Adeno-associated virus interactions with B23/Nucleophosmin: Identification of sub-nucleolar virion regions

Bevington, Joyce; Needham, Patrick G.; Verrill, Kristin C.; Collaco, Roy F.; Basrur, Venkatesh; Trempe, James P.

doi:10.1016/j.virol.2006.07.050

Cited by 49 publications

(55 citation statements)

References 79 publications

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“…They concluded that this AAV2 PDZ binding domain might initiate signaling pathways within the cell to facilitate nuclear entry (49). Moreover, previous work has shown that AAV2 can interact with the nucleolar proteins nucleophosmin and nucleolin (27,90,91), suggesting that rAAV2 can translocate into the nucleus through interaction with these proteins. In addition to the observations of nuclear envelope breakdown by parvoviruses, including AAV2, Porwal et al showed that AAV2 can directly interact with nucleoporins that comprise the nuclear pore complex (50).…”

Section: Discussionmentioning

confidence: 97%

Recombinant Adeno-Associated Virus Utilizes Host Cell Nuclear Import Machinery To Enter the Nucleus

Nicolson

Samulski

2014

J Virol

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Recombinant adeno-associated viral (rAAV) vectors have garnered much promise in gene therapy applications. However, widespread clinical use has been limited by transduction efficiency. Previous studies suggested that the majority of rAAV accumulates in the perinuclear region of cells, presumably unable to traffic into the nucleus. rAAV nuclear translocation remains illdefined; therefore, we performed microscopy, genetic, and biochemical analyses in vitro in order to understand this mechanism. Lectin blockade of the nuclear pore complex (NPC) resulted in inhibition of nuclear rAAV2. Visualization of fluorescently labeled particles revealed that rAAV2 localized to importin-␤-dense regions of cells in late trafficking steps. Additionally, small interfering RNA (siRNA) knockdown of importin-␤ partially inhibited rAAV2 nuclear translocation and inhibited transduction by 50 to 70%. Furthermore, coimmunopreciptation (co-IP) analysis revealed that capsid proteins from rAAV2 could interact with importin-␤ and that this interaction was sensitive to the small GTPase Ran. More importantly, mutations to key basic regions in the rAAV2 capsid severely inhibited interactions with importin-␤. We tested several other serotypes and found that the extent of importin-␤ interaction varied, suggesting that different serotypes may utilize alternative import proteins for nuclear translocation. Co-IP and siRNA analyses were used to investigate the role of other karyopherins, and the results suggested that rAAV2 may utilize multiple import proteins for nuclear entry. Taken together, our results suggest that rAAV2 interacts with importin-␤ alone or in complex with other karyopherins and enters the nucleus via the NPC. These results may lend insight into the design of novel AAV vectors that have an enhanced nuclear entry capability and transduction potential. IMPORTANCEUse of recombinant adeno-associated viral (rAAV) vectors for gene therapy applications is limited by relatively low transduction efficiency, in part due to cellular barriers that hinder successful subcellular trafficking to the nucleus, where uncoating and subsequent gene expression occur. Nuclear translocation of rAAV has been regarded as a limiting step for successful transduction but it remains ill-defined. We explored potential nuclear entry mechanisms for rAAV2 and found that rAAV2 can utilize the classical nuclear import pathway, involving the nuclear pore complex, the small GTPase Ran, and cellular karyopherins. These results could lend insight into the rational design of novel rAAV vectors that can more efficiently translocate to the nucleus, which may lead to more efficient transduction.

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Section: Discussionmentioning

confidence: 97%

Recombinant Adeno-Associated Virus Utilizes Host Cell Nuclear Import Machinery To Enter the Nucleus

Nicolson

Samulski

2014

J Virol

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“…Assuming that nucleolar accumulation is determined by the abundance of the net positive charges within this region, AAP5 would likely be devoid of an NoLS and excluded from the nucleolus. If this is true, AAP5 would not be able to transport AAV5 capsid VP proteins to the nucleolus, the organelle that is believed to be important for AAV capsid assembly, at least for AAV2 (24)(25)(26)(27). How would AAV5 viral capsid proteins assemble if they are not transported to the nucleolus?…”

Section: Discussionmentioning

confidence: 99%

“…Since we analyzed only 18 combinations out of all 25 possible combinations of AAP2BR mutations that leave at least 2 intact AAP2BRs (10 combinations of 2 intact BRs with 3 mutated BRs, 10 combinations of 3 intact BRs with 2 mutated BRs, and 5 combinations of 4 intact BRs with 1 mutated BR), a range of P values are given in which all the possible outcomes from the 7 AAP2BR mutants that were not assessed are taken into account. The P values that would be obtained if all 25 AAP2BR mutants were analyzed are expressed as P 25 . The P values obtained from the 18 AAP2BR mutants are expressed as P 18 .…”

Section: Cellsmentioning

confidence: 99%

Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein

Earley

Kawano

Adachi

et al. 2015

J Virol

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Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2 144 -184 ). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and ␤-galactosidase-AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2 144 -184 has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly. IMPORTANCEAdeno-associated virus (AAV) has become a popular and successful vector for in vivo gene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of the assembly-activating protein (AAP), a nonstructural, nucleolarlocalizing AAV protein essential for viral capsid assembly, has provided us a new opportunity to better understand the fundamental processes required for virion formation. Here, we identify clusters of basic amino acids in the carboxy terminus of AAP from AAV serotype 2 (AAV2) that act as nuclear and nucleolar localization signals. We also demonstrate their importance in maintaining AAP expression levels and efficient production of viral capsids. Insights into the functions of AAP can elucidate the requirements and process for AAV capsid assembly, which may lead to improved vector production for use in gene therapy. This study also contributes to the growing body of work on nuclear and nucleolar localization signals.A deno-associated virus (AAV) is a small, single-stranded DNA virus from the parvovirus family that has become a successful vector for gene delivery. The recent achievements in the field of AAV vector research have called attention to the incompletely understood life cycle of the v...

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“…A recent report using proteomics analysis of AAV vectors showed the association of 10 cellular proteins, for example, Nucleolin (NCL) and nucleophosmin (NPM1), with AAV vectors (Dong et al, 2014). NCL and NPM1 are nucleolus proteins with many functions, which bind to AAV2 capsids and shuttle the AAV proteins between the cytoplasm and nucleus (Qiu and Brown, 1999;Bevington et al, 2007;Johnson and Samulski, 2009), demonstrating an important role of cellular proteins in AAV capsid assembly or vector genome encapsidation.…”

Section: Introductionmentioning

confidence: 99%

Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy

et al. 2014

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Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, preexisting immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAVassociated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45-and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat (ITR) sequence. The significance and implications of our findings in future improvement of AAV production are discussed.

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Adeno-associated virus interactions with B23/Nucleophosmin: Identification of sub-nucleolar virion regions

Cited by 49 publications

References 79 publications

Recombinant Adeno-Associated Virus Utilizes Host Cell Nuclear Import Machinery To Enter the Nucleus

Recombinant Adeno-Associated Virus Utilizes Host Cell Nuclear Import Machinery To Enter the Nucleus

Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein

Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy

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