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...

During the life cycle of human papillomaviruses (HPVs), the L1 capsid proteins seem to enter the nucleus twice: once after the virions infect the cells, and later during the productive phase when they assemble the replicated HPV genomic DNA into infectious virions. We established for the high-risk HPV45 that when digitonin-permeabilized HeLa cells were incubated with L1 homopentameric capsomers, the HPV45 L1 protein was imported into the nucleus in a receptor-mediated manner. In contrast, intact capsids were not able to enter the nucleus. Immunoisolation assays showed that HPV45 L1 capsomers interact with cytosolic karyopherin alpha 2 beta 1 heterodimers. HPV45 L1 bound strongly to karyopherin alpha 2, and weakly to karyopherin beta 1, as did its nuclear localization signal (NLS). Nuclear import of HPV45 L1, or of a GST-NLS(HPV45L1) fusion protein was efficiently mediated by karyopherin alpha 2 beta 1 heterodimers, and only weakly by karyopherin beta 1. Nuclear import required RanGDP, but was independent of GTP hydrolysis by Ran. Together, these data suggest that the major nuclear import pathway for HPV45 L1 major capsid protein in infected host cells is mediated by karyopherin alpha 2 beta 1 heterodimers and that GTP hydrolysis by Ran is not required for import. Remarkably, HPV45 L1 capsomers can interact nonspecifically with different types of HPV-DNA, and the DNA binding region of HPV45 L1 overlaps with its NLS sequence.

We have previously shown that the L1 major capsid protein of low-risk HPV11 binds to the Kap alpha2 adapter and enters the nucleus via a Kap alpha2beta1-mediated pathway. In this study, we discovered that HPV11 L1 capsomeres bind to Kap beta2 import receptor, known to mediate nuclear import of hnRNP A1 via interaction with its nuclear localization signal termed M9. Significantly, binding of HPV11 L1 capsomeres to Kap beta2 inhibited the nuclear import of Kap beta2, and its specific M9-containing cargo. Interestingly, HPV11 L1 capsomeres also interacted with Kap beta3 import receptor and inhibited Kap beta3 nuclear import. Moreover, the L1 capsomeres of high-risk HPV-16 shared these activities. These data suggest that HPV L1 major capsid proteins interact with Kap beta2 and Kap beta3, and they may inhibit the Kap beta2- and Kap beta3-mediated nuclear import pathways during the productive phase of the viral life cycle when the virions are assembled and released.

During the life cycle of human papillomaviruses (HPVs), the L1 capsid proteins seem to enter the nucleus twice: once after the virions infect the cells, and later during the productive phase when they assemble the replicated HPV genomic DNA into infectious virions. We established for the high-risk HPV45 that when digitonin-permeabilized HeLa cells were incubated with L1 homopentameric capsomers, the HPV45 L1 protein was imported into the nucleus in a receptor-mediated manner. In contrast, intact capsids were not able to enter the nucleus. Immunoisolation assays showed that HPV45 L1 capsomers interact with cytosolic karyopherin alpha 2 beta 1 heterodimers. HPV45 L1 bound strongly to karyopherin alpha 2, and weakly to karyopherin beta 1, as did its nuclear localization signal (NLS). Nuclear import of HPV45 L1, or of a GST-NLS(HPV45L1) fusion protein was efficiently mediated by karyopherin alpha 2 beta 1 heterodimers, and only weakly by karyopherin beta 1. Nuclear import required RanGDP, but was independent of GTP hydrolysis by Ran. Together, these data suggest that the major nuclear import pathway for HPV45 L1 major capsid protein in infected host cells is mediated by karyopherin alpha 2 beta 1 heterodimers and that GTP hydrolysis by Ran is not required for import. Remarkably, HPV45 L1 capsomers can interact nonspecifically with different types of HPV-DNA, and the DNA binding region of HPV45 L1 overlaps with its NLS sequence.