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• W2026008221 abstract "During the late phase of human papillomavirus (HPV) infection, the L1 major capsid proteins enter the nuclei of host epithelial cells and, together with the L2 minor capsid proteins, assemble the replicated viral DNA into virions. We investigated the nuclear import of the L1 major capsid protein of high risk HPV16. When digitonin-permeabilized HeLa cells were incubated with HPV16 L1 capsomeres, the L1 protein was imported into the nucleus in a receptor-mediated manner. HPV16 L1 capsomeres formed complexes with Kap α2β1 heterodimers via interaction with Kap α2. Accordingly, nuclear import of HPV16 L1 capsomeres was mediated by Kap α2β1 heterodimers, required RanGDP and free GTP, and was independent of GTP hydrolysis. Remarkably, HPV16 L1 capsomeres also interacted with Kap β2 and binding of RanGTP to Kap β2 did not dissociate the HPV16 L1·Kap β2 complex. Significantly, HPV16 L1 capsomeres inhibited the nuclear import of Kap β2 and of a Kap β2-specific M9-containing cargo. These data suggest that, during the productive stage of infection, while the HPV16 L1 major capsid protein enters the nucleus via the Kap α2β1-mediated pathway to assemble the virions, it also inhibits the Kap β2-mediated nuclear import of host hnRNP A1 protein and, in this way, favors virion formation. During the late phase of human papillomavirus (HPV) infection, the L1 major capsid proteins enter the nuclei of host epithelial cells and, together with the L2 minor capsid proteins, assemble the replicated viral DNA into virions. We investigated the nuclear import of the L1 major capsid protein of high risk HPV16. When digitonin-permeabilized HeLa cells were incubated with HPV16 L1 capsomeres, the L1 protein was imported into the nucleus in a receptor-mediated manner. HPV16 L1 capsomeres formed complexes with Kap α2β1 heterodimers via interaction with Kap α2. Accordingly, nuclear import of HPV16 L1 capsomeres was mediated by Kap α2β1 heterodimers, required RanGDP and free GTP, and was independent of GTP hydrolysis. Remarkably, HPV16 L1 capsomeres also interacted with Kap β2 and binding of RanGTP to Kap β2 did not dissociate the HPV16 L1·Kap β2 complex. Significantly, HPV16 L1 capsomeres inhibited the nuclear import of Kap β2 and of a Kap β2-specific M9-containing cargo. These data suggest that, during the productive stage of infection, while the HPV16 L1 major capsid protein enters the nucleus via the Kap α2β1-mediated pathway to assemble the virions, it also inhibits the Kap β2-mediated nuclear import of host hnRNP A1 protein and, in this way, favors virion formation. human papillomavirus nuclear localization signal glutathioneS-transferase guanosine 5′-O-(thiotriphosphate) guanylyl imidodiphosphate heterogeneous nuclear ribonucleoprotein isopropyl-1-thio-β-d-galactopyranoside monopartite NLS bipartite/monopartite NLS nuclear pore complex Human papillomaviruses (HPVs)1 are thought to be the primary causative agent in more than 90% of all cervical cancers, with HPV16 being the type most frequently found in these tumors. Approximately 500,000 new cases of cervical cancer are identified each year globally with nearly 300,000 deaths annually. About 85 genotypically distinct HPV genotypes have been isolated and characterized, with roughly half infecting the skin and the other half preferentially infecting oral/anogenital mucosal epithelial tissues. Mucosal HPVs have demonstrated varying degrees of oncogenic potential; high risk HPVs, such as types 16, 18, 31, and 45, are frequently detected in invasive cervical carcinomas, whereas the low risk types, such as types 6 and 11, are more often associated with benign exophytic condylomas (1zur Hausen H. J. Natl. Cancer Inst. 2000; 92: 690-698Crossref PubMed Google Scholar). HPVs are small, nonenveloped, icosahedral DNA viruses that replicate in the nucleus of squamous epithelial cells. The virion particles (52–55 nm in diameter) consist of a single molecule comprising 8 kb of double-stranded circular DNA contained within a spherical capsid composed of 72 homopentameric L1 capsomeres and of L2 minor capsid protein. The number of L2 molecules per capsid has been estimated at 36 (2Doorbar J. Gallimore P.H. J. Virol. 1987; 61: 2793-2799Crossref PubMed Google Scholar) or 12 (3Trus B.L. Roden R.B. Greenstone H.L. Vrhel M. Schiller J.T. Booy F.P. Nat. Struct. Biol. 1997; 4: 413-420Crossref PubMed Scopus (158) Google Scholar). L1 protein is capable of self-assembly both in vivo and in vitro into capsid-like structures, referred to as virus-like particles (4Kirnbauer R. Booy F. Cheng N. Lowy D.R. Schiller J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12180-12184Crossref PubMed Scopus (938) Google Scholar, 5Rose R.C. Bonnez W. Reichman R.C. Garcea R.L. J. Virol. 1993; 67: 1936-1944Crossref PubMed Google Scholar, 6Zhou J. Stenzel D.J. Sun X.Y. Frazer I.H. J. Gen. Virol. 1993; 74: 763-768Crossref PubMed Scopus (107) Google Scholar, 7Hagensee M.E. Olson N.H. Baker T.S. Galloway D.A. J. Virol. 1994; 68: 4503-4505Crossref PubMed Google Scholar, 8Volpers C. Schirmacher P. Streeck R.E. Sapp M. Virology. 1994; 200: 504-512Crossref PubMed Scopus (130) Google Scholar, 9Belnap D.M. Olson N.H. Cladel N.M. Newcomb W.W. Brown J.C. Kreider J.W. Christensen N.D. Baker T.S. J. Mol. Biol. 1996; 259: 249-263Crossref PubMed Scopus (99) Google Scholar). L1 is stable in two oligomeric configurations: homopentameric capsomeres and capsids composed of 72 capsomeres. The capsids can be disassembled into capsomeres quantitatively by an agent that reduces disulfide bonds and reassembled by removing the reducing agent (10Li M. Beard P. Estes P.A. Lyon M.K. Garcea R.L. J. Virol. 1998; 72: 2160-2167Crossref PubMed Google Scholar, 11McCarthy M.P. White W.I. Palmer-Hill F. Koenig S. Suzich J.A. J. Virol. 1998; 72: 32-41Crossref PubMed Google Scholar). The L1 capsid proteins of HPVs seem to enter the nucleus of host cells twice during the virus life cycle: immediately after the virions infect the undifferentiated proliferating epithelial cells and again during the late productive phase when the newly synthesized L1 and L2 proteins assemble the replicated HPV genomic DNA into infectious virions inside the nuclei of terminally differentiated epithelial cells. Proteins must carry nuclear localization signals (NLSs) to enter the nucleus. The first identified NLSs, now referred to as classic NLSs, fall into two categories: a monopartite NLS consisting of a simple sequence of 3–5 basic amino acid residues and a bipartite NLS consisting of a basic dipeptide upstream from a simple basic sequence. Other types of NLSs have since been identified in hnRNP proteins, ribosomal proteins, and U small nuclear RNPs. NLSs are recognized by adapters of the karyopherin α/importin α (Kap α/Imp α) family and by import receptors of the karyopherin β/importin β (Kap β/Imp β) family that shuttle between the nucleus and the cytoplasm. The basic paradigm for nuclear import is that the NLS cargo is bound (either directly or indirectly via an adapter) by an import receptor in the cytoplasm, translocated through the nuclear pore complex (NPC) and released inside the nucleus. The receptor and the adapter are then recycled back to the cytoplasm in a form competent for another round of import (12Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar, 13Moroianu J. J. Cell. Biochem. 1999; 32–33 (suppl.): 76-83Crossref Google Scholar, 14Wente S.R. Science. 2000; 288: 1374-1377Crossref PubMed Scopus (214) Google Scholar). The first identified nuclear import receptor was Kap β1, which functions together with a Kap α adapter in nuclear import of proteins that contain classic monopartite or bipartite NLSs. Kap α binds to the NLS of the cargo, whereas Kap β1 mediates docking at the NPC (15Gorlich D. Prehn S. Laskey R.A. Hartmann E. Cell. 1994; 79: 767-778Abstract Full Text PDF PubMed Scopus (597) Google Scholar, 16Gorlich D. Kostka S. Kraft R. Dingwall C. Laskey R.A. Hartmann E. Prehn S. Curr. Biol. 1995; 5: 383-392Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 17Radu A. Blobel G. Moore M.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1769-1773Crossref PubMed Scopus (381) Google Scholar, 18Moroianu J. Hijikata M. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6532-6536Crossref PubMed Scopus (248) Google Scholar, 19Moroianu J. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2008-2011Crossref PubMed Scopus (249) Google Scholar, 20Weis K. Mattaj I.W. Lamond A.I. Science. 1995; 268: 1049-1053Crossref PubMed Scopus (306) Google Scholar, 21Imamoto N. Shimamoto T. Takao T. Tachibana T. Kose S. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. EMBO J. 1995; 14: 3617-3626Crossref PubMed Scopus (269) Google Scholar). There are six human Kap α adapters, and it is likely that they have both distinct and overlapping specificities for classic basic NLSs. Kap β1 can also function without adapters when importing ribosomal proteins, cyclin B1, and viral proteins. Other members of the Kap β family have been identified and shown to function in nuclear import of specific cargoes. All Kap β receptors shuttle between the nucleus and the cytoplasm and bind to nucleoporins at the NPC and to the GTPase Ran in its GTP-bound form. The nucleotide state of Ran is regulated by cytoplasmic RanGAP and RanBP1, which accelerate GTP hydrolysis to form cytoplasmic RanGDP, and by the nuclear RanGEF (RCC1), which catalyzes nucleotide exchange to regenerate nuclear RanGTP. Binding of nuclear RanGTP to Kap βs (importins) causes the dissociation of the import complexes with release of the cargoes inside the nucleus. The Kap βs (importins/exportins) exit the nucleus in complex with RanGTP and, therefore, constantly deplete Ran from the nucleus (12Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar, 13Moroianu J. J. Cell. Biochem. 1999; 32–33 (suppl.): 76-83Crossref Google Scholar, 14Wente S.R. Science. 2000; 288: 1374-1377Crossref PubMed Scopus (214) Google Scholar). Ran is actively re-imported into the nucleus via the transport factor p10/NTF2, which binds specifically to RanGDP but not to RanGTP (22Ribbeck K. Lipowsky G. Kent H.M. Stewart M. Gorlich D. EMBO J. 1998; 17: 6587-6598Crossref PubMed Scopus (351) Google Scholar). In this study we investigated the nuclear import of the L1 major capsid protein of high risk HPV16. We found that HPV16 L1 capsomeres enter the nucleus via the classic Kap α2β1-mediated nuclear import pathway. Importantly, we also discovered that both HPV16 and HPV45 L1 capsomeres interact with Kap β2 and inhibit nuclear import of Kap β2 and its specific M9-containing cargo. These data suggest that during the late productive stage of infection the newly synthesized L1 major capsid proteins can interact with Kap β2 and may inhibit major Kap β2-mediated nuclear import pathways and, thus, inhibit other nuclear events so as to optimize virion formation. His-tagged Kap α2 (20Weis K. Mattaj I.W. Lamond A.I. Science. 1995; 268: 1049-1053Crossref PubMed Scopus (306) Google Scholar), His-tagged Kap β1 (23Chi N.C. Adam E.J. Visser G.D. Adam S.A. J. Cell Biol. 1996; 135: 559-569Crossref PubMed Scopus (157) Google Scholar), and His-tagged p10 (18Moroianu J. Hijikata M. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6532-6536Crossref PubMed Scopus (248) Google Scholar) were expressed in Escherichia coliBL21(DE3) (3-h induction with 2 mm IPTG at 30 °C), and the soluble His-tagged proteins were purified in their native state on Talon beads using a standard procedure. GST-Kap β1 (23Chi N.C. Adam E.J. Visser G.D. Adam S.A. J. Cell Biol. 1996; 135: 559-569Crossref PubMed Scopus (157) Google Scholar) and GST-Kap β2 (24Chook Y.M. Blobel G. Nature. 1999; 399: 230-237Crossref PubMed Scopus (287) Google Scholar) were expressed in E. coli BL21(DE3) (3-h induction with 1 mm IPTG at 30 °C), and the soluble GST fusion proteins were purified in their native state on glutathione-Sepharose beads using a standard procedure. To obtain cleaved Kap β2, the GST-Kap β2 fusion protein was incubated for 2 h at room temperature with a Tev enzyme that has a His tag (Invitrogen), and after cleavage the GST was removed by binding to glutathione-Sepharose beads and the Tev enzyme by binding to Talon beads, using standard procedures. Human Ran (25Coutavas E. Ren M. Oppenheim J.D. D'Eustachio P. Rush M.G. Nature. 1993; 366: 585-587Crossref PubMed Scopus (226) Google Scholar) was prepared as described previously (26Floer M. Blobel G. J. Biol. Chem. 1996; 271: 5313-5316Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). All proteins were checked for purity and lack of proteolytic degradation by SDS-PAGE and Coomassie Blue staining. The purified proteins were dialyzed in buffer A (20 mm HEPES-KOH, pH 7.3, 110 mm potassium acetate, 2 mm magnesium acetate, 1 mm EGTA, 2 mm dithiothreitol) containing protease inhibitors and stored in aliquots at −80 °C until use. The L1 capsids of HPV16 and HPV45 were generated in insect cells, purified as described (27Rose R.C. Bonnez W., Da Rin C. McCance D.J. Reichman R.C. J. Gen. Virol. 1994; 75: 2445-2449Crossref PubMed Scopus (87) Google Scholar, 28Rose R.C. Reichman R.C. Bonnez W. J. Gen. Virol. 1994; 75: 2075-2079Crossref PubMed Scopus (148) Google Scholar), and stored at 4 °C until use. Purity and lack of proteolytic degradation of the L1 proteins were always checked by SDS-PAGE, Coomassie Blue staining, and immunoblotting prior to use. L1 capsomeres were obtained by incubating the L1 capsids with 5% mercaptoethanol overnight at 4 °C (11McCarthy M.P. White W.I. Palmer-Hill F. Koenig S. Suzich J.A. J. Virol. 1998; 72: 32-41Crossref PubMed Google Scholar). Rabbit polyclonal antiserum was raised against HPV16 L1 capsids as described (27Rose R.C. Bonnez W., Da Rin C. McCance D.J. Reichman R.C. J. Gen. Virol. 1994; 75: 2445-2449Crossref PubMed Scopus (87) Google Scholar, 28Rose R.C. Reichman R.C. Bonnez W. J. Gen. Virol. 1994; 75: 2075-2079Crossref PubMed Scopus (148) Google Scholar). Murine antibodies raised against Kap α2/Rch-1 and Kap β2/transportin were from Transduction Laboratories; a mouse monoclonal antibody to Kap β1/p97 was from Affinity Bioreagents, Inc., and the goat anti-GST antibody was fromAmersham Biosciences. HeLa cytosol from Cellex Biosciences Inc. was centrifuged and stored in aliquots at −80 °C. The L1 major capsid protein of HPV16 contains two classic NLSs at its C terminus: a monopartite NLS (AKRKKRKL) and a bipartite type that overlaps with the monopartite NLS (KRKATPTTSSTSTTAKRKKRKL) (29Zhou J. Doorbar J. Sun X.Y. Crawford L.V. McLean C.S. Frazer I.H. Virology. 1991; 185: 625-632Crossref PubMed Scopus (69) Google Scholar). The monopartite NLS (mpNLS) of HPV16 L1 and the overlapping bipartite/monopartite NLS (bpNLS) of HPV16 L1 were separately fused to a GST reporter protein. For making the GST-bpNLS construct the forward and reverse oligonucleotides, phosphorylated at 5′-ends, were annealed, and the overhanging areas were filled in using a DNA polymerase I, large Klenow fragment. The resulting DNA fragments were blunt end-ligated with T4 DNA ligase, double-digested withEcoRI and XhoI, and inserted into pGEX4T-1 (Amersham Biosciences) that was double cut with the same enzymes. For making the GST-mpNLS construct, unphosphorylated oligonucleotides were designed with the corresponding restriction-cutting sites, annealed, and inserted directly into the pGEX4T-1 vector as described above. The GST-bpNLS and GST-mpNLS constructs were transformed in XL1 blue bacteria and confirmed by automated sequence analysis (BioServe Biotechnologies Sequencing Department). The M9-GST construct was a gift from Dr. Gideon Dreyfuss. For protein expression, the constructs were used to transform E. coli BL21(DE3) bacteria. After induction of E. coli BL21(DE3) with 1 mm IPTG for 3 h at 37 °C, the GST-NLS fusion proteins were purified in their native state on glutathione-Sepharose using a standard procedure. The purified proteins were checked by SDS-PAGE and Coomassie Blue staining, dialyzed in buffer A, and stored in aliquots at −80 °C until use. Digitonin-permeabilized HeLa cells have been extensively used by many laboratories to investigate different nuclear import pathways mediated by mammalian Kap βs/importins (20Weis K. Mattaj I.W. Lamond A.I. Science. 1995; 268: 1049-1053Crossref PubMed Scopus (306) Google Scholar, 22Ribbeck K. Lipowsky G. Kent H.M. Stewart M. Gorlich D. EMBO J. 1998; 17: 6587-6598Crossref PubMed Scopus (351) Google Scholar, 30Adam E.J. Adam S.A. J. Cell Biol. 1994; 125: 547-555Crossref PubMed Scopus (256) Google Scholar, 31Chi N.C. Adam E.J. Adam S.A. J. Cell Biol. 1995; 130: 265-274Crossref PubMed Scopus (246) Google Scholar, 32Gorlich D. Vogel F. Mills A.D. Hartmann E. Laskey R.A. Nature. 1995; 377: 246-248Crossref PubMed Scopus (405) Google Scholar, 33Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (339) Google Scholar, 34Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 35Kataoka N. Bachorik J.L. Dreyfuss G. J. Cell Biol. 1999; 145: 1145-1152Crossref PubMed Scopus (174) Google Scholar, 36Ribbeck K. Kutay U. Paraskeva E. Gorlich D. Curr. Biol. 1999; 9: 47-50Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 37Schwoebel E.D. Talcott B. Cushman I. Moore M.S. J. Biol. Chem. 1998; 273: 35170-35175Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 38Nelson L.M. Rose R.C. LeRoux L. Lane C. Bruya K. Moroianu J. J. Cell. Biochem. 2000; 79: 225-238Crossref PubMed Scopus (30) Google Scholar). HeLa cells are cervical carcinoma cells that contain HPV18 DNA integrated into the cellular genome with consequent disruption of several viral genes but preservation of theE6 and E7 genes. Although HeLa cells are HPV18-positive, this does not affect the import properties of their nuclear pore complexes, as demonstrated by the fact that the majority of nuclear import pathways have been identified and characterized with HeLa cells (20Weis K. Mattaj I.W. Lamond A.I. Science. 1995; 268: 1049-1053Crossref PubMed Scopus (306) Google Scholar, 22Ribbeck K. Lipowsky G. Kent H.M. Stewart M. Gorlich D. EMBO J. 1998; 17: 6587-6598Crossref PubMed Scopus (351) Google Scholar, 30Adam E.J. Adam S.A. J. Cell Biol. 1994; 125: 547-555Crossref PubMed Scopus (256) Google Scholar, 31Chi N.C. Adam E.J. Adam S.A. J. Cell Biol. 1995; 130: 265-274Crossref PubMed Scopus (246) Google Scholar, 32Gorlich D. Vogel F. Mills A.D. Hartmann E. Laskey R.A. Nature. 1995; 377: 246-248Crossref PubMed Scopus (405) Google Scholar, 33Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (339) Google Scholar, 34Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 35Kataoka N. Bachorik J.L. Dreyfuss G. J. Cell Biol. 1999; 145: 1145-1152Crossref PubMed Scopus (174) Google Scholar, 36Ribbeck K. Kutay U. Paraskeva E. Gorlich D. Curr. Biol. 1999; 9: 47-50Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 37Schwoebel E.D. Talcott B. Cushman I. Moore M.S. J. Biol. Chem. 1998; 273: 35170-35175Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 38Nelson L.M. Rose R.C. LeRoux L. Lane C. Bruya K. Moroianu J. J. Cell. Biochem. 2000; 79: 225-238Crossref PubMed Scopus (30) Google Scholar). We have previously used these in vitro nuclear import assays in HeLa cells to investigate import of HPV11 L1 and HPV45 L1 major capsid proteins (38Nelson L.M. Rose R.C. LeRoux L. Lane C. Bruya K. Moroianu J. J. Cell. Biochem. 2000; 79: 225-238Crossref PubMed Scopus (30) Google Scholar, 39Merle E. Rose R.C. LeRoux L. Moroianu J. J. Cell. Biochem. 1999; 74: 628-637Crossref PubMed Scopus (53) Google Scholar). In the present study, subconfluent HeLa cells, grown on poly-l-lysine-coated glass coverslips for 24 h, were permeabilized with 70 μg/ml digitonin for 5 min on ice and washed with buffer A. Digitonin permeabilizes the plasma membrane but leaves the nuclear envelope intact. As a consequence, digitonin-permeabilized HeLa cells retain intact import-competent nuclei but are largely depleted of cytosolic transport factors, as indicated by the absence of NLS-mediated nuclear import in the presence of buffer alone and in the absence of either exogenous cytosol or recombinant Kaps plus RanGDP. It should be noted that p10 is retained in the digitonin-permeabilized HeLa cells in sufficient amounts to support NLS-mediated nuclear import (Refs. 32Gorlich D. Vogel F. Mills A.D. Hartmann E. Laskey R.A. Nature. 1995; 377: 246-248Crossref PubMed Scopus (405) Google Scholar, 34Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, and 35Kataoka N. Bachorik J.L. Dreyfuss G. J. Cell Biol. 1999; 145: 1145-1152Crossref PubMed Scopus (174) Google Scholar and Fig. 2). Unless otherwise specified all import reactions contained the energy-regenerating system (0.5 mm GTP, 5 mm phosphocreatine, and 0.4 unit of creatine phosphokinase), plus various transport factors (1 μm Kap α2, 0.5 μm Kap β1, 0.5 μm Kap β2, 3 μm RanGDP, 0.2 μm p10), plus the L1 capsomeres (0.25 μm), or the GST-NLS fusion proteins (0.5 μm). Final import reaction volume was adjusted to 20 μl with buffer A. For visualization of nuclear import, the HPV16 L1 protein and the GST-NLS fusion proteins were detected by immunofluorescence with specific antibodies, as previously described (38Nelson L.M. Rose R.C. LeRoux L. Lane C. Bruya K. Moroianu J. J. Cell. Biochem. 2000; 79: 225-238Crossref PubMed Scopus (30) Google Scholar). The nuclei were identified by DAPI staining. Nuclear import was analyzed with a Nikon Eclipse TE 300 microscope, which has a fluorescence attachment, and a Sony DKC-5000 charge-coupled device camera. Two types of binding assays were performed. First, GST-NLSHPV16L1 fusion proteins, immobilized on glutathione-Sepharose beads (2 μg of protein/10 μl of beads) were incubated under rotation for 30 min at room temperature with the purified Kaps (2 μg of protein) in buffer A (total volume, 40 μl). Control experiments for binding specificity consisted of incubating GST immobilized on glutathione-Sepharose beads with the purified Kaps. After incubation, the beads were washed three times with buffer A, and the bound proteins were eluted with SDS-PAGE sample buffer and analyzed by SDS-PAGE followed by Coomassie Blue staining. Second, GST-Kap β1 or GST-Kap β2 immobilized on glutathione-Sepharose beads (2 μg of protein/10 μl of beads) was incubated for 30 min under rotation at room temperature with HPV16 L1 or HPV45 L1 capsomeres (2 μg of protein) in buffer A containing 0.25% Tween 20 (binding buffer). In some experiments, 2 μg of either Kap α2 or RanGTP was added to the incubation mixture, as indicated in the figure legends. Control experiments for binding specificity consisted of incubating GST immobilized on glutathione-Sepharose beads with the L1 capsomeres. The bound proteins were eluted with SDS-PAGE sample buffer and analyzed by SDS-PAGE followed by Coomassie Blue staining. We had previously established for the L1 major capsid proteins of HPV11 and HPV45 that capsid disassembly is required for their nuclear import (38Nelson L.M. Rose R.C. LeRoux L. Lane C. Bruya K. Moroianu J. J. Cell. Biochem. 2000; 79: 225-238Crossref PubMed Scopus (30) Google Scholar, 39Merle E. Rose R.C. LeRoux L. Moroianu J. J. Cell. Biochem. 1999; 74: 628-637Crossref PubMed Scopus (53) Google Scholar). Therefore, the nuclear import of the L1 major capsid protein of HPV16 was investigated in either capsid or capsomere conformation. Digitonin-permeabilized HeLa cells were incubated with either HPV16 L1 capsomeres (Fig.1 A) or HPV16 L1 capsids (Fig. 1 B) in the presence of cytosol and an energy-regenerating system. As previously found for HPV11 and HPV45, the HPV16 L1 capsomeres were imported into the nuclei of digitonin-permeabilized cells in the presence of cytosol (Fig. 1 A), whereas the HPV16 L1 capsids were not (Fig. 1 B). It has been shown previously that the L1 major capsid protein of HPV16 contains two classic NLSs at the C terminus: a monopartite NLS and a bipartite type that overlaps with the monopartite NLS (29Zhou J. Doorbar J. Sun X.Y. Crawford L.V. McLean C.S. Frazer I.H. Virology. 1991; 185: 625-632Crossref PubMed Scopus (69) Google Scholar). Because HPV16 L1 contains classic basic NLSs, we investigated whether nuclear import of HPV16 L1 major capsid protein can be mediated by the Kap α2β1 heterodimers. Digitonin-permeabilized HeLa cells were incubated with HPV16 L1 capsomeres in the presence of either Kap β1 + RanGDP (A), Kap β1 + RanGDP + p10 (B), Kap β1 + Kap α2 + RanGDP (C), or Kap β1 + Kap α2 + RanGDP + p10 (D). We found that nuclear import of HPV16 L1 was efficiently mediated by Kap α2β1 heterodimers (Fig. 2,C and D) but not by Kap β1 (Fig. 2,A and B). Addition of exogenous p10 did not increase nuclear import of HPV16 L1 capsomeres in agreement with previous results that sufficient endogenous p10 is retained in the digitonin-permeabilized HeLa cells to support NLS-mediated import (32Gorlich D. Vogel F. Mills A.D. Hartmann E. Laskey R.A. Nature. 1995; 377: 246-248Crossref PubMed Scopus (405) Google Scholar,34Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 35Kataoka N. Bachorik J.L. Dreyfuss G. J. Cell Biol. 1999; 145: 1145-1152Crossref PubMed Scopus (174) Google Scholar). We next tested for nuclear import two GST fusion proteins containing either the overlapping bipartite/monopartite NLS of HPV16 L1 (GST-bpNLSHPV16L1) or the monopartite NLS of HPV16 L1 (GST-mpNLSHPV16L1). Both the GST-bpNLSHPV16L1and the GST-mpNLSHPV16L1 were imported into the nucleus in the presence of cytosol or in the presence of Kap α2β1 heterodimers plus RanGDP but not in the presence of transport buffer alone or Kap β1 plus RanGDP (Fig. 3 and data not shown). As a control, GST itself was not imported into the nucleus in the presence of cytosol (data not shown). In agreement with the nuclear import data, the bpNLSHPV16L1interacted directly with Kap α2 (Fig.4 A, lane 1) and formed a trimeric complex with the Kap α2/Kap β1 heterodimer (Fig.4 A, lane 2). The weak binding of Kap β1 alone to the GST-bpNLSHPV16L1 (Fig. 4 A, lane 3) is assumed to be nonspecific, because it is equivalent to the interaction of Kap β1 with the GST alone (Fig. 4 A,lane 4). The mpNLSHPV16L1 also interacted directly with Kap α2 and formed a trimeric complex with the Kap α2/Kap β1 heterodimer (data not shown). To investigate the direct interactions between HPV16 L1 capsomeres and Kap α2β1 heterodimers, we used in-solution binding assays with the GST-Kap β1 immobilized on glutathione-Sepharose beads via the GST (Fig. 4 B). The GST-Kap β1-containing beads were incubated for 30 min at room temperature with HPV16 L1 capsomeres alone or in the presence of Kap α2. Binding of HPV16 L1 capsomeres to the Kap β1-containing beads was strongly increased in the presence of Kap α2 (Fig. 4 B, compare lanes 1 and 2) indicating the formation of a complex: L1 capsomere·Kap α2·Kap β1. Moreover, binding of RanGTP to Kap β1 inhibited binding of the Kap α2·L1 capsomere complex to Kap β1 or promoted its dissociation (Fig. 4 B, compare lanes 2 and 3). These binding results support the nuclear import data indicating that the Kap α2β1 heterodimers can mediate nuclear import of HPV16 L1 capsomeres. Previous studies have suggested that GTP hydrolysis by Ran is not required for Kap α2β1-mediated nuclear import of specific cargoes (37Schwoebel E.D. Talcott B. Cushman I. Moore M.S. J. Biol. Chem. 1998; 273: 35170-35175Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 38Nelson L.M. Rose R.C. LeRoux L. Lane C. Bruya K. Moroianu J. J. Cell. Biochem. 2000; 79: 225-238Crossref PubMed Scopus (30) Google Scholar). We investigated whether nuclear import of HPV16 L1 capsomeres and GST-mpNLSHPV16L1 are also independent of GTP hydrolysis by Ran by comparing their nuclear import in the presence of Kap α2 plus Kap β1 plus RanGDP and either GTP or the nonhydrolyzable GTP analogues, GTPγS and GMP-PNP. Both HPV16 L1 capsomeres and the GST-mpNLSHPV16L1 were efficiently imported in the presence of either GTP, GTPγS, or GMP-PNP (Fig.5). These data suggest that GTP hydrolysis by Ran is not required for the nuclear import of HPV16 L1 major capsid protein. Human Kap β2/transportin is known to mediate nuclear import of hnRNP A1 and A2 via interaction with their Gly/Asn-rich NLS termed M9 (24Chook Y.M. Blobel G. Nature. 1999; 399: 230-237Crossref PubMed Scopus (287) Google Scholar, 34Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 40Michael W.M. Choi M. Dreyfuss G. Cell. 1995; 83: 415-422Abstract Full Text PDF PubMed Scopus (468) Google Scholar, 41Siomi H. Dreyfuss G. J. Cell Biol. 1995; 129: 551-560Crossref PubMed Scopus (433) Google Scholar, 42Bonifaci N. Moroianu J. Radu A. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5055-5060Crossref PubMed Scopus (147) Google Scholar, 43Siomi M.C. Eder P.S. Kataoka N. Wan L. Liu Q. Dreyfuss G. J. Cell Biol. 1997; 138: 1181-1192Crossref PubMed Scopus (202) Google Scholar). The yeast homologue, Kap104p, mediates nuclear import of two abundant nuclear mRNA binding proteins, Nab2p and Nab4p, via interaction with their Arg/Gly-rich NLSs (44Lee D.C. Aitchison J.D. J. Biol. Chem. 1999; 274: 29031-29037Abstract Full Text Full Text PDF PubMed Scopus (85) Goo" @default.
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• W2026008221 title "Nuclear Import Strategies of High Risk HPV16 L1 Major Capsid Protein" @default.
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