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Biological differences between recombinant adeno-associated virus (rAAV) serotypes define their efficiencies in expressing a transgene in a particular target cell. Few studies have directly compared how differences in viral entry, intracellular trafficking, and nuclear import of rAAV serotypes influence the effectiveness of transduction in the same cell type. We evaluated these characteristics for three rAAV serotypes in HeLa cells, using biochemical techniques and fluorescence-based detection of multiple serotypes in the same cell. Although rAAV2 exhibited the slowest entry, intracellular trafficking, and nuclear import among the three serotypes, it elicited the highest levels of transduction. Conversely, rAAV1 exhibited more rapid entry and nuclear import than the other serotypes, yet was ineffective at transducing HeLa cells due to impaired capsid disassembly in the nucleus. rAAV5, which entered the cell less rapidly than rAAV1, was imported efficiently into the nucleus, but then rapidly degraded, resulting in poor transduction of HeLa cells. We conclude that rAAV1, 2, and 5 utilize distinct mechanisms for intracellular trafficking, and that post-nuclear events play an important role in determining the efficiency of HeLa cell transduction by these serotypes. Thus, overcoming post-nuclear barriers that limit uncoating and/or promote virion degradation may enhance the efficiency of certain AAV serotypes.

Unique Characteristics of AAV1, 2, and 5 Viral Entry, Intracellular Trafficking, and Nuclear Import Define Transduction Efficiency in HeLa Cells