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: 9023618
• 负责人: Mavis Agbandje-Mckenna
• 金额: $ 9万
• 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/9023618
• 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，在递送方面显示出巨大的前景。 基因治疗的载体或载体。我们小组的重点一直是结构-功能 甲型肝炎病毒衣壳的分析。我们有相结合的结构分析(X射线结晶学和 冷冻-EM)，进行诱变和生化分析以确定衣壳的 对感染是必不可少的。这导致了重要的信息，使 开发新的载体生产策略和目标载体的承诺。我们有 使用X射线结晶学鉴定AAV衣壳的结构区域 衣壳在酸性PHS中的变化及圆二色谱(CD)显示 微小衣壳病毒蛋白VP1(VP1u)的独特区域，含有磷脂酶 A2(PLA2)功能，在类似条件下展开。这些PHS(pH 4-6)一直是 被证明是生产性AAV感染所必需的，并且与衣壳病毒的衣壳相当 在细胞进入和运输过程中在内体隔间相遇。我们的研究得出了两个结论 意想不到的新发现。第一种是衣壳有一种以前未知的酶 活性：一种对pH敏感的酶，可以催化衣壳的自溶裂解以及 外部衬底。该酶的活性和功能的机制尚不清楚。 与其他病毒编码的蛋白水解酶相比似乎是独一无二的。第二个就是 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在细胞核中没有被包被之后，这表明衣壳在 DNA去包被后，细胞核内的基因表达。此外，裁谈会的研究表明， VP1u外化的机制，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。在这项提案中，我们希望 通过以下方式探索这些新的发现：(1)确定蛋白酶(S)的活性部位以及它的 切割靶标；(2)确定pH敏感衣壳区域在基因表达中的作用 (3)检测pH值和阳离子对其他酶的影响。 在衣壳内活动，与VP1u相关的PLA2。