847. Adeno-Associated Virus Capsids from Divergent AAV Clades and Subgroup C Adenovirus Capsids Share a Common Microtubule-Binding Mechanism Relevant to Virus Trafficking
Authors: Samir A. Kelkar, Bishnu P. De, Guangping Gao, James M. Wilson, Ronald G. Crystal, Philip L. Leopold
Journal: Molecular Therapy (2005)

The importance of adeno-associated virus (AAV)-based and adenovirus (Ad)-based gene therapy vectors as therapeutic vehicles is derived in part from their ability to efficiently penetrate cell membranes and translocate rapidly to the nucleus where their genetic cargo is delivered. Both Ad and AAV are known to utilize the microtubule cytoskeleton and the molecular motor, cytoplasmic dynein, to move in an energy-dependent manner toward the nucleus. We have previously described an in vitro microtubule-binding assay that demonstrates the interaction between cytoplasmic dynein, microtubule, and viral capsids. This assay was used to test the hypothesis that divergent members of the AAV family and Ad share a common microtubule binding mechanism. The experimental strategy involves the production of AAV2 capsids by infecting SF9 insect cells with a baculovirus expressing AAV2 capsid proteins VP1, VP2, and VP3. The AAV2 capsid produced by baculovirus exhibited microtubule-binding comparable to microtubule binding by AAV2 produced by standard techniques in 293 cells. To examine competition among the various capsids for microtubule binding, a fixed amount of Cy3 fluorophore-conjugated AAV2 was introduced into the microtubule binding assay in the presence of SF9 cell lysate containing baculovirus-derived AAV2 or SF9 cell lysate following infection with a null baculovirus. Typically, >80% of Cy3-AAV2 capsid pelleted with microtubules in the binding assay. However, in the presence of SF9 cell lysate containing the baculovirus-derived AAV2 capsid protein, but not null baculovirus lysate, competitive inhibition of Cy3-AAV2 microtubule binding was observed (<1% Cy3-AAV2 in the pellet). To determine whether the AAV2 (clade B) binding mechanism was shared by AAV capsids from divergent clades, two additional AAV capsids were evaluated. AAV5 (the most evolutionarily divergent human AAV, not currently included in a clade) and AAVrh.10 (a primate AAV from clade B) were evaluated in the competition assay. Whereas Cy3-AAV5 and Cy3AAVRh.10 efficiently bound to microtubules in the binding assay, <1% of the Cy3 capsids were found in the pellet when a lysate containing baculovirus-derived AAV2 capsid proteins was included, suggesting that AAV2, AAV5, and AAVRh.10 share a common mechanism for interaction with the microtubule cytoskeleton. To determine whether the AAV2 binding mechanism is similar to the adenovirus-microtubule binding mechanism to microtubules, SF9 cell lysate containing baculovirus-derived AAV2 capsid proteins or null baculovirus was added to a Cy3 adenovirus-microtubule binding assay. As observed in the AAV competition experiments, the interaction of Cy3-adenovirus and microtubules was inhibited in the presence of excess AAV2 capsid proteins. Thus, genotypically diverse viruses utilize the same molecular mechanisms to interact with the microtubule cytoskeleton, providing a potential unifying mechanism for the efficient translocation of many different viral capsids to nucleus during infection.