Impact of VP1-Specific Protein Sequence Motifs on Adeno-Associated Virus Type 2 Intracellular Trafficking and Nuclear Entry

Popa-Wagner, Ruth; Porwal, Manvi; Kann, Michael; Reuß, Matthias; Weimer, Marc; Florin, Luise; Kleinschmidt, Jürgen A.

doi:10.1128/jvi.00282-12

Cited by 90 publications

(97 citation statements)

References 64 publications

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“…S5). Of note, silencing of these genes also increased transduction of ΔVP1-ssAAV2 particles, which are strongly impaired in intracellular trafficking (30). However, transduction was significantly lower than that of wild-type AAV vectors ( Fig.…”

Section: Resultsmentioning

confidence: 98%

Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction

Mano

Ippodrino

Zentilin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors' broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency.adeno-associated virus | DNA-damage response | high-throughput screening | self-complementary vectors | RNA interference V iral vectors based on the adeno-associated virus (AAV) have received incremental attention over the past two decades as effective tools for in vivo gene transfer. The vectors' intrinsic structural simplicity, lack of pathogenicity, low immunogenicity, and ability to mediate long-term, episomal expression in nondividing cells in vitro and, most notably, postmitotic organs in vivo are desirable characteristics for several gene therapy applications (reviewed in ref. 1). Indeed, to date, over 90 clinical trials using these vectors have been carried out (www.abedia.com/wiley/index. html); the first commercial gene therapy product (Glybera) is an AAV1 vector for the treatment of familial lipoprotein lipase deficiency (2).Despite these achievements, AAV vectors still display an undeniable number of limitations in terms of restricted cellular permissivity and efficacy of transgene expression. Efficient transduction in vivo is essentially limited to postmitotic cells [in particular, cardiomyocytes, neurons, retinal cells, and skeletal muscle fibers (1)] and requires infection at a relatively high multiplicity of infection; in addition, vector-driven gene expression is achieved only after a relatively long lag period (3). These characteristics are still incompletely understood in molecular terms but certainly reside within the intrinsic biological properties of target cells. This finding is also highlighted by the peculiar biology of wild-type AAV, which, for the completion of its biological cycle, requires cellular coinfection by adenovirus or other viruses that modify the cellular environment of the target cells, rendering them permissive for viral replication (4-6).The efficient internalization of AAV vectors into different cell types is mediated by the binding of virions to ubiquitously expressed cell surface receptors (...

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Section: Resultsmentioning

confidence: 98%

Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction

Mano

Ippodrino

Zentilin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…In fact, recent work by Popa-Wagner et al identified a PDZ-binding motif on VP1 that, when mutated, rendered AAV2 defective in nuclear entry. 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.…”

Section: Discussionmentioning

confidence: 96%

“…In a study utilizing wt AAV2 in the presence of adenovirus, inhibition of nuclear entry via the NPC through use of the calcium channel inhibitor thapsigargin did not prevent the appearance of viral genomes in the nucleus but did affect the level of nuclear accumulation and replication (48). Recently, a PDZbinding domain was discovered on the N terminal of VP1 that was shown to be important for nuclear entry of wt AAV2 (49). While the current study was under review, a recent study revealed that AAV2 that had been acidified and then neutralized could cause nuclear envelope breakdown in permeabilized HeLa cells (50).…”

Section: A Deno-associated Virus (Aav) Is a Dependovirus In The Familymentioning

confidence: 99%

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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“…Interaction with cellular proteases and pH-activated autohydrolysis have also been implicated in the entry process (39). In vitro studies typically use a heat pulse as a surrogate for cellular triggers of VP1u and VP1/2 externalization, although characterization of capsids after heating has primarily been limited to antibody detection and electron microscopy (EM) studies (34,(40)(41)(42)75).…”

mentioning

confidence: 99%

Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

Rayaprolu

Kruse

Kant

et al. 2013

J Virol

128

108

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bIcosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production.

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Impact of VP1-Specific Protein Sequence Motifs on Adeno-Associated Virus Type 2 Intracellular Trafficking and Nuclear Entry

Cited by 90 publications

References 64 publications

Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction

Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction

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

Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

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