Dataset Information

A Generic Method for Fast and Sensitive Detection of Adeno-Associated Viruses Using Modified AAV Receptor Recombinant Proteins.

ABSTRACT: Adeno-Associated Viruses (AAV) are widely used gene-therapy vectors for both clinical applications and laboratory investigations. The titering of different AAV preparations is important for quality control purposes, as well as in comparative studies. However, currently available methods are limited in their ability to detect various serotypes with sensitivity and convenience. Here, we took advantage of a newly discovered AAV receptor protein with high affinity to multiple AAV serotypes, and developed an ELISA-like method named "VIRELISA" (virus receptor-linked immunosorbent assay) by adopting fusion with a streptavidin-binding peptide (SBP). It was demonstrated that optimized VIRELISA assays exhibited satisfactory performance for the titering of AAV2. The linear range of AAV2 was 1 × 105 v.g. to 5 × 109 v.g., with an LOD (limit of detection) of 5 × 104 v.g. Testing of VIRELISA for the quantification of AAV1 was also successful. Our study indicated that a generic protocol for the quantification of different serotypes of AAVs was feasible, reliable and cost-efficient. The applications of VIRELISA will not only be of benefit to laboratory research due to its simplicity, but could also potentially be used for monitoring the circulation AAV loads both in clinical trials and in wild type infection of a given AAV serotype.

SUBMITTER: Cui M

PROVIDER: S-EPMC6864843 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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A Generic Method for Fast and Sensitive Detection of Adeno-Associated Viruses Using Modified AAV Receptor Recombinant Proteins.

Cui Mengtian M Lu Yabin Y Tang Can C Zhang Ran R Wang Jing J Si Yang Y Cheng Shan S Ding Wei W

Molecules (Basel, Switzerland) 20191103 21

Adeno-Associated Viruses (AAV) are widely used gene-therapy vectors for both clinical applications and laboratory investigations. The titering of different AAV preparations is important for quality control purposes, as well as in comparative studies. However, currently available methods are limited in their ability to detect various serotypes with sensitivity and convenience. Here, we took advantage of a newly discovered AAV receptor protein with high affinity to multiple AAV serotypes, and deve ...[more]

PMID: 31684125

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Project description:Post-translational modifications in viral capsids are known to fine-tune and regulate several aspects of the infective life cycle of several viruses in the host. Recombinant viruses that are generated in a specific producer cell line are likely to inherit unique post-translational modifications during intra-cellular maturation of its capsid proteins. Data on such post-translational modifications in the capsid of recombinant adeno-associated virus serotypes (AAV1-rh10) is limited. We have employed liquid chromatography and mass spectrometry analysis to characterize post-translational modifications in AAV1-rh10 capsid protein. Our analysis revealed a total of 52 post-translational modifications in AAV2-AAVrh10 capsids, including ubiquitination (17%), glycosylation (36%), phosphorylation (21%), SUMOylation (13%) and acetylation (11%). While AAV1 had no detectable post-translational modification, at least four AAV serotypes had >7 post-translational modifications in their capsid protein. About 82% of these post-translational modifications are novel. A limited validation of AAV2 capsids by MALDI-TOF and western blot analysis demonstrated minimal glycosylation and ubiquitination of AAV2 capsids. To further validate this, we disrupted a glycosylation site identified in AAV2 capsid (AAV2-N253Q), which severely compromised its packaging efficiency (~ 100-fold vs. AAV2 wild-type vectors). In order to confirm other post-translational modifications detected such as SUMOylation, mutagenesis of a SUMOylation site(K258Q) in AAV2 was performed. This mutant vector demonstrated reduced levels of SUMO-1/2/3 proteins and negligible transduction, 2 weeks after ocular gene transfer. Our study underscores the heterogeneity of post-translational modifications in AAV vectors. The data presented here, should facilitate further studies to understand the biological relevance of post-translational modifications in AAV life cycle and the development of novel bioengineered AAV vectors for gene therapy applications. ENZYMES: Trypsin, EC 3.4.21.4.

Project description:UnlabelledAdeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5' rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad.ImportanceNext-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study AAV in vivo, combined in cellulae and in silico studies will help to forward the understanding of the unique, bipartite AAV life cycle.

Dataset Information

A Generic Method for Fast and Sensitive Detection of Adeno-Associated Viruses Using Modified AAV Receptor Recombinant Proteins.

Publications

A Generic Method for Fast and Sensitive Detection of Adeno-Associated Viruses Using Modified AAV Receptor Recombinant Proteins.

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