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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：
9341366
负责人：
Mavis Agbandje-Mckenna
金额：
$ 47.72万
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 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9341366
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 Transcription Genome Infection Learning Light Liposomes Location Messenger RNA Minor Mutation Nature Negative Staining Nuclear Pattern Peptide Hydrolases Peptides Phospholipase A2 Phospholipases A Play Process Production Protease Inhibitor RNA Splicing Research Role Sampling Serotyping Site Structure Techniques Testing Transcript Viral Viral Proteins Virus Virus Diseases Viviparous-1 protein X-Ray Crystallography adeno-associated viral vector chromatin immunoprecipitation experimental study gene therapy inhibitor/antagonist mutant next generation novel promoter public health relevance reconstruction response trafficking transcription factor vector viral DNA

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：腺相关病毒（AAV）是一种非致病性病毒，作为基因治疗的递送载体或载体显示出巨大的前景。我们小组的重点是 AAV 衣壳的结构功能分析。我们将结构分析（X 射线晶体学和冷冻电镜）与诱变和生化分析相结合，以确定对感染至关重要的衣壳区域。这带来了重要的信息，使新的载体生产策略的开发和目标载体的前景成为可能。我们使用 X 射线晶体学来识别 AAV 衣壳在酸性 pH 条件下发生结构变化的区域，并使用圆二色性 (CD) 来显示含有磷脂酶 A2 (PLA2) 功能的次要衣壳病毒蛋白 VP1 (VP1u) 的独特区域在类似条件下会展开。这些 pH 值 (pH 4-6) 已被证明对于有效的 AAV 感染至关重要，并且与细胞进入和运输过程中衣壳在内体区室中遇到的 pH 值相当。我们的研究带来了两个意想不到的新发现。首先，衣壳具有以前未知的酶活性：一种 pH 敏感的蛋白酶，可以催化衣壳以及外部底物的自溶裂解。该蛋白酶活性的机制及其功能都是未知的，并且与其他病毒编码的蛋白酶相比似乎是独特的。其次，衣壳 pH 敏感区域的突变对基因表达具有深远的影响，即使病毒 DNA 在细胞核中脱壳后，这表明衣壳在 DNA 在细胞核中脱壳后在基因表达中发挥作用。此外，CD研究提出了VP1u外化的机制，VP1u通常埋藏在衣壳内部，但在通过酸性内体区室的运输过程中被挤出。在本提案中，我们希望通过以下方式探索这些新发现：（1）确定蛋白酶的活性位点及其切割靶点； (2) 确定 pH敏感衣壳区在核脱壳后基因表达中的作用； (3) 检查 pH 值和阳离子对衣壳中其他酶活性（VP1u 相关 PLA2）的影响。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Structural Dynamics and Activity of B19V VP1u during the pHs of Cell Entry and Endosomal Trafficking.

DOI：
10.3390/v14091922
发表时间：
2022-08-30
期刊：
Viruses
影响因子：
0
作者：
Lakshmanan RV;Hull JA;Berry L;Burg M;Bothner B;McKenna R;Agbandje-McKenna M
通讯作者：
Agbandje-McKenna M

Defining the stoichiometry and cargo load of viral and bacterial nanoparticles by Orbitrap mass spectrometry.

DOI：
10.1021/ja502616y
发表时间：
2014-05-21
期刊：
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
影响因子：
15
作者：
Snijder, Joost;van de Waterbeemd, Michiel;Damoc, Eugen;Denisov, Eduard;Grinfeld, Dmitry;Bennett, Antonette;Agbandje-McKenna, Mavis;Makarov, Alexander;Heck, Albert J. R.
通讯作者：
Heck, Albert J. R.

Adeno-associated virus capsid assembly is divergent and stochastic.

DOI：
10.1038/s41467-021-21935-5
发表时间：
2021-03-12
期刊：
Nature communications
影响因子：
16.6
作者：
Wörner TP;Bennett A;Habka S;Snijder J;Friese O;Powers T;Agbandje-McKenna M;Heck AJR
通讯作者：
Heck AJR

Endogenous amdoparvovirus-related elements reveal insights into the biology and evolution of vertebrate parvoviruses.