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）是一种非致病病毒，显示出作为基因治疗的递送工具或载体的巨大希望。我们小组的重点是对AAV CAPSID的结构 - 功能分析。我们将结构分析（X射线晶体学和冷冻EM）与诱变和生化分析结合在一起，以识别山皮动物的感染所必需的区域。这导致了重要的信息，从而制定了新的向量生产策略和目标向量的希望。我们已经使用X射线晶体学来识别AAV CapSID的区域，该区域在经过酸性pHS并使用圆形二科主义（CD）进行结构变化，以表明其中包含磷脂酶A2（pla2）功能的次要Capsid病毒蛋白VP1（VP1）的独特区域，该区域均与phospholipase A2（Pla2）功能相似。这些pH值（pH 4-6）已被证明对于生产性AAV感染至关重要，并且与在细胞进入和运输过程中内体隔室相遇的衣壳遇到的pH值可比。我们的研究导致了两个意外的新发现。首先是衣壳具有先前未知的酶活性：一种pH敏感蛋白酶，可以催化衣壳和外部底物的自溶性裂解。蛋白酶活性及其功能的机制均尚不清楚，并且与其他编码蛋白酶相比，似乎是独一无二的。第二个是，即使在细胞核中未涂抹病毒DNA之后，衣壳的pH敏感区域中的突变也对基因表达具有深远的作用，这表明衣壳在原子核中DNA脱落后在基因表达中起作用。此外，CD研究提出了一种通常埋在衣壳内部的VP1U外部化的机制，但在通过酸性内体隔室进行运输过程中被挤出。在此提案中，我们希望通过（1）识别蛋白酶的活跃部位及其裂解靶标来探索这些新颖的发现；（2）确定 pH敏感的衣壳区域在核脱被涂抹后基因表达中的作用；（3）检查pH和阳离子对CAPSID中其他酶活性的影响，VP1U相关的PLA2。

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.