1013. Comparison of VP22 Intercellular Spreading in Live Versus Fixed Cells

Top of pageAbstract The property of the HSV-1 tegument protein VP22 of intercellular trafficking makes it a promising tool to overcome low transduction efficiencies in gene therapy. Over the last decade this distinctive ability of VP22 has been a prominent object of numerous investigations. However, recent reports not only proclaim (i) that VP22 cannot facilitate intercellular spreading, but (ii) that trafficking of VP22 fusion proteins results from artefacts of cell fixation only. To unravel this situation and to provide direct evidence for the presence or absence of VP22 mediated intercellular trafficking we generated an adenoviral vector with a dual expression cassette for VP22-GFP and DsRed both being under the control of distinct hCMV-promoters. Using this new vector type we were able to investigate not only the extent of VP22 mediated intercellular spreading by live fluorescence microscopy and FACS, but also onone and the same cells whether or not fixation (e.g., with paraformaldehyde) does influence VP22 mediated intercellular spreading. Our results clearly demonstrate that VP22-GFP can spread from less than 4% primary transduced cells to more than 70% cells of a cellular monolayer and that fixation with paraformaldehyde has no influence on VP22 mediated spreading. Thereby, we confirm the unique ability of VP22 mediated intercellular trafficking.