Fiber swap between adenovirus subgroups B and C alters intracellular trafficking of adenovirus gene transfer vectors

Following receptor binding and internalization, intracellular trafficking o f adenovirus (Ad) among subgroups B and C is different, with significant am ounts of Ad serotype 7 (Ad7) (subgroup B) virions found in cytoplasm during the initial hours of infection while Ad5 (subgroup C) virions rapidly tran slocate to the nucleus. To evaluate the role of the fiber in these differen ces, we examined intracellular trafficking of Ad5, Ad7, and Ad5f7 (a chimer ic vector composed of the Ad5 capsid with the fiber replaced by the Ad7 fib er) by conjugating Ad capsids directly with Cy3 fluorescent dye, permitting the trafficking of the capsids to be examined by fluorescence microscopy. The human lung carcinoma cell line A549 was infected with Cy3-conjugated vi ruses for 10 min followed by a 1-h incubation. Ad5 virions rapidly transloc ated to the nucleus (within 1 h of infection), while Ad7 virions were widel y distributed in the cytoplasm at the same time point. Interestingly, chime ric Ad5f7 virions behaved similarly to Ad7 but not Ad5. In this regard, the percentages of nuclear localization of Ad5, Ad7, and Ad5f7 at 1 h followin g infection were 72% +/- 4%, 32% +/- 6%, and 38% +/-2%, respectively. Consi stent with these observations, fluorescence in situ hybridization demonstra ted that most of the Ad5 DNA was detected at the nucleus after 1 h, but at the same time point, DNA of Ad7 and Ad5f7 was distributed in both the nucle us and cytoplasm. Quantification of the kinetics of Ad genomic DNA delivery to the nucleus using a fluorogenic probe-based PCR assay (TaqMan PCR) demo nstrated that the percentages of nuclear association of Ad5 DNA and Ad5f7 D NA at 1 h postinfection were 80% +/- 13% and 43% +/- 1%, respectively. Alth ough it has been generally accepted that Ad fiber protein mediates attachme nt of virions to cells and that fibers dissociate during endocytic uptake, these data suggest that in addition to mediating binding to the cell surfac e, fiber likely modulates intracellular trafficking as well.