Enhancement of Adeno-Associated Virus Infection by Mobilizing Capsids into and Out of the Nucleolus

Gene Therapy Center and Department of Pharmacology, The University of North Carolina at Chapel Hill, 7119 Thurston Bowles, CB 7352, Chapel Hill, North Carolina 27599-7352, USA.
Journal of Virology (Impact Factor: 4.44). 03/2009; 83(6):2632-44. DOI: 10.1128/JVI.02309-08
Source: PubMed


Adeno-associated virus (AAV) serotypes are being tailored for numerous therapeutic applications, but the parameters governing the subcellular fate of even the most highly characterized serotype, AAV2, remain unclear. To understand how cellular conditions control capsid trafficking, we have tracked the subcellular fate of recombinant AAV2 (rAAV2) vectors using confocal immunofluorescence, three-dimensional infection analysis, and subcellular fractionation. Here we report that a population of rAAV2 virions enters the nucleus and accumulates in the nucleolus after infection, whereas empty capsids are excluded from nuclear entry. Remarkably, after subcellular fractionation, virions accumulating in nucleoli were found to retain infectivity in secondary infections. Proteasome inhibitors known to enhance transduction were found to potentiate nucleolar accumulation. In contrast, hydroxyurea, which also increases transduction, mobilized virions into the nucleoplasm, suggesting that two separate pathways influence vector delivery in the nucleus. Using a small interfering RNA (siRNA) approach, we then evaluated whether nucleolar proteins B23/nucleophosmin and nucleolin, previously shown to interact with AAV2 capsids, affect trafficking and transduction efficiency. Similar to effects observed with proteasome inhibition, siRNA-mediated knockdown of nucleophosmin potentiated nucleolar accumulation and increased transduction 5- to 15-fold. Parallel to effects from hydroxyurea, knockdown of nucleolin mobilized capsids to the nucleoplasm and increased transduction 10- to 30-fold. Moreover, affecting both pathways simultaneously using drug and siRNA combinations was synergistic and increased transduction over 50-fold. Taken together, these results support the hypothesis that rAAV2 virions enter the nucleus intact and can be sequestered in the nucleolus in stable form. Mobilization from the nucleolus to nucleoplasmic sites likely permits uncoating and subsequent gene expression or genome degradation. In summary, with these studies we have refined our understanding of AAV2 trafficking dynamics and have identified cellular parameters that mobilize virions in the nucleus and significantly influence AAV infection.

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    • "Cathepsins B and L, which have been found to cleave AAV8 in vitro with higher efficiency than AAV2 (Akache et al., 2007), may also be contributing to capsid dissociation. Finally, there is the possibility that AAV particles that enter the nucleus first localize to nucleoli, necessitating an additional transport step prior to capsid uncoating in the nucleoplasm (Johnson and Samulski, 2009). "
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    ABSTRACT: Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8׳s robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII & IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype.
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    • "General IS (taken from treatment of autoimmune disorders or transplantations) (9) and more specific interventions for AAV have been proposed and tested in pre-clinical models, such as proteasomal inhibitors (PI) (e.g., bortezomib, MG 132, carfilzomib). Many groups focus on increasing AAV transduction levels in various tissues using PI (47–50), and other compounds like arsenic trioxide (51), rather than their influence on the immune response. At the same time, another group observed reduced CTL responses as well (42). "
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    • "Interestingly, a combination of GeLC-MS and 2DE identified thirteen cellular proteins (Table 1 and 2), including nucleolin [17], [18] and nucleophosm [17], [19] which have been reported to bind to the capsid during AAV packaging or infection process. However, the remaining 11 appear to represent novel cellular AAV capsid-binding proteins as evidenced by one such protein SET. "
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