(392e) Development of Quantum Dot Labeling of Gene Delivery Vectors for Visualization of Viral Transduction Pathways

γ-Retroviral and lentiviral vectors are currently the most commonly used gene delivery vehicles due to their ability to permanently integrate a therapeutic transgene into a target cell chromosome. Improved understanding of virus-host cell interactions can provide crucial insights for enhancing the efficacy of virus-mediated gene delivery as well as preventing virus-triggered diseases. The ability to track individual viruses is a powerful tool for investigating viral infection routes and characterizing the dynamic interactions between viruses and target cells. We report here a general method of labeling enveloped viruses with semiconductor quantum dots (QDs) for use in single virus trafficking studies. Retroviruses, including human immunodeficiency virus (HIV), could be successfully tagged with QDs through the membrane incorporation of a short acceptor peptide (AP) that is susceptible to site-specific biotinylation and attachment of streptavidin-conjugated QDs. We showed that this AP tag-based QD labeling had little effect on the viral infectivity and allowed for the study of the kinetics of the internalization of the recombinant lentivirus enveloped with vesicular stomatitis virus glycoprotein (VSVG) into the early endosomes. We further demonstrated by direct visualization of QD-labeled virus that VSVG-pseudotyped lentivirus enters cells independent of clatherin- and caveolin-pathways, while the entry of VSVG-pseudotyped retrovirus occurs via the clathrin pathway.

Adeno-associated virus (AAV), a member of the non-envelope parvoviridae family, has attracted considerable interest because it shows great promise for use in human gene therapy. However, the membrane penetration and uncoating mechanisms are poorly understood for these viruses, even though they are relatively small and simple in structure. Since the AAV virion is only about 20 nm in diameter, the number of dye molecules that can be attached to a single virus without causing self-quenching or affecting viral infectivity is very limited. Quantum dots are much brighter than the conventional fluorophores, which can allow the detection of viruses with much lower amounts of labeling molecules. This study also reports the general strategy for linking AAV virion with quantum dots through a coupling reaction for a single-virus tracking in target cells. Intracellular trafficking of AAV, including endosomal tracking and cytoskelton-dependent transport will be addressed in more detail.