing CD4 and CCR5 or gp120 and gp41, in the presence or absence of PKC inhibitors rottlerin, hispidin, Ro318220, Co6976, or the fusion inhibitor C34 as the control. (D), (E) and (F) Phase contrast microscopy images of syncytia in a coculture of cells expressing either CD4 and CCR5 or gp120 and gp41, in the presence of C34 (D), rotllerin 10 μM (F) or without inhibitors (E). Arrows indicate syncytium formation. (G) Macrophages were pretreated or not with rottlerin or C34 fusion inhibitor for 30 minutes and infected with HIV-1 BaL for 3 h, washed and treated with trypsin. After extensive washing, cells were lysed and p24 quantified using p24 ELISA.