Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.

Adeno-associated virus (AAV) is a small human Dependovirus whose low immunogenicity and capacity for long-term persistence have led to its widespread use as vector for gene therapy. Despite great recent successes in AAV-based gene therapy, further improvements in vector technology may be hindered by an inadequate understanding of various aspects of basic AAV biology. AAV is unique in that its replication is largely dependent on a helper virus and cellular factors. In the absence of helper virus coinfection, wild-type AAV establishes latency through mechanisms that are not yet fully understood. Challenging the currently held model for AAV latency, we show here that the corepressor Krüppel-associated box domain-associated protein 1 (KAP1) binds the latent AAV2 genome at the rep ORF, leading to trimethylation of AAV2-associated histone 3 lysine 9 and that the inactivation of KAP1 repression is necessary for AAV2 reactivation and replication. We identify a viral mechanism for the counteraction of KAP1 in which interference with the KAP1 phosphatase protein phosphatase 1 (PP1) by the AAV2 Rep proteins mediates enhanced phosphorylation of KAP1-S824 and thus relief from KAP1 repression. Furthermore, we show that this phenomenon involves recruitment of the NIPP1 (nuclear inhibitor of PP1)–PP1α holoenzyme to KAP1 in a manner dependent upon the NIPP1 FHA domain, identifying NIPP1 as an interaction partner for KAP1 and shedding light on the mechanism through which PP1 regulates cellular KAP1 activity.

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Helper Functions Required for Wild Type and Recombinant Adeno- Associated Virus Growth

Current Gene Therapy ◽

10.2174/1566523054064977 ◽

2005 ◽

Vol 5 (3) ◽

pp. 265-271 ◽

Cited By ~ 45

Author(s):

Marie-Claude Geoffroy ◽

Anna Salvetti

Keyword(s):

Wild Type ◽

Adeno Associated Virus ◽

Virus Growth

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Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions

Journal of Virology ◽

10.1128/jvi.00580-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 7833-7843 ◽

Cited By ~ 43

Author(s):

Joshua C. Grieger ◽

Jarrod S. Johnson ◽

Brittney Gurda-Whitaker ◽

Mavis Agbandje-McKenna ◽

R. Jude Samulski

Keyword(s):

Conformational Changes ◽

Structural Changes ◽

Wild Type ◽

Dot Blot ◽

Adeno Associated Virus ◽

N Terminus ◽

Localization Signal ◽

Significant Effort

ABSTRACT Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.

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Presence of atrs-Like Motif Promotes Rep-Mediated Wild-Type Adeno-Associated Virus Type 2 Integration

Journal of Virology ◽

10.1128/jvi.00426-15 ◽

2015 ◽

Vol 89 (14) ◽

pp. 7428-7432 ◽

Cited By ~ 9

Author(s):

Karl Petri ◽

Richard Gabriel ◽

Leticia Agundez ◽

Raffaele Fronza ◽

Saira Afzal ◽

...

Keyword(s):

Virus Type ◽

Integration Site ◽

Dermal Fibroblasts ◽

Wild Type ◽

Mobility Shift ◽

Adeno Associated Virus ◽

Rep Protein ◽

Electrophoretic Mobility Shift Assays ◽

First Time

High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.

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Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

Journal of Virology ◽

10.1128/jvi.01198-17 ◽

2017 ◽

Vol 91 (20) ◽

Cited By ~ 23

Author(s):

Stefanie Grosse ◽

Magalie Penaud-Budloo ◽

Anne-Kathrin Herrmann ◽

Kathleen Börner ◽

Julia Fakhiri ◽

...

Keyword(s):

Protein Stability ◽

Capsid Protein ◽

Insect Cells ◽

Critical Factor ◽

Capsid Proteins ◽

Wild Type ◽

Particle Assembly ◽

Adeno Associated Virus ◽

Recombinant Vectors

ABSTRACT The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

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High-Titer, Wild-Type Free Recombinant Adeno-Associated Virus Vector Production Using Intron-Containing Helper Plasmids

Journal of Virology ◽

10.1128/jvi.74.24.11456-11463.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11456-11463 ◽

Cited By ~ 39

Author(s):

Lei Cao ◽

Yuhong Liu ◽

Matthew J. During ◽

Weidong Xiao

Keyword(s):

Gene Expression ◽

High Titer ◽

Infectious Clone ◽

Wild Type ◽

Helper Plasmid ◽

Intron Insertion ◽

Adeno Associated Virus ◽

Vector Systems ◽

Simplex Virus ◽

High Level

ABSTRACT Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate therep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.

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Site-Specific Integration of an Adeno-Associated Virus Vector Plasmid Mediated by Regulated Expression of Rep Based on Cre-loxP Recombination

Journal of Virology ◽

10.1128/jvi.74.22.10631-10638.2000 ◽

2000 ◽

Vol 74 (22) ◽

pp. 10631-10638 ◽

Cited By ~ 14

Author(s):

Wataru Satoh ◽

Yukihiko Hirai ◽

Kenji Tamayose ◽

Takashi Shimada

Keyword(s):

Transient Expression ◽

Expression System ◽

Wild Type ◽

Chromosome 19 ◽

Aav Vector ◽

Adeno Associated Virus ◽

Site Specific ◽

Coding Sequence ◽

Site Specific Integration ◽

Regulated Expression

ABSTRACT Recombinant adeno-associated virus (AAV) type 2 has attracted attention because it appears to have the potential to serve as a vector for human gene therapy. An interesting feature of wild-type AAV is its site-specific integration into AAVS1, a defined locus on chromosome 19. This reaction requires the presence of two viral elements: inverted terminal repeats and Rep78/68. Accordingly, current AAV vectors lacking the rep gene lack the capacity for site-specific integration. In this report, we describe the use of Cre-loxP recombination in a novel system for the regulated, transient expression of Rep78, which is potentially cytotoxic when synthesized constitutively. We constructed a plasmid in which the p5 promoter was situated downstream of the rep coding sequence; in this configuration, rep expression is silent. However, Cre circularizes the rep expression unit, directly joining the p5 promoter to the 5′ end of the rep78 coding sequence, resulting in expression of Rep78. Such structural and functional changes were confirmed by detailed molecular analysis. A key feature of this system is that Rep expression was terminated when the circular molecule was linearized and integrated into the chromosome. Using this regulated expression system, we attempted site-specific integration of AAV vector plasmids. A PCR-based assay and analysis of fluorescence in situ hybridization showed that the AAV vector sequence was integrated into chromosome 19. Sequence analysis also confirmed that transient expression of Rep78 was sufficient for site-specific integration at the AAVS1 locus, as is observed with integration of wild-type AAV.

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Feasibility of Generating Adeno-Associated Virus Packaging Cell Lines Containing Inducible Adenovirus Helper Genes

Journal of Virology ◽

10.1128/jvi.76.4.1904-1913.2002 ◽

2002 ◽

Vol 76 (4) ◽

pp. 1904-1913 ◽

Cited By ~ 27

Author(s):

Chunping Qiao ◽

Juan Li ◽

Anna Skold ◽

Xudong Zhang ◽

Xiao Xiao

Keyword(s):

Cell Line ◽

Cell Lines ◽

Cost Effective ◽

Helper Virus ◽

Vector System ◽

Wild Type ◽

Aav Vector ◽

Adeno Associated Virus ◽

Packaging Cell ◽

Producer Cell

ABSTRACT The adeno-associated virus (AAV) vector system is based on nonpathogenic and helper-virus-dependent parvoviruses. The vector system offers safe, efficient, and long-term in vivo gene transfer in numerous tissues. Clinical trials using AAV vectors have demonstrated vector safety as well as efficiency. The increasing interest in the use of AAV for clinical studies demands large quantities of vectors and hence a need for improvement in vector production. The commonly used transient-transfection method, although versatile and free of adenovirus (Ad), is not cost-effective for large-scale production. While the wild-type-Ad-dependent AAV producer cell lines seem to be cost-effective, this method faces the problem of wild-type Ad contamination. To overcome these shortcomings, we have explored the feasibility of creating inducible AAV packaging cell lines that require neither transfection nor helper virus infection. As a first step toward that goal, we have created a cell line containing highly inducible Ad E1A and E1B genes, which are essential for AAV production. Subsequently, the AAV Rep and Cap genes and an AAV vector containing a green fluorescent protein (GFP) reporter gene were stably introduced into the E1A-E1B cell line, generating inducible AAV-GFP packaging cell lines. Upon induction of E1A and E1B genes and infection with replication-defective Ad with E1A, E1B, and E3 deleted, the packaging cells yielded high-titer AAV-GFP vectors. Finally, the E2, E4, and VA genes of Ad, under the control of their endogenous promoters, were also introduced into these cells. A few producer cell lines were obtained, which could produce AAV-GFP vectors upon simple drug induction. Although future improvement is necessary to increase the stability and vector yield of the cells, our study has nonetheless demonstrated the feasibility of generating helper-virus-free inducible AAV producer cell lines.

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Addition of Six-His-Tagged Peptide to the C Terminus of Adeno-Associated Virus VP3 Does Not Affect Viral Tropism or Production

Journal of Virology ◽

10.1128/jvi.76.23.12023-12031.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 12023-12031 ◽

Cited By ~ 25

Author(s):

Huang-Ge Zhang ◽

Jinfu Xie ◽

Igor Dmitriev ◽

Elena Kashentseva ◽

David T. Curiel ◽

...

Keyword(s):

Capsid Protein ◽

Affinity Purification ◽

High Titer ◽

Plasmid Vector ◽

Nitrilotriacetic Acid ◽

Start Codon ◽

Wild Type ◽

Adeno Associated Virus ◽

Viral Tropism ◽

His Tag

ABSTRACT Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6×His tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6×His)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6×His-tagged VP3mutant recombinant AAV. The 6×His-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6×His-modified AAV were equivalent to those of wild-type particles. The 6×His-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6×His tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6×His-tagged AAV VP3 capsid protein and to utilize the 6×His-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

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