Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis

Modified viruses are used as gene transfer vectors because of their ability to transfer genetic material efficiently to the nucleus of a target cell, To better understand intracellular translocation of adenovirus serotype 5 ( Ad), fluorophores were covalently conjugated to Ad capsids, and movement of fluorescent Ad within the cytoplasm was observed during the first hour of infection of a human lung epithelial carcinoma cell line (A549). Ad translo cation was characterized with respect to its ability to achieve nuclear env elope localization as well as directed movement in the cytoplasm, Whereas A d achieved efficient nuclear localization 60 min after infection of A549 ce lls under control conditions, depolymerization of the microtubule cytoskele ton by addition of 25 mu M nocodazole reversibly inhibited development of n uclear localization, In contrast, depolymerization of microfilaments by add ition of 1 mu M cytochalasin D had no effect on nuclear localization, Direc t video observation of Ad motility showed that nocodazole, but not cytochal asin D, caused a reversible decrease in rapid linear translocations of Ad i n the cytoplasm of A549 cells, Microinjection of function-blocking antibodi es against the microtubule-dependent motor protein, cytoplasmic dynein, but not kinesin, blocked nuclear localization of Ad, consistent with net minus end-directed motility indicated by accumulation of Ad at mitotic spindles, Fluorescence ratio imaging revealed a neutral pH in the environment of tra nslocating Ad, leading to a model in which the interaction of Ad with an in tact microtubule cytoskeleton and functional cytoplasmic dynein occurs afte r escape from endosomes and is a necessary prerequisite to nuclear localiza tion of adenovirus serotype 5.