Nuclear import of HPV11 L1 capsid protein is mediated by karyopherin α2β1 heterodimers

Merle, Eric; Rose, Robert C.; Roux, Lucia Le; Moroianu, Junona

doi:10.1002/(sici)1097-4644(19990915)74:4<628::aid-jcb12>3.3.co;2-9

Cited by 17 publications

(22 citation statements)

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“…Our first goals were to examine the nuclear entry of the PV genome and its possible continued association with L1 and͞or L2 as well as where in the cell the initial uncoating took place. We expected that at least partial disassembly of the viral particle would occur before nuclear import, because it has been reported that intact human PV capsids are unable to enter the nucleus (27,28). Consistent with this possibility, initial analyses of HeLa cells infected with BPV-1 virus or pseudovirus showed no evidence of nuclear L1 with any of several tested L1 antibodies or fixation conditions (data not shown).…”

Section: Resultsmentioning

confidence: 55%

Establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (PML) expression

Day

Baker

Lowy

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

218

277

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Previous studies have suggested that most papillomaviruses enter the host cell via clathrin-dependent receptor-mediated endocytosis but have not addressed later steps in viral entry. To examine these events, we followed the localization of L2 and packaged DNA after entry of infectious virions or L1͞L2 pseudovirions. Confocal microscopic analyses of HeLa cells showed a time-dependent uncoating of capsids in cytoplasmic vesicles and the accumulation of both L2 and viral DNA at distinct nuclear domains identified as nuclear domain 10 (ND10). Both L2 and the pseudogenome had a punctate distribution and localized to ND10 in promyelocytic leukemia protein (PML)-expressing cells, whereas L2 had a diffuse nuclear distribution in PML؊͞؊ cells. The number of pseudovirus-infected cells was an order of magnitude higher in the PML؉ cells compared with the PML؊͞؊ cells, and viral genome transcription after infection with authentic bovine papillomavirus virions was similarly elevated in PML؉ cells. The results identify a role for PML in the enhancement of viral infectivity in the early part of the life cycle. We propose a model in which L2 chaperones the viral genome to ND10 to efficiently initiate viral transcription.

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Section: Resultsmentioning

confidence: 55%

Establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (PML) expression

Day

Baker

Lowy

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

218

277

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“…In the nucleus, binding of RanGTP to Kap ␤1 dissociates the import complex with release of HPV16 L1⅐Kap ␣2. We had previously established that the L1 proteins of low risk HPV11 and high risk HPV45 can interact with Kap ␣2 and enter the nucleus via the classic Kap ␣2␤1-mediated pathway (38,39). Six Kap ␣ isoforms have been identified in higher eukaryotes, and they all bind to the same Kap ␤1.…”

Section: Discussionmentioning

confidence: 99%

“…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 (20, 22, 30 -38). 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 (38,39). In the present study, subconfluent HeLa cells, grown on poly-L-lysine-coated glass coverslips for 24 h, were permeabilized with 70 g/ml digitonin for 5 min on ice and washed with buffer A. Digitonin permeabilizes the plasma membrane but leaves the nuclear envelope intact.…”

Section: Methodsmentioning

confidence: 99%

“…Major Capsid Protein-We had previously established for the L1 major capsid proteins of HPV11 and HPV45 that capsid disassembly is required for their nuclear import (38,39). Therefore, the nuclear import of the L1 major capsid protein of HPV16 was investigated in either capsid or capsomere conformation.…”

Section: Disassembly Of L1 Capsids Is Required For Nuclear Import Of mentioning

confidence: 99%

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Nuclear Import Strategies of High Risk HPV16 L1 Major Capsid Protein

Nelson¹,

Rose²,

Moroianu³

2002

Journal of Biological Chemistry

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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 ␣2␤1 heterodimers via interaction with Kap ␣2. Accordingly, nuclear import of HPV16 L1 capsomeres was mediated by Kap ␣2␤1 heterodimers, required RanGDP and free GTP, and was independent of GTP hydrolysis. Remarkably, HPV16 L1 capsomeres also interacted with Kap ␤2 and binding of RanGTP to Kap ␤2 did not dissociate the HPV16 L1⅐Kap ␤2 complex. Significantly, HPV16 L1 capsomeres inhibited the nuclear import of Kap ␤2 and of a Kap ␤2-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 ␣2␤1-mediated pathway to assemble the virions, it also inhibits the Kap ␤2-mediated nuclear import of host hnRNP A1 protein and, in this way, favors virion formation.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 (1).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 (2) or 12 (3). L1 protein is capable of self-assembly both in vivo and in vitro into capsid-like structures, referred to as virus-like particles (4 -9). 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 (10, 11). The L1 capsid proteins of HPVs seem to enter the nucleus of host cells twice during the virus life cycle: immediately after the vi...

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“…For reconstitution experiments, purified recombinant importin-␣2 (0.125 mg/ml) and/or importin-␤ (0.125 mg/ml) were added to the cytosol depleted of the importins. For the docking assay, recombinant importin-␤ was used as a source of import factor instead of HeLa cell cytosol, as previously described (30,33,34).…”

Section: Methodsmentioning

confidence: 99%

Importin-β Mediates Cdc7 Nuclear Import by Binding to the Kinase Insert II Domain, Which Can Be Antagonized by Importin-α

Kim

Lee

2006

Journal of Biological Chemistry

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We investigated the nuclear import mechanism of Cdc7, which is essential for the initiation of DNA replication. Here we report that importin-␤ binds directly to Cdc7 via the Kinase Insert II domain, promoting its nuclear import. Although both importin-␣ and -␤ bind to Cdc7 via the Kinase Insert II domain in a mutually independent manner, the binding affinity of Cdc7 for importin-␤ is ϳ10 times higher than for importin-␣ at low protein concentrations of an equimolar ratio. Immunodepletion of importin-␤, but not importin-␣, abrogates Cdc7 nuclear import, and the addition of importin-␤ to the importin-depleted cytosol restores Cdc7 nuclear import. Furthermore, transduction of anti-importin-␤, but not anti-importin-␣ antibodies, into live cells inhibits Cdc7 nuclear import. Unexpectedly, we found that Cdc7 nuclear import is inhibited by competitive binding of importin-␣ to Cdc7. Further studies by site-directed mutagenesis suggest that Lys 306 and Lys 309 within the Kinase Insert II domain are critical for Cdc7 nuclear localization.

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Nuclear import of HPV11 L1 capsid protein is mediated by karyopherin α2β1 heterodimers

Cited by 17 publications

References 0 publications

Establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (PML) expression

Establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (PML) expression

Nuclear Import Strategies of High Risk HPV16 L1 Major Capsid Protein

Importin-β Mediates Cdc7 Nuclear Import by Binding to the Kinase Insert II Domain, Which Can Be Antagonized by Importin-α

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