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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 revea...

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Autores principales: Hoad, Mikayla, Cross, Emily M., Donnelly, Camilla M., Sarker, Subir, Roby, Justin A., Forwood, Jade K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9964314/
https://www.ncbi.nlm.nih.gov/pubmed/36851528
http://dx.doi.org/10.3390/v15020315
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author Hoad, Mikayla
Cross, Emily M.
Donnelly, Camilla M.
Sarker, Subir
Roby, Justin A.
Forwood, Jade K.
author_facet Hoad, Mikayla
Cross, Emily M.
Donnelly, Camilla M.
Sarker, Subir
Roby, Justin A.
Forwood, Jade K.
author_sort Hoad, Mikayla
collection PubMed
description 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 (IMPα) 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 IMPα binding. Using a high-resolution crystal structure, we identified that the IMPα binding determinants of the porcine AAV Cap comprise a bipartite NLS with an N-terminal BR binding at the minor site of IMPα, 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 IMPα using the classical nuclear localization pathway.
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spelling pubmed-99643142023-02-26 Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal Hoad, Mikayla Cross, Emily M. Donnelly, Camilla M. Sarker, Subir Roby, Justin A. Forwood, Jade K. Viruses Article 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 (IMPα) 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 IMPα binding. Using a high-resolution crystal structure, we identified that the IMPα binding determinants of the porcine AAV Cap comprise a bipartite NLS with an N-terminal BR binding at the minor site of IMPα, 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 IMPα using the classical nuclear localization pathway. MDPI 2023-01-23 /pmc/articles/PMC9964314/ /pubmed/36851528 http://dx.doi.org/10.3390/v15020315 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hoad, Mikayla
Cross, Emily M.
Donnelly, Camilla M.
Sarker, Subir
Roby, Justin A.
Forwood, Jade K.
Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal
title Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal
title_full Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal
title_fullStr Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal
title_full_unstemmed Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal
title_short Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal
title_sort structural characterization of porcine adeno-associated virus capsid protein with nuclear trafficking protein importin alpha reveals a bipartite nuclear localization signal
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9964314/
https://www.ncbi.nlm.nih.gov/pubmed/36851528
http://dx.doi.org/10.3390/v15020315
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