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

• 批准号: 9134791
• 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
• 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/9134791
• 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

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(申请人提供)：腺相关病毒(AAV)是一种非致病病毒，作为基因治疗的载体或载体显示出巨大的前景。我们小组的重点是对AAV衣壳的结构和功能进行分析。我们将结构分析(X射线结晶学和低温电子显微镜)与诱变和生化分析相结合，以确定衣壳中对感染至关重要的区域。这导致了重要的信息，使新的媒介生产战略的开发和靶向媒介的承诺成为可能。我们使用X射线结晶学来确定AAV衣壳在酸性PHS下经历结构变化的区域，并使用圆二色谱(CD)来表明包含磷脂酶A2(PLA2)功能的次要衣壳蛋白VP1(VP1u)的独特区域在类似条件下变得展开。这些PHS(PH值4-6)已被证明是产生AAV感染所必需的，并与衣壳在细胞进入和运输过程中在内体隔间遇到的PHS相当。我们的研究导致了两项意想不到的新发现。首先，衣壳具有一种以前未知的酶活性：一种对pH敏感的蛋白酶，可以催化衣壳和外部底物的自溶切割。该酶的活性机制和功能尚不清楚，与其他病毒编码的蛋白水解酶相比似乎是独一无二的。第二，衣壳pH敏感区的突变对基因表达有深远的影响，即使在病毒DNA在核内去涂层后也是如此，这表明在DNA在核内去涂层后，衣壳在基因表达中发挥了作用。此外，CD研究提出了VP1u外化的机制，它通常埋在衣壳内部，但在运输过程中通过酸性内体隔间挤出。在这个提议中，我们希望通过(1)确定蛋白酶(S)的活性部位及其切割靶标来探索这些新的发现；(2)确定 (3)检测pH和阳离子对衣壳中其他酶活性的影响，即VP1u相关的PLA2。