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: 8926457
• 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/8926457
• 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

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）是一种非致病病毒，显示出很大的希望作为递送 基因治疗的车辆或载体。我们小组的重点是结构功能 AAV CAPSID的分析。我们已经结合了结构分析（X射线晶体学和 cryo-em）具有诱变和生化分析，以识别capsid的区域 对于感染至关重要。这导致了重要的信息，使 制定新的向量生产策略和目标向量的承诺。我们有 使用X射线晶体学来识别经历结构性的AAV CAPSID的区域 当将衣壳经受酸性pHS并使用圆形二色性（CD）时，会改变 含有磷脂酶的次要衣壳病毒蛋白VP1（VP1U）的独特区域 A2（PLA2）函数在相似条件下展开。这些pH（pH 4-6）已经 证明是生产性AAV感染至关重要的，并且与Capsid的感染相当 在细胞进入和贩运过程中，内体隔室遇到。我们的研究导致了两个 意外的新颖发现。首先是衣壳具有先前未知的酶促 活动：一种pH敏感蛋白酶，可以催化衣壳的自溶性裂解以及 外部基材。蛋白酶活性及其功能的机制均未知 与其他编码的蛋白酶相比，似乎是独一无二的。第二个是 衣壳的pH敏感区域中的突变对基因表达有深远的影响， 即使在细胞核中也未涂覆病毒DNA之后，也表明衣壳在 DNA在细胞核中脱落后的基因表达。此外，CD研究表明 VP1U外部化的机制，该机制通常埋在衣壳内部，但 在通过酸性内体室的运输过程中被挤出。在此提案中，我们希望 通过（1）识别蛋白酶的活性位点及其 切割目标； （2）确定pH敏感的衣壳区域在基因表达中的作用 核涂覆后； （3）检查pH和阳离子对其他酶促的影响 Capsid中的活性，VP1U相关的PLA2。