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，显示出巨大的潜力，作为一个交付 载体或载体。我们小组的重点一直是结构-功能 AAV衣壳的分析。我们结合了结构分析（X射线晶体学和 cryo-EM）与诱变和生物化学分析一起鉴定衣壳的区域， 是感染的关键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射线晶体学鉴定AAV衣壳的区域， 当衣壳经受酸性pH时发生变化，并使用圆二色性（CD）显示 次要衣壳病毒蛋白VP 1（VP 1u）的独特区域，含有磷脂酶 A2（PLA 2）功能，在类似条件下展开。这些pH值（pH 4-6）已被 显示出对于生产性AAV感染是必需的，并且与衣壳 在细胞进入和运输过程中遇到内体隔室。我们的研究导致了两个 意想不到的新发现。第一个是衣壳有一种以前未知的酶 活性：一种pH敏感性蛋白酶，可催化衣壳的自溶裂解以及 外部基板。该蛋白酶的活性机制及其功能尚不清楚 并且与其它病毒编码的蛋白酶相比似乎是独特的。第二是 衣壳的pH敏感区的突变对基因表达具有深远的影响， 甚至在病毒DNA在细胞核中未被包覆之后，这表明衣壳在以下方面起作用： 基因表达后，DNA在细胞核中的脱壳。此外，CD研究表明， VP 1u的外化机制，VP 1u通常埋在衣壳内部， 在运输过程中通过酸性内体区室被挤出。在这一建议中，我们希望 为了探索这些新的发现，通过（1）鉴定蛋白酶的活性位点以及其 切割靶点;（2）确定pH敏感衣壳区在基因表达中的作用 （3）研究pH和阳离子对其他酶促反应的影响， 活性的衣壳，VP 1u相关的PLA 2。