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（由申请人提供）：腺相关病毒（AAV）是一种非致病性病毒，作为基因治疗的递送载体或载体显示出巨大的前景。我们小组的重点是AAV衣壳的结构-功能分析。我们结合结构分析（X射线晶体学和冷冻EM）与诱变和生化分析，以确定感染所必需的衣壳区域。这导致了重要的信息，使新的载体生产战略的发展和有希望的目标载体。我们已经使用X射线晶体学来鉴定当衣壳经受酸性pH时经历结构变化的AAV衣壳的区域，并且使用圆二色性（CD）来显示包含磷脂酶A2（PLA 2）功能的次要衣壳病毒蛋白VP 1（VP 1u）的独特区域在类似条件下展开。这些pH（pH 4-6）已被证明是生产性AAV感染所必需的，并且与衣壳在细胞进入和运输期间在内体区室中遇到的那些相当。我们的研究带来了两个意想不到的新发现。首先是衣壳具有先前未知的酶活性：pH敏感性蛋白酶，其可以催化衣壳以及外部底物的自溶裂解。蛋白酶活性的机制及其功能都是未知的，并且与其他病毒编码的蛋白酶相比似乎是独特的。第二个是衣壳的pH敏感区域中的突变对基因表达具有深远的影响，甚至在病毒DNA在核中未被包覆之后，这表明衣壳在核中DNA未被包覆之后在基因表达中起作用。此外，CD研究提出了VP 1u外化的机制，VP 1u通常埋在衣壳内部，但在运输过程中通过酸性内体区室被挤出。在这个提议中，我们希望通过以下方式探索这些新发现：（1）鉴定蛋白酶的活性位点以及其切割靶点;（2）确定蛋白酶的活性位点。 pH敏感性衣壳区在核去包被后基因表达中的作用;和（3）检测pH和阳离子对衣壳中其它酶活性，即VP 1u相关的PLA 2的影响。