Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript

Becerra, Soraya; Koczot, F.; Fabisch, P; Rose, James A.

doi:10.1128/jvi.62.8.2745-2754.1988

Cited by 138 publications

(85 citation statements)

References 29 publications

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“…1. Seed [20] 15-cm plates with 293 cells so that they will be 75-80% confluent the following day (12-16 h prior to transfection). 293 cells are cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin.…”

Section: Transfection Protocolmentioning

confidence: 99%

“…VP1 is translated from the minor spliced mRNA, yielding less VP1 protein. VP2 and VP3 are both translated from the more abundant major spliced mRNA; however, VP2 is translated less efficiently because it initiates at an ACG codon, while VP3 is translated very efficiently because of a favorable Kozak context [20]. As a result, the AAV capsid proteins, which differ only in their N-terminal region, are present in the mature virion in a ratio of 1:1:10 (VP1:VP2:VP3).…”

Section: Introductionmentioning

confidence: 99%

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The Role of the Adeno-Associated Virus Capsid in Gene Transfer

Vliet

Blouin

Brument

et al. 2008

Methods in Molecular Biology™

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Adeno-associated virus (AAV) is one of the most promising viral gene transfer vectors that has been shown to effect long-term gene expression and disease correction with low toxicity in animal models, and is well tolerated in human clinical trials. The surface of the AAV capsid is an essential component that is involved in cell binding, internalization, and trafficking within the targeted cell. Prior to developing a gene therapy strategy that utilizes AAV, the serotype should be carefully considered since each capsid exhibits a unique tissue tropism and transduction efficiency. Several approaches have been undertaken in an effort to target AAV vectors to specific cell types, including utilizing natural serotypes that target a desired cellular receptor, producing pseudotyped vectors, and engineering chimeric and mosaic AAV capsids. These capsid modifications are being incorporated into vector production and purification methods that provide for the ability to scale-up the manufacturing process to support human clinical trials. Protocols for small-scale and large-scale production of AAV, as well as assays to characterize the final vector product, are presented here. The structures of AAV2, AAV4, and AAV5 have been solved by X-ray crystallography or cryo-electron microscopy (cryo-EM), and provide a basis for rational vector design in developing customized capsids for specific targeting of AAV vectors. The capsid of AAV has been shown to be remarkably stable, which is a desirable characteristic for a gene therapy vector; however, recently it has been shown that the AAV serotypes exhibit differential susceptibility to proteases. The capsid fragmentation pattern when exposed to various proteases, as well as the susceptibility of the serotypes to a series of proteases, provides a unique fingerprint for each serotype that can be used for capsid identity validation. In addition to serotype identification, protease susceptibility can also be utilized to study dynamic structural changes that must occur for the AAV capsid to perform its various functions during the virus life cycle. The use of proteases for structural studies in solution complements the crystal structural studies of the virus. A generic protocol based on proteolysis for AAV serotype identification is provided here.

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Section: Transfection Protocolmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The Role of the Adeno-Associated Virus Capsid in Gene Transfer

Vliet

Blouin

Brument

et al. 2008

Methods in Molecular Biology™

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“…The Cap ORF encodes the coat proteins that assemble to produce a mature capsid. The Rep ORF carries the protein sequence information needed for AAV replication and gene regulation (5). Four mRNAs from Rep ORF give rise to 4 AAV nonstructural proteins: Rep78, Rep68, Rep52, and Rep40 (6,7).…”

Section: Introductionmentioning

confidence: 99%

Adeno‐Associated Virus Rep78 Binds to E2‐Responsive Element 1 of Bovine Papillomavirus Type 1

Chon

Rim

1999

IUBMB Life

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Adeno-associated virus type-2 (AAV-2) is a helper-dependent parvovirus that has been implicated in the inhibition of replication and oncogenic transformation of bovine papillomavirus type-1 (BPV-1) and other transforming DNA viruses. Previous studies have suggested that the Rep78 protein of AAV-2 is a key player mediating this effect. In this report we have analyzed the effect of AAV-2 Rep78 protein on the regulation of gene expression of a reporter gene under the control of the long control region (LCR) of BPV-1. Our results show that Rep78 is capable of down-regulating the promoter activity of the LCR in vivo in tissue culture cells. Inhibition of LCR activity in vivo suggested the need for Rep78 to bind to a region of the LCR promoter spanning the E2-responsive elements of BPV-1. This observation was further confirmed in vitro with gel shift assays showing specific binding of Rep78 to DNA oligonucleotides containing E2-responsive element 1 (E2RE1) sequences of BPV-1 LCR. Our results expand the understanding of the mechanism of trans-regulation mediated by Rep78 and involving this protein and DNA sequences with complex secondary structure.

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“…14) Transcription from the p40 promoter generates two alternatively spliced forms, the minor transcript encoding VP1 and the major transcript for VP2 and VP3. 15,16) One of the limiting factors for AAV vector-mediated gene therapy is the difficulty in producing high-titer AAV vector stocks. The conventional method for AAV vector production is based on cotransfection of a helper plasmid expressing the viral genes, rep and cap, without ITRs and a vector plasmid consisting of a foreign gene flanked by ITRs into 293 cells infected with adenoviruses.…”

mentioning

confidence: 99%

Efficient Production of Adeno‐associated Virus Vectors Using Split‐type Helper Plasmids

Ogasawara

Urabe

Kogure

et al. 1999

Japanese Journal of Cancer Research

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Adeno-associated virus (AAV) vectors are potentially useful vehicles for the delivery of therapeutic genes into human cells. To determine the optimal expression pattern of AAV proteins (Rep78, Rep68, Rep52, Rep40, and Cap proteins) for packaging the recombinant AAV genome, helper plasmids were split into two portions. In this study, two sets of split-type helper plasmids were prepared; i.e., 1) a Rep expression plasmid (pRep) and Cap expression plasmid (pCap), and 2) a large Rep expression plasmid (pR78/68) and small Rep plus Cap expression plasmid (pR52/ 40Cap). When AAV vectors were produced using these sets of split-type helper plasmids at various ratios, the optimal ratio of (large) Rep expression plasmid and Cap expression plasmid was 1 to 9 for both sets. More importantly, the titers were comparable to or even higher than that of a conventional helper plasmid (pIM45) (4.9 ± ± ± ±2.1× × × ×10 11 vector particles/10 cm dish for pRep and pCap; 2.9 ± ± ± ±1.6× × × ×10 11 vector particles/10 cm dish for pR78/68 and pR52/40Cap; and 1.8 ± ± ± ±0.16× × × ×10 11 particles/10 cm dish for pIM45). Western analysis of AAV proteins suggests that the expression of a relatively small amount of large Rep and a large amount of Cap is important for optimal vector production. The present study shows that the AAV helper plasmid can be split without losing the ability to package the recombinant AAV genome, and provides us with valuable basic information for the development of efficient AAV packaging cell lines.

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Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript

Cited by 138 publications

References 29 publications

The Role of the Adeno-Associated Virus Capsid in Gene Transfer

The Role of the Adeno-Associated Virus Capsid in Gene Transfer

Adeno‐Associated Virus Rep78 Binds to E2‐Responsive Element 1 of Bovine Papillomavirus Type 1

Efficient Production of Adeno‐associated Virus Vectors Using Split‐type Helper Plasmids

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