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

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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.