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Adeno-associated virus (AAV) is a single-stranded DNA parvovirus that causes no currently known pathology in humans. Despite the fact that this virus is of increasing interest to molecular medicine as a vector for gene delivery, relatively little is known about the cellular mechanisms controlling infection. In this study, we have examined endocytic and intracellular trafficking of AAV-2 using fluorescent (Cy3)-conjugated viral particles and molecular techniques. Our results demonstrate that internalization of heparan sulfate proteoglycan-bound AAV-2 requires ␣V␤5 integrin and activation of the small GTP-binding protein Rac1. Following endocytosis, activation of a phosphatidylinositol-3 (PI3) kinase pathway was necessary to initiate intracellular movement of AAV-2 to the nucleus via both microfilaments and microtubules. Inhibition of Rac1 using a dominant N17Rac1 mutant led to a decrease in AAV-2-mediated PI3 kinase activation, indicating that Rac1 may act proximal to PI3 kinase during AAV-2 infection. In summary, our results indicate that ␣V␤5 integrinmediated endocytosis of AAV-2 occurs through a Rac1 and PI3 kinase activation cascade, which directs viral movement along the cytoskeletal network to the nucleus.

Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.

. Nox2-containing NADPH oxidase and Akt activation play a key role in angiotensin II-induced cardiomyocyte hypertrophy. Physiol Genomics 26: 180 -191, 2006. First published May 2, 2006 doi:10.1152/physiolgenomics.00029.2005.-Angiotensin II (ANG II) has profound effects on the development and progression of pathological cardiac hypertrophy; however, the intracellular signaling mechanisms are not fully understood. In this study, we used genetic tools to test the hypothesis that increased formation of superoxide (O 2 Ϫ ⅐) radicals from a Rac1-regulated Nox2-containing NADPH oxidase is a key upstream mediator of ANG II-induced activation of serine-threonine kinase Akt, and that this signaling cascade plays a crucial role in ANG II-dependent cardiomyocyte hypertrophy. ANG II caused a significant time-dependent increase in Rac1 activation and O 2 Ϫ ⅐ production in primary neonatal rat cardiomyocytes, and these responses were abolished by adenoviral (Ad)-mediated expression of a dominant-negative Rac1 (AdN17Rac1) or cytoplasmic Cu/ZnSOD (AdCu/ZnSOD). Moreover, both AdN17Rac1 and AdCu/ZnSOD significantly attenuated ANG II-stimulated increases in cardiomyocyte size. Quantitative real-time PCR analysis demonstrated that Nox2 is the homolog expressed at highest levels in primary neonatal cardiomyocytes, and small interference RNA (siRNA) directed against it selectively decreased Nox2 expression by Ͼ95% and abolished both ANG II-induced O 2 Ϫ ⅐ generation and cardiomyocyte hypertrophy. Finally, ANG II caused a time-dependent increase in Akt activity via activation of AT 1 receptors, and this response was abolished by Ad-mediated expression of cytosolic human O 2 Ϫ ⅐ dismutase (AdCu/ ZnSOD). Furthermore, pretreatment of cardiomyocytes with dominant-negative Akt (AdDNAkt) abolished ANG II-induced cellular hypertrophy. These findings suggest that O 2 Ϫ ⅐ generated by a Nox2-containing NADPH oxidase is a central mediator of ANG II-induced Akt activation and cardiomyocyte hypertrophy, and that dysregulation of this signaling cascade may play an important role in cardiac hypertrophy. superoxide radicals; renin-angiotensin system; dominant-negative Rac1; small interference RNA; oxidative stress CARDIAC HYPERTROPHY is one of the most significant sequelae of ischemic heart disease, hypertension, and valvular disease (14). Initially an adaptive response that preserves cardiac output and minimizes wall tension, cardiac hypertrophy predisposes to ischemia, arrhythmia, and heart failure. Signal transduction pathways important in the pathogenesis of hypertrophy have been the target of many recent investigations, with angiotensin II (ANG II) emerging as a key molecule both in compensatory cardiac hypertrophy and in the pathogenesis of progressive myocardial dysfunction leading to heart failure (48, 54). Despite the importance of ANG II in cardiac hypertrophy, the signaling cascades downstream of ANG II receptor activation remain to be fully elucidated.The small GTP-binding protein Rac1, a member of the Rho family of GTPases (51), has eme...

Long-term recombinant AAV (rAAV) transgene expression in muscle has been associated with the molecular conversion of single-stranded rAAV genomes to high-molecular-weight head-to-tail circular concatamers. However, the mechanisms by which these large multimeric concatamers form remain to be defined. To this end, we tested whether concatamerization of rAAV circular intermediates occurs through intra- or intermolecular mechanisms of amplification. Coinfection of the tibialis muscle of mice with rAAV alkaline phosphatase (Alkphos)- and green fluorescent protein (GFP)-encoding vectors was used to evaluate the frequency of circular concatamer formation by intermolecular recombination of independent viral genomes. The GFP shuttle vector also encoded ampicillin resistance and contained a bacterial origin of replication to allow for bacterial rescue of circular intermediates from Hirt DNA of infected muscle samples. The results demonstrated a time-dependent increase in the abundance of rescued plasmids encoding both GFP and Alkphos, which reached 33% of the total circular intermediates by 120 days postinfection. Furthermore, these large circular concatamers were capable of expressing both GFP- and Alkphos-encoding transgenes following transient transfection in cell lines. These findings demonstrate that concatamerization of AAV genomes in vivo occurs through intermolecular recombination of independent monomer circular viral genomes and suggest new viable strategies for delivering multiple DNA segments at a single locus. Such developments will expand the utility of rAAV for splicing large gene inserts or large promoter-gene combinations carried by two or more independent rAAV vectors.