Fig. 2.
Clathrin/caveolin-dependent entry of engineered lentiviruses. GFP-Vpr-labeled lentiviruses (green) enveloped with αCD20 and SINmu (A and B) or HAmu (C and D) were incubated with 293T/CD20 cells at 4°C for 30 min. The cells were warmed to 37°C for various time periods (0, 10, and 30 min), fixed, permeabilized, and immunostained with anti-clathrin (A and C; red) or anti-caveolin-1 (B and D; red) antibodies. The boxed regions are magnified and shown below in individual panels. Scale bar represents 5 μm. Inhibition of clathrin-dependent internalization by chlorpromazine (E) or caveolin-dependent internalization by filipin (F). 293T/CD20 cells were preincubated with chlorpromazine or filipin for 30 min at 37°C. The cells (2 × 105) were then spin-infected with supernatants of lentiviruses (FUGW/αCD20+SINmu, FUGW/αCd20+HAmu, FUGW/VSVG, or MIG/VSVG). Both drug concentrations were maintained during the spin-infection as well as for the additional 3 h incubation, after which the drug was removed and replaced with fresh media. The percentage of GFP+ cells was analyzed by FACS.