Nuclear translocation of adeno-associated virus type 2 capsid proteins for virion assembly

Adeno-associated virus (AAV) capsid assembly occurs in the nucleus. Newly synthesized capsid proteins VP1, VP2 and VP3 contain several basic regions (BRs), which may act as nuclear localization signals (NLSs). Mutation of BR2 and BR3, located at the VP1 and VP2 N termini, marginally reduced nuclear uptake of VP1 or VP2, but not of VP3, when expressed in the context of the whole AAV type 2 (AAV2) genome. Combined mutation of BR1, BR2 and BR3 resulted in capsids with slightly reduced amounts of VP1. Expression of isolated VP1/2 N termini revealed an influence of BR3 on nuclear transport, whilst BR1 or BR2 had no effect. However, deletion of an N-terminal fragment in front of the BR elements strongly reduced nuclear uptake of VP1/2 N termini. Mutation of BR4, present in all three capsid proteins, led to their retention in the cytoplasm and to the formation of speckles, resulting in a lack of capsid formation and a significant reduction in VP levels. In a VP fragment comprising BR2, BR3 and BR4, the BR4 element was not necessary for nuclear localization. Mutation of BR5 in the C-terminal part of the VPs resulted in a speckled protein distribution in the nucleus, strongly reduced capsid assembly, and low VP1 and VP2 levels. Taken together, these results showed that BR2 and BR3 have a weak influence on nuclear transport of VP1 and VP2, whilst combined mutation of BR1, BR2 and BR3 influences the stoichiometry of VPs in assembled capsids. BR4 and BR5 play a crucial role in capsid assembly but have no NLS activity.