Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal

Adeno-associated viruses (AAV) are important vectors for gene therapy, and accordingly, many aspects of their cell transduction pathway have been well characterized. However, the specific mechanisms that AAV virions use to enter the host nucleus remain largely unresolved. We therefore aimed to reveal the interactions between the AAV Cap protein and the nuclear transport protein importin alpha (IMP alpha) at an atomic resolution. Herein we expanded upon our earlier research into the Cap nuclear localization signal (NLS) of a porcine AAV isolate, by examining the influence of upstream basic regions (BRs) towards IMP alpha binding. Using a high-resolution crystal structure, we identified that the IMP alpha binding determinants of the porcine AAV Cap comprise a bipartite NLS with an N-terminal BR binding at the minor site of IMP alpha, and the previously identified NLS motif binding at the major site. Quantitative assays showed a vast difference in binding affinity between the previously determined monopartite NLS, and bipartite NLS described in this study. Our results provide a detailed molecular view of the interaction between AAV capsids and the nuclear import receptor, and support the findings that AAV capsids enter the nucleus by binding the nuclear import adapter IMP alpha using the classical nuclear localization pathway.

Automated summary

This study reveals the interaction between the AAV Cap protein and the nuclear transport protein importin alpha (IMP alpha) at an atomic resolution. The research identifies a bipartite nuclear localization signal (NLS) in the porcine AAV Cap that binds to IMP alpha. The findings provide a detailed molecular view of how AAV virions enter the host nucleus.