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Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus

Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5′end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS mo...

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Autores principales: Al-Wassiti, Hareth A., Thomas, David R., Wagstaff, Kylie M., Fabb, Stewart A., Jans, David A., Johnston, Angus P., Pouton, Colin W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036708/
https://www.ncbi.nlm.nih.gov/pubmed/33804953
http://dx.doi.org/10.3390/ijms22073310
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author Al-Wassiti, Hareth A.
Thomas, David R.
Wagstaff, Kylie M.
Fabb, Stewart A.
Jans, David A.
Johnston, Angus P.
Pouton, Colin W.
author_facet Al-Wassiti, Hareth A.
Thomas, David R.
Wagstaff, Kylie M.
Fabb, Stewart A.
Jans, David A.
Johnston, Angus P.
Pouton, Colin W.
author_sort Al-Wassiti, Hareth A.
collection PubMed
description Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5′end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP–host interactions further, we expressed TP in Escherichia coli (E. coli). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp β, and a complex of Imp α/β but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp β or Imp α/β. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.
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spelling pubmed-80367082021-04-12 Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus Al-Wassiti, Hareth A. Thomas, David R. Wagstaff, Kylie M. Fabb, Stewart A. Jans, David A. Johnston, Angus P. Pouton, Colin W. Int J Mol Sci Article Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5′end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP–host interactions further, we expressed TP in Escherichia coli (E. coli). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp β, and a complex of Imp α/β but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp β or Imp α/β. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA. MDPI 2021-03-24 /pmc/articles/PMC8036708/ /pubmed/33804953 http://dx.doi.org/10.3390/ijms22073310 Text en © 2021 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 (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ).
spellingShingle Article
Al-Wassiti, Hareth A.
Thomas, David R.
Wagstaff, Kylie M.
Fabb, Stewart A.
Jans, David A.
Johnston, Angus P.
Pouton, Colin W.
Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus
title Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus
title_full Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus
title_fullStr Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus
title_full_unstemmed Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus
title_short Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus
title_sort adenovirus terminal protein contains a bipartite nuclear localisation signal essential for its import into the nucleus
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036708/
https://www.ncbi.nlm.nih.gov/pubmed/33804953
http://dx.doi.org/10.3390/ijms22073310
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