Trafficking and Propagation of Canine Adenovirus Vectors Lacking a Known Integrin-Interacting Motif

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Abstract
Canine adenovirus type 2 (CAV-2) vectors may be attractive tools for gene transfer thanks to the lack of pre-existing immunity in humans, and because of the preferential transduction of neurons when the vector is injected into the brain and some innervated tissues. The coxsackievirus-adenovirus receptor (CAR) appears to play a major role during infection of most human serotypes, whereas the αvβ3/5 integrins have been reported to play a significant auxiliary role. We showed that CAV-2 also attaches to and uses CAR to enter cells, but CAV-2 transduction could be notably different from that of the prototype human adenovirus serotype 5 (Ad5). Initially, the CAV-2 capsid appears to be 10-fold less negatively charged than Ad5. Second, the CAV-2 penton, hexon, and fiber proteins do not contain a known integrin-interacting motif. Because of its potential interest in the clinic, we analyzed the different steps of cellular trafficking and the propagation kinetics of CAV-2 vectors. We found that Ad5 and CAV-2 vectors have comparable kinetics of binding (10 min), internalization (10 min), endosomal escape (17 min), attachment to the nuclear membrane (35 min), and formation (18 hr) and release (34 hr) of functional virions. Surprisingly, the RGD- CAV-2 capsid also induced the reorganization of actin filaments in HeLa cells. Actin reorganization is thought to be dependent on αvβ3/5 integrin stimulation.