{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T22:59:30Z","timestamp":1790636370278,"version":"4.1.0"},"reference-count":34,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2021,3,24]],"date-time":"2021-03-24T00:00:00Z","timestamp":1616544000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5\u2032end. 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\u2013host 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 \u03b2, and a complex of Imp \u03b1\/\u03b2 but not Imp \u03b1 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 \u03b2 or Imp \u03b1\/\u03b2. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.<\/jats:p>","DOI":"10.3390\/ijms22073310","type":"journal-article","created":{"date-parts":[[2021,3,24]],"date-time":"2021-03-24T15:42:19Z","timestamp":1616600539000},"page":"3310","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8840-8654","authenticated-orcid":false,"given":"Hareth A.","family":"Al-Wassiti","sequence":"first","affiliation":[{"name":"Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne 3800, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David R.","family":"Thomas","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Melbourne 3800, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kylie M.","family":"Wagstaff","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Melbourne 3800, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stewart A.","family":"Fabb","sequence":"additional","affiliation":[{"name":"Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne 3052, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5115-4745","authenticated-orcid":false,"given":"David A.","family":"Jans","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Melbourne 3800, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angus P.","family":"Johnston","sequence":"additional","affiliation":[{"name":"Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne 3800, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Colin W.","family":"Pouton","sequence":"additional","affiliation":[{"name":"Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne 3800, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,3,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"277","DOI":"10.3390\/cells4030277","article-title":"Misdelivery at the Nuclear Pore Complex\u2014Stopping a Virus Dead in Its Tracks","volume":"4","author":"Flatt","year":"2015","journal-title":"Cells"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1128\/JVI.01571-10","article-title":"Stepwise Loss of Fluorescent Core Protein V from Human Adenovirus during Entry into Cells","volume":"85","author":"Puntener","year":"2010","journal-title":"J. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/j.jviromet.2009.04.010","article-title":"Accurate single-day titration of adenovirus vectors based on equivalence of protein VII nuclear dots and infectious particles","volume":"159","author":"Walkiewicz","year":"2009","journal-title":"J. Virol. Methods"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1101\/SQB.1974.039.01.064","article-title":"A circular DNA-protein complex adenoviruses and its possible role in DNA replication","volume":"39","author":"Robinson","year":"1975","journal-title":"Cold Spring Harb. Symp. Quant. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6381","DOI":"10.1128\/jvi.71.9.6381-6389.1997","article-title":"Role of preterminal protein processing in adenovirus replication","volume":"71","author":"Webster","year":"1997","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1197","DOI":"10.1101\/gad.4.7.1197","article-title":"Adenovirus terminal protein mediates both nuclear matrix association and efficient transcription of adenovirus DNA","volume":"4","author":"Schaack","year":"1990","journal-title":"Genes Dev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1313","DOI":"10.1111\/j.1600-0854.2007.00618.x","article-title":"A Role for Transportin in the Nuclear Import of Adenovirus Core Proteins and DNA","volume":"8","author":"Hindley","year":"2007","journal-title":"Traffic"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1005","DOI":"10.1016\/0092-8674(88)90245-0","article-title":"Nuclear transport of adenovirus DNA polymerase is facilitated by interaction with preterminal protein","volume":"55","author":"Zhao","year":"1988","journal-title":"Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1562","DOI":"10.1016\/j.bbamcr.2010.10.013","article-title":"Molecular basis for specificity of nuclear import and prediction of nuclear localization","volume":"1813","author":"Marfori","year":"2011","journal-title":"Biochim. Biophys. Acta Bioenerg."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1025","DOI":"10.1016\/S1097-2765(01)00240-4","article-title":"HIV-1 Infection Requires a Functional Integrase NLS","volume":"7","author":"Dvorin","year":"2001","journal-title":"Mol. Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1038\/20367","article-title":"Structure of importin-\u03b2 bound to the IBB domain of importin-\u03b1","volume":"399","author":"Cingolani","year":"1999","journal-title":"Nature"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1345","DOI":"10.1016\/S1097-2765(02)00727-X","article-title":"Molecular Basis for the Recognition of a Nonclassical Nuclear Localization Signal by Importin \u03b2","volume":"10","author":"Cingolani","year":"2002","journal-title":"Mol. Cell"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1016\/S0092-8674(00)80173-7","article-title":"A Novel Receptor-Mediated Nuclear Protein Import Pathway","volume":"86","author":"Pollard","year":"1996","journal-title":"Cell"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1016\/0092-8674(91)90245-T","article-title":"Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: Identification of a class of bipartite nuclear targeting sequence","volume":"64","author":"Robbins","year":"1991","journal-title":"Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"D506","DOI":"10.1093\/nar\/gky1049","article-title":"UniProt: A worldwide hub of protein knowledge","volume":"47","author":"Bateman","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1083\/jcb.104.2.189","article-title":"Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores","volume":"104","author":"Finlay","year":"1987","journal-title":"J. Cell Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"851","DOI":"10.1042\/BJ20120150","article-title":"Ivermectin is a specific inhibitor of importin \u03b1\/\u03b2-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus","volume":"443","author":"Wagstaff","year":"2012","journal-title":"Biochem. J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"700","DOI":"10.1021\/cb2000296","article-title":"Importazole, a Small Molecule Inhibitor of the Transport Receptor Importin-\u03b2","volume":"6","author":"Soderholm","year":"2011","journal-title":"ACS Chem. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/S0168-1702(01)00263-5","article-title":"Inhibition of nuclear export of ribonucleoprotein complexes of influenza virus by leptomycin B","volume":"77","author":"Watanabe","year":"2001","journal-title":"Virus Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/S1074-5521(97)90257-X","article-title":"Leptomycin B is an inhibitor of nuclear export: Inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA","volume":"4","author":"Wolff","year":"1997","journal-title":"Chem. Biol."},{"key":"ref_21","first-page":"9155","article-title":"An Importin \u03b1\/\u03b2-Recognized Bipartite Nuclear Localization Signal Mediates Targeting of the Human Herpes Simplex Virus Type 1 DNA Polymerase Catalytic Subunit pUL30 to the Nucleus\u2020","volume":"46","author":"Alvisi","year":"2007","journal-title":"Biochimica"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1111\/j.1600-0854.2009.01028.x","article-title":"Expanding the Definition of the Classical Bipartite Nuclear Localization Signal","volume":"11","author":"Lange","year":"2010","journal-title":"Traffic"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1578","DOI":"10.1016\/j.bbamcr.2010.10.012","article-title":"The importin \u03b2 binding domain as a master regulator of nucleocytoplasmic transport","volume":"1813","author":"Lott","year":"2011","journal-title":"Biochim. Biophys. Acta Bioenerg."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1038\/sj.mt.6300093","article-title":"Histone-mediated Transduction as an Efficient Means for Gene Delivery","volume":"15","author":"Wagstaff","year":"2007","journal-title":"Mol. Ther."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Khanal, S., Ghimire, P., and Dhamoon, A.S. (2018). The Repertoire of Adenovirus in Human Disease: The Innocuous to the Deadly. Biomedicines, 6.","DOI":"10.20944\/preprints201801.0267.v1"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"18482","DOI":"10.1073\/pnas.1216635109","article-title":"Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages","volume":"109","author":"Holguera","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e62","DOI":"10.1093\/nar\/gkm1170","article-title":"Terminal proteins of Streptomyces chromosome can target DNA into eukaryotic nuclei","volume":"36","author":"Tsai","year":"2008","journal-title":"Nucleic Acids Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"246","DOI":"10.1016\/j.jbiotec.2012.10.001","article-title":"AdHTS: A high-throughput system for generating recombinant adenoviruses","volume":"162","author":"Choi","year":"2012","journal-title":"J. Biotechnol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1007\/978-1-60327-461-6_11","article-title":"Reconstitution of nuclear import in permeabilized cells","volume":"464","author":"Cassany","year":"2008","journal-title":"Methods Mol. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"22191","DOI":"10.1074\/jbc.272.35.22191","article-title":"SV40 Large Tumor Antigen Nuclear Import Is Regulated by the Double-stranded DNA-dependent Protein Kinase Site (Serine 120) Flanking the Nuclear Localization Sequence","volume":"272","author":"Xiao","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4899","DOI":"10.1074\/jbc.M211512200","article-title":"Role of Prodomain in Importin-mediated Nuclear Localization and Activation of Caspase-2","volume":"278","author":"Baliga","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Lieu, K.G., Shim, E.H., Wang, J., Lokareddy, R.K., Tao, T., Cingolani, G., Zambetti, G.P., and Jans, D.A. (2014). The p53-induced factor Ei24 inhibits nuclear import through an importin \u03b2-binding-like domain. J. Cell Biol.","DOI":"10.1083\/jcb.201304055"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.ab.2005.10.029","article-title":"Intramolecular masking of nuclear localization signals: Analysis of importin binding using a novel AlphaScreen-based method","volume":"348","author":"Wagstaff","year":"2006","journal-title":"Anal. Biochem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1177\/1087057110390360","article-title":"An AlphaScreen\u00ae-based assay for high-throughput screening for specific inhibitors of nuclear import","volume":"16","author":"Wagstaff","year":"2011","journal-title":"J. Biomol. Screen."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/7\/3310\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:40:14Z","timestamp":1760161214000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/7\/3310"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,3,24]]},"references-count":34,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2021,4]]}},"alternative-id":["ijms22073310"],"URL":"https:\/\/doi.org\/10.3390\/ijms22073310","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,3,24]]}}}