Fig 2.

MT disruption does not impair the attachment and internalization of AAV2. HeLa cells were preincubated with DMSO or nocodazole for 30 to 60 min before pulse infection with Cy5-AAV2. (A) To determine the binding efficiency, cells were harvested at 0 h p.i. for viral genome extraction. The number of viral genomes per cell (vgs/cell) was determined by quantitative PCR. (B and C) Cells were fixed at 0 h or 1 h p.i. without permeabilization. Nuclei were stained with DAPI (blue), and AAV2 capsids were stained with monoclonal antibody A20 (green). Before membrane permeabilization, A20 antibody is not able to access the viral particles inside the cells and thus binds only to the uninternalized AAV2 virions (yellow in the merged figures). (B) Representative images from maximum-intensity projection (MIP) show the costaining between A20 and Cy5 signals. Several representative uninternalized AAV2 particles are highlighted by arrowheads. (C) Quantification of colocalization between Cy5 and A20 as a measurement of viral particles remaining on cell surface and those which have internalized.