Structure-function relationships of AAV capsids in post entry trafficking ans gene transfer

重组腺相关病毒(RAAV)介导的基因转移在细胞水平上受到三个主要障碍的限制：(A)细胞结合和进入，(B)进入后运输和脱壳，(C)单链(Ss)到双链(DS)基因组转化。这项赠款申请侧重于甲型肝炎病毒衣壳结构变化的作用，这种变化发生在进入后，是病毒脱壳和基因转导所必需的。具体的构象变化是由隐藏的衣壳蛋白(VP1/VP2)N-末端通过衣壳的五重对称轴上的孔暴露而定义的。这进而导致病毒磷脂酶活性的激活，并可能导致位于VP1/VP2N末端的更多功能元件的激活。该项目的目的是(1)描述体内发生构象变化的时间点，(2)确定引起构象变化的生理触发因素，(3)确定VP1/VP2暴露对AAV进入后运输和脱壳的影响，(4)描述这些进入后事件对靶向载体发展的影响，以及(5)确定位于VP1/2N-末端的短基本序列元件是否对病毒的核摄取是必需的。

Recombinant adeno-associated virus (rAAV) mediated gene transfer is limited at the cellular level by three major barriers: (a) by cell binding and entry, (b) by post entry trafficking and uncoating and (c) by single-strand (ss) to doublestrand (ds) genome conversion. This grant application focuses on the role of a conformational change in the AAV capsid which occurs post entry and is required for viral uncoating and gene transduction. The specific conformational change is defined by the exposure of hidden capsid protein (VP1/VP2) N-termini through pores at the five-fold symmetry axes of the capsid. This in turn leads to the activation of a viral phospholipase activity and possibly further functional elements located on the VP1/VP2 N-termini. The project aims (1) to describe the time point when and in which cellular compartment the conformational change occurs in vivo, (2) to identify the physiological trigger that induces the conformational change, (3) to identify the consequences of VP1/VP2 exposure for AAV post entry trafficking and uncoating, (4) to describe the implications of these post entry events for the development of targeted vectors and (5) to determine if short basic sequence elements located on the VP1/2 N-termini are required for nuclear uptake of the virus.