Adeno-associated virus type 5 exploits two different entry pathways in human embryo fibroblasts.

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.

Physical and biological characterization of superparamagnetic iron oxide- and ultrasmall superparamagnetic iron oxide-labeled cells: a comparison.

RATIONALE: Superparamagnetic iron-oxide particles are used frequently for cellular magnetic resonance imaging and in vivo cell tracking. The purpose of this study was to compare the labeling characteristics and efficiency as well as toxicity of superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) for 3 cell lines. METHODS: Using human fibroblasts, immortalized rat progenitor cells and HEP-G2-hepatoma cells, dose- and time-dependence of SPIO and USPIO uptake were evaluated. The amount of intracellular (U)SPIO was monitored over 2 weeks after incubation by T2-magnetic resonance relaxometry, ICP-mass-spectrometry, and histology. Transmission-electronmicroscopy was used to specify the intracellular localization of the endocytosed iron particles. Cell death-rate and proliferation-index were assessed as indicators of cell-toxicity. RESULT: For all cell lines, SPIO showed better uptake than USPIO, which was highest in HEP-G2 cells (110 +/- 2 pg Fe/cell). Cellular iron concentrations in progenitor cells and fibroblasts were 13 +/- 1pg Fe/cell and 7.2 +/- 0.3pg Fe/cell, respectively. For all cell lines T2-relaxation times in cell pellets were below detection threshold (<3 milliseconds) after 5 hours of incubation with SPIO (3.0 micromol Fe/mL growth medium) and continued to be near the detection for the next 6 days. For both particle types and all cell lines cellular iron oxide contents decreased after recultivation and surprisingly were found lower than in unlabeled control cells after 15 days. Viability and proliferation of (U)SPIO-labeled and unlabeled cells were not significantly different. CONCLUSIONS: The hematopoetic progenitor, mesenchymal fibroblast and epithelial HEP-G2 cell lines accumulated SPIO more efficiently than USPIO indicating SPIO to be better suited for cell labeling. However, the results indicate that there may be an induction of forced cellular iron elimination after incubation with (U)SPIO.

Endocytosis of adeno-associated virus type 5 leads to accumulation of virus particles in the Golgi compartment.

Among the adeno-associated virus (AAV) serotypes which are discussed as vectors for gene therapy AAV type 5 (AAV5) represents a candidate with unique advantages. To further our knowledge on AAV5-specific characteristics, we studied the entry pathway of wild-type virus in HeLa cells in the absence of helper virus by immunofluorescence and electron microscopy and by Western blot analysis. We found virus binding at the apical cell surface, especially at microvilli and, with increasing incubation time, virus accumulation at cell-cell boundaries. The different binding kinetics suggest different binding properties at apical versus lateral plasma membranes. Endocytosis of viruses was predominantly by clathrin-coated vesicles from both membrane domains; however, particles were also detected in noncoated pits. AAV5 particles were mainly routed to the Golgi area, where they could be detected within cisternae of the trans-Golgi network and within vesicles associated with cisternae and with the dictyosomal stacks of the Golgi apparatus. These data suggest that AAV5 makes use of endocytic routes that have hitherto not been described as pathways for virus entry.