The role of pH and protease activity in AAV viral transduction

描述（由申请人提供）：腺相关病毒（AAV）是一种非致病性病毒，作为基因治疗的运载工具或载体具有很大的前景。我们小组的重点是AAV衣壳的结构-功能分析。我们将结构分析（x射线晶体学和低温电镜）与诱变和生化分析相结合，以确定衣壳中对感染至关重要的区域。这带来了重要的信息，使我们能够制定新的病媒生产战略和目标病媒的前景。我们使用x射线晶体学来识别当衣壳受到酸性ph时发生结构变化的AAV衣壳区域，并使用圆二色性（CD）来显示含有磷脂酶A2 （PLA2）功能的次要衣壳病毒蛋白VP1 （VP1u）的独特区域在类似条件下变得展开。这些pH值（pH值4-6）已被证明是产生性AAV感染所必需的，与衣壳在细胞进入和运输过程中在内体室中遇到的pH值相当。我们的研究有两个意想不到的新发现。首先，衣壳具有一种以前未知的酶活性：一种pH敏感的蛋白酶，可以催化衣壳和外部底物的自溶裂解。该蛋白酶活性的机制和功能都是未知的，与其他病毒编码的蛋白酶相比似乎是独特的。二是衣壳pH敏感区突变对基因表达有深远影响，即使病毒DNA在细胞核中未被包衣，这表明衣壳在DNA在细胞核中未被包衣后对基因表达起作用。此外，CD研究提出了VP1u外化的机制，VP1u通常埋在衣壳内部，但在通过酸性内体室运输时被挤出。在本提案中，我们希望通过(1)确定蛋白酶的活性位点及其切割靶点来探索这些新发现；(2)确定

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.