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）是一种非致病性病毒，作为传递病毒具有巨大的前景 用于基因治疗的载体或载体。我们小组的重点是结构功能 AAV 衣壳的分析。我们结合了结构分析（X 射线晶体学和 冷冻电镜）通过诱变和生化分析来识别衣壳区域 对于感染来说是必不可少的。这导致了重要的信息，使 新载体生产策略的开发和靶向载体的前景。我们有 使用 X 射线晶体学来识别 AAV 衣壳中经历结构性变化的区域 当衣壳处于酸性 pH 条件下时发生变化，并使用圆二色性 (CD) 来显示 小衣壳病毒蛋白 VP1 (VP1u) 的独特区域，其中含有磷脂酶 A2 (PLA2) 功能在类似条件下展开。这些 pH 值（pH 4-6）已 已被证明对于生产性 AAV 感染至关重要，并且与衣壳所产生的感染相当 在细胞进入和运输过程中在内体区室中相遇。我们的研究得出了两个结论 意想不到的新发现。第一个是衣壳具有以前未知的酶 活性：一种 pH 敏感的蛋白酶，可以催化衣壳的自溶裂解以及 外部基板。蛋白酶活性的机制及其功能尚不清楚 与其他病毒编码的蛋白酶相比似乎是独特的。第二个是 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 在细胞核中脱去外壳后，这表明衣壳在 DNA在细胞核中脱壳后基因表达。此外，CD 研究表明 VP1u 外化的机制，通常埋在衣壳内部，但 在通过酸性内体区室的运输过程中被挤出。在这个提案中，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敏感衣壳区在基因表达中的作用 核去涂层后； (3) 检查 pH 值和阳离子对其他酶的影响 衣壳中的活性，VP1u 相关的 PLA2。