755. Cytoplasmic Dynein-Dependent Adeno-Associated Virus Interaction with Microtubules
Authors: Samir Kelkar, Ronald G. Crystal, Philip L. Leopold
Journal: Molecular Therapy (2004)

The importance of adeno-associated virus (AAV)-based gene therapy vectors as therapeutic vehicles is derived in part from efficient cell membrane penetration and translocation of the adeno-associated virus capsid to the nucleus where its genetic cargo is delivered. Other viruses have enhanced intracellular trafficking to the nucleus through interaction with the microtubule cytoskeleton with subsequent energy-dependent translocation in a retrograde manner towards the nucleus. The retrograde molecular motor, cytoplasmic dynein, participates in the microtubule-based nuclear-directed translocation of numerous viruses. Based on the hypothesis that the cytoplasmic dynein motor complex mediates a biochemical linkage of the AAV capsid with microtubules, a microtubule binding assay was developed in which binding of fluorophore-conjugated AAV capsids to microtubules was evaluated by mixing fluorophore-conjugated AAV capsid with polymerized microtubules, centrifuging to pellet the microtubules, and measuring the presence of the AAV-associated fluorescence in either the microtubule-free supernatant or microtubule-containing pellet. SDS-polyacrylamide gel electrophoresis of supernatant and pellet fractions showed that fluorophore-labeled AAV capsid was located predominantly in the supernatant when AAV was incubated with taxol-stabilized microtubules. However, when purified bovine microtubule-associated proteins were combined with microtubules, the majority of AAV was found in the pellet after centrifugation (69 ± 4 % vs 23 ± 4% in the absence of microtubule-associated proteins). The interaction of microtubule-associated proteins and AAV with microtubules did not occur in the presence of 500 mM NaCl. Using freshly isolated A549 lung epithelial cell lysate, the direct involvement of cytoplasmic dynein was evaluated. A549 cell lysate was depleted of endogenous cytoplasmic dynein by immunoprecipitation with a monoclonal antibody against the 74.1 kDa intermediate chain of cytoplasmic dynein. Microtubule-associated proteins isolated from whole A549 cell lysate versus cytoplasmic dynein-depleted A549 cell lysate were compared for the ability to support AAV-microtubule interaction. Whereas AAV capsid pelleted with microtubules when incubated with microtubule-associated proteins from whole A549 cell lysate, AAV capsid was found predominantly in the supernatant when cytoplasmic dynein-depleted cell lysate was used. No effect was observed when an irrelevant primary antibody was used for immunprecipitation. To confirm the involvement of cytoplasmic dynein in AAV-microtubule interaction, the nucleotide-sensitive binding property of cytoplasmic dynein was utilized. Under conditions that are known to disrupt cytoplasmic dynein interaction with microtubules (10 mM ATP), the efficiency of the AAV-microtubule interaction was reduced. However, the presence of an identical concentration of AMP-PNP, a non-hydrolyzable analog of ATP, had no effect on AAV-microtubule interactions. The results of this study suggest that the AAV capsid interacts with microtubules in a cytoplasmic dynein-dependent manner, providing a potential mechanism for the efficient translocation of the AAV capsid to the nucleus during infection.