Figure 2.
Knockdown of syndecan-2 expression with siRNA inhibits viral binding and infection. (A) Cells were transfected with nonspecific, HVEM-specific, or syndecan-2–specific siRNA. Forty-eight hours after transfection, they were stained with anti-syndecan Abs (green), and then they were examined by confocal microscopy; plasma membranes were stained with EZ-Link (red) and nucleic acids were stained with DAPI (blue) (left). Alternatively, cell lysates were prepared 48 h after transfection, and then proteins were separated, transferred by Western blotting, and incubated with anti-syndecan mAb. Blots were then stripped and reprobed with mAb to β-actin. Representative blots from three independent experiments are shown (middle). Blots were scanned and relative amounts of syndecan-2 compared by optical densitometry (right). For each confocal experiment, 100 cells were counted, and the percentage of cells expressing syndecan in siRNA-transfected cells relative to expression in cells transfected with nonspecific siRNA was compared (right). (B) Transfected CaSki cells were exposed to serial dilutions of HSV-2(G) at the moi indicated for 5 h at 4°C. The cell-bound viral particles were detected by analyzing Western blots of cell lysates for gD; blots were also probed with a mAb to β-actin to control for cell loading. The graph shows relative viral particles bound at each moi, and it is representative of results obtained in three independent experiments conducted in duplicate. (C) Transfected CaSki cells were infected with K26GFP virus (moi = 5) and 1- and 4 h pi, the cells were fixed, stained, and viewed by confocal microscopy (red; plasma membrane, blue, nuclei; green, viral VP26). Results are representative of three independent experiments. The relative amount of intracellular GFP was compared by collecting data from ∼100 cells from different fields using NIH Image software. Results are expressed as the pixel intensity per cell in relative units. (D) Cells transfected with nonspecific, syndecan-2 or syndecan-1 specific siRNA were exposed to serial dilutions of HSV-2(G). Plaques were counted 48 h pi and viral titer (pfu/ml) determined. Results are means ± SD of three independent experiments conducted in duplicate.