Interaction between importin alpha and BR1-3 of VP1 From Divergent Adeno Associated Virus Serotypes is a crucial determinant of Viral Capsids Nuclear Targeting

Thanks to their non-pathogenic nature, broad tropism, and design versatility, Adeno-associated viruses (AAVs) have gained popularity among gene therapy vectors, with successful applications in FDA-approved treatment for two genetic diseases. However, limitations such as low gene capacity and transduction efficiency have prevented them from reaching their full potential. AAV genome nuclear transport is a critical step, strongly limiting transduction efficacy. Even though the process is believed to rely on the interaction between four basic regions (BRs) in AAV VP1 capsid proteins and members of the importin (IMP) superfamily, the molecular and structural details of such interaction are not thoroughly understood. Furthermore, little is known regarding differences between different AAV serotypes in terms of capsid nuclear import. In this inter-laboratory study, the nuclear import of VP1 from multiple AAV serotypes was investigated from a molecular, biochemical, structural, and functional point of view. Structural and biophysical approaches performed by our collaborators at Charles Sturt University revealed that the BR1-3 from all AAV-tested serotypes (2, 3, 5, 6, 8, 11) interact with several IMPalpha isoforms, although with different affinity. Furthermore, structural analyses of VP1:IMPalpha2 complexes revealed, in all cases, direct interaction of BR3 with IMPalpha major binding site. For AAV3, no further interaction could be visualized with the IMPalpha minor binding site, suggesting a monopartite cNLS. On the contrary, for all other serotypes, BR1 could be visualized in the IMPalpha minor site, suggesting the presence of a bipartite NLS. At the University of Padova, we validated such results by means of quantitative confocal laser scanning microscopy in cells expressing fusion proteins between GFP and VP1 from several AAVs. Overall, our results indicate that all tested capsid proteins strongly accumulate in the cell nucleus when expressed in the absence of additional viral proteins, although to different extents, which correlated with differences in IMPalpha binding affinities. Intriguingly, such accumulation was strongly impaired by co-expression with IMPalpha inhibitor Bimax2, implying that capsid nuclear import is an active process mediated by IMPalpha. The role of individual BR domains for capsid nuclear transport was investigated by substitution of basic residues with alanine and revealed important differences across the AAV serotypes analyzed, with BR3 playing a major role, complemented by BR2 and BR1. Overall, our data indicate that the levels of nuclear accumulation of VP1 BR mutants varied significantly across different genotypes and correlated with binding affinity to IMPalpha isoforms. Our data suggests that engineering AAV capsids to maximize genome nuclear transport could provide a new venue for AAV vector optimization and obtain higher transduction efficiency, lower toxicity, and broaden the tissue specificity and tropism.