18. Incorporation of the Green Fluorescent Protein into the Adeno-Associated Virus Type 2 Capsid

Abstract

Adeno-Associated Virus type 2 (AAV) is a small, nonenveloped, icosahedral virus of approximately 25 nm in diameter that packages a single-stranded DNA. Until now, no human disease caused by AAV has been detected. This and other features as e.g. its ability to transduce both dividing and non-dividing cells, its low immunogenicity and its broad tropism make of AAV a promising system for the development as gene therapy vector. However, many aspects of its infectious biology still remain to be elucidated. Green fluorescent protein (GFP) has been extensively used to study intracellular trafficking of proteins. Therefore, we incorporated GFP into the AAV capsid in order to allow a direct visualization of the infectious process. Based on earlier results, obtained by Yang et al. (1998), we generated an N-terminal fusion of the GFP protein with the second largest capsid protein, named VP2. We could show by transient transfection assays that this fusion protein is tranported into the nucleus like wild type capsid protein. Next, we generated viral particles containing the GFP-VP2 fusion protein. Viral progeny was obtained with titers comparable to wild type AAV and could be purified by iodixanol gradient centrifugation or heparin affinity chromatography. The GFP-VP2 fusion protein was detected together with the other wild type capsid proteins in Western Blot analysis of purified viral preparations. The particle to capsid ratio showed that the fusion protein does not interfere with viral genome packaging. Furthermore, HeLa cells infected with GFP-VP2 containing virions resulted in eGFP positive cells measurable by FACS analysis. Such infections could be inhibited by the addition of heparin. Finally, fluorescent viral particles could be visualized by live cell imaging and fluorescent microscopy. First results will be presented.