| Adeno-associated virus (AAV) is an attractive vehicle for gene therapy. Serotypes of this non-pathogenic parvovirus exhibit both diversity in cellular tropism and long-term transgene expression; however, their medical application has been limited due to inefficient transduction of target tissues. In this dissertation we have explored how virion components and host factors at the subcellular level help determine whether a vector can transduce a target cell. Transduction is only successful when virions attach to host cell receptors, internalize into an endosome, escape from a subcellular compartment, traffic through the cytosol, target the nucleus, and release their genomes during favorable conditions. Prior to this work, it was unclear if capsids could enter the nucleus intact during infection. Additionally, specific mutations in the two larger capsid proteins, VP1 and VP2, were known to reduce AAV infectivity. These proteins possess unique N-termini that contain a putative phospholipase A2 domain and potential nuclear localization signals, but their roles in directing subcellular trafficking have not been well defined. Moreover, induction of cellular stress by genotoxic agents, heat shock, or proteasome inhibition, dramatically increases the transduction efficiency of AAV, although the mechanisms responsible for this effect remain unknown. Therefore, we set out to build upon this foundation of AAV subcellular biology and hypothesized that AAV virions preferentially transduce during conditions of cellular stress. With this work, we have elucidated that: (1) AAV capsids are capable of entering the nucleus as an infectious virion; (2) basic regions near the N-terminus of VP1 direct steps in subcellular trafficking and may play a role in uncoating; and (3) AAV exploits conditions of cellular stress related to expression of misfolded proteins, which may be a generalized phenomenon having positive implications for targeting diseased cells with these vectors. With its focus on how AAV navigates the subcellular environment, this work intersects areas of cell biology, virology, and gene delivery. Ultimately, by analyzing how capsid composition and host parameters influence infection we have addressed central concepts at the level of subcellular trafficking that help advance the design of viral vectors for gene therapy. |
