The helper-dependent adeno-associated viruses (AAVs) have attracted great interest as vectors for gene therapy. Uptake and intracellular trafficking pathways of AAV are of importance, since they are often rate-limiting steps in infection. Here, we have investigated the entry of AAV type 5 (AAV5) in primary human embryo fibroblasts. At low binding temperatures, numerous virions are concentrated between cells, at contact points between cells and cellular protrusions, and at filopodia. When the temperature is raised to 37 degrees C, uptake of AAV5 takes place but up to 80 % of the bound virions dissociate from the cells. Uptake is achieved by cellular structures that are part of at least two different entry pathways. In addition to the common clathrin-dependent route, caveolar endocytosis and caveosome-like organelles are involved in a second pathway not yet described for parvoviruses. Both pathways can be used in parallel to enter an individual cell.
