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The role of pH and protease activity in AAV viral transduction

pH 值和蛋白酶活性在 AAV 病毒转导中的作用

基本信息

批准号：
9023618
负责人：
Mavis Agbandje-Mckenna
金额：
$ 9万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-15 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9023618
关键词：
Active Sites Affect Binding Biochemical Capsid Cations Cell Nucleus Cells Circular Dichroism Clinical Trials Cryoelectron Microscopy Cysteine DNA Defect Dependovirus Development Disease Electron Spin Resonance Spectroscopy Gene Expression Gene Transduction Agent Genetic Genetic Transcription Genome Infection Lead Learning Light Liposomes Location Messenger RNA Minor Mutation Nature Negative Staining Nuclear Pattern Peptide Hydrolases Peptides Phospholipase A2 Play Process Production Protease Inhibitor RNA Splicing Research Role Sampling Serotyping Site Structure Techniques Testing Transcript Viral Viral Proteins Virus Virus Diseases X-Ray Crystallography adeno-associated viral vector chromatin immunoprecipitation gene therapy inhibitor/antagonist mutant next generation novel promoter public health relevance reconstruction research study response trafficking transcription factor vector viral DNA

项目摘要

Adeno-associated virus (AAV) is a non-pathogenic virus that shows great promise as a delivery vehicle or vector for gene therapy. The focus of our group has been a structure-function analysis of the AAV capsid. We have combined structural analysis (X-ray crystallography and cryo-EM) with mutagenic and biochemical analysis toward identifying regions of the capsid that are essential for infection. This has led to important information that has allowed the development of new vector production strategies and the promise of targeted vectors. We have used X-ray crystallography to identify regions of the AAV capsid that undergo a structural change when the capsid is subjected to acidic pHs and used circular dichroism (CD) to show that unique region of the minor capsid viral protein VP1 (VP1u), which contains a phospholipase A2 (PLA2) function, becomes unfolded under similar conditions. These pHs (pH 4-6) have been shown to be essential for productive AAV infections and are comparable to those that the capsid encounters in endosomal compartments during cell entry and trafficking. Our studies led to two unexpected novel discoveries. The first is that the capsid has a previously unknown enzymatic activity: a pH sensitive protease that can catalyze autolytic cleavage of the capsid as well as external substrates. Both the mechanism of the protease activity and its function are unknown and appear to be unique compared to other virus encoded proteases. The second is that mutations in the pH sensitive region of the capsid have a profound effect on gene expression, even after the viral DNA is uncoated in the nucleus, suggesting that the capsid plays a role in gene expression after DNA uncoating in the nucleus. Furthermore, the CD studies suggested a mechanism for the externalization of the VP1u which is normally buried in the capsid interior but is extruded during trafficking through acidic endosomal compartments. In this proposal, we wish to explore these novel findings by (1) identifying the active site of the protease(s) as well as its cleavage targets; (2) determining the role of the pH sensitive capsid region in gene expression after nuclear uncoating; and (3) examining the effect of pH and cations on the other enzymatic activity in the capsid, the VP1u associated PLA2.

腺相关病毒（AAV）是一种非致病性病毒，作为一种运载工具显示出很大的希望