FIG 3.

SGIV entry depends on clathrin-mediated endocytosis and dynamin. (A) CPZ and dynasore inhibit SGIV uptake. GS cells were pretreated with CPZ or dynasore for 120 min, or were left untreated, and then were incubated with Cy5-labeled SGIV (red) for 120 min. Infected cells were labeled with DiO (green), and more than 100 cells were analyzed by CLSM. (B) Detection of VP19 expression in mock-treated or CPZ-treated cells by immunostaining. Cells were pretreated with different amounts of CPZ for 120 min and infected with SGIV. The infected cells were then analyzed at 48 hpi by IFA using anti-VP19 antibody (green). (C to E) SGIV infectivity and viral protein synthesis are impaired by CPZ (C), sucrose (D), and dynasore (E). GS cells were pretreated with different drugs at the indicated concentrations for 120 min and were then infected with SGIV for 120 min. After being washed in citrate buffer to remove noninternalized viruses, the cells were collected at 48 hpi. The infection rate was examined by IFA and was quantified as the percentage of treated cells with incorporated viruses relative to that for untreated cells. The viral infection rate of untreated cells was arbitrarily set as 100%. The data shown are the means and standard deviations (SD) of the results from three independent experiments. Endogenous β-actin was used as an internal loading control for Western blotting. (F) SGIV particles colocalize with clathrin. GS cells were transfected with pEGFP-LCa prior to incubation with Cy5-labeled SGIVs. The arrow indicates an individual SGIV particle (red) colocalized with clathrin (green). Scale bars are 10 μm (A and F) and 50 μm (B). *, P < 0.05. DMSO, dimethyl sulfoxide.