Nuclear Entry of High-Risk Human Papillomavirus Type 16 E6 Oncoprotein Occurs via Several Pathways

Human papillomaviruses (HPVs) are icosahedral DNA tumor viruses that infect squamous epithelial cells of the skin or of the anogenital and oropharyngeal mucosa. About 85 distinct HPV genotypes have been identified and fully sequenced, and more than 120 types have been partially characterized. Mucosal HPVs have demonstrated various degrees of oncogenic potential, with some classified as high risk, such as types 16, 18, 31, and 45, and others as low risk, such as types 6 and 11 (60). High-risk HPVs are frequently detected in invasive cervical carcinomas, whereas the low-risk types are more often associated with benign exophytic condylomas. HPV infection is associated with more than 90% of all cervical cancers, which is the second leading cause of cancer death among women worldwide (60). Integration of the viral genome, resulting in deletion of several genes including E2 and maintenance of only the E6 and E7 genes, is a hallmark of malignant progression. Abrogation of E2 expression as a result of integration leads to uncontrolled expression of the E6 and E7 oncoproteins, which is crucial for the establishment and progression of the tumors. High-risk HPV E6 and E7 proteins cooperatively induce cellular immortalization and transformation (32, 40). Studies with transgenic mice suggest that whereas E7 promotes the formation of benign tumors, E6 acts primarily to accelerate progression of these benign tumors to the malignant stage (52). HPV16 E6 is a basic protein of 151 amino acids that is able to form a complex with the p53 tumor suppressor protein targeting p53 degradation (47). The E6-AP ubiquitin ligase facilitates the formation of the E6-p53 complex and functions together with E6 in the ubiquitination and subsequent degradation of p53 via the ubiquitin-dependent proteolytic system (19, 45). As a consequence, the level and half-life of p53 in E6 immortalized cell lines or in HPV-positive carcinoma cells are decreased (46). High-risk HPV E6 oncoproteins are multifunctional, and several other E6-associated proteins have been identified, including the Ca2+-binding protein E6-BP (3), the focal adhesion protein paxilin (55, 56), the transcription factor c-Myc (17), the coactivators CBP and p300 (38), and the transcriptional activator IRF-3 (44). Interaction of HPV16 E6 with cellular proteins results in modification of several cellular activities, including differentiation, signal transduction, cytoskeletal structure, cell polarity, and DNA replication (54). In the nucleus, high-risk HPV E6 proteins can also bind DNA and activate transcription of several genes (16, 24, 27). HPV16 E6 upregulates the expression of human telomerase reverse transcriptase through a combination of Myc and GC-rich Sp1 binding sites (36). High-risk HPV16 E6 oncoprotein localizes mainly to the nucleus when expressed in COS cells or insect cells (10, 16, 24, 33, 49). In cervical lesions, HPV16 E6 was found in both the nucleus and the cytoplasm (57), suggesting that the HPV16 E6 oncoprotein may shuttle between the two compartments. The basic paradigm for nuclear import is that the NLS cargo is bound in the cytoplasm directly, or via an adapter, by an import receptor belonging to the karyopherin β (Kap β)/importin β superfamily, translocated through the nuclear pore complex, and released inside the nucleus. Binding of nuclear RanGTP to Kap βs causes the dissociation of the import complexes with release of the cargoes inside the nucleus (14, 30, 53, 59). Several members of the mammalian Kap β superfamily have been identified and shown to function in nuclear import of specific cargoes. For example, Kap β1/importin β functions together with a Kap α/importin α adapter in nuclear import of proteins that contain classic monopartite or bipartite NLSs (14, 30). Kap β1 can also function without adapters in import of different cargoes, including ribosomal proteins L23a, S7, and L5, cyclin B1, Smad, human immunodeficiency virus TAT and Rev proteins, and human T-cell leukemia virus Rex protein (30, 53). Mammalian Kap β2/transportin mediates the nuclear import of hnRNP A1 and A2 via interaction with their Gly/Asn-rich NLS, termed M9 (2, 39, 50, 51), and of ribosomal proteins via interaction with their arginine-rich NLS (22). Ribosomal proteins L23a, S7, and L5 can interact with Kap β1, Kap β2/transportin, Kap β3/Imp5, and Imp7 and be imported alternatively by these Kaps (22). Many studies have focused on elucidating the molecular mechanisms of E6-mediated transformation. Nevertheless, there is virtually no information regarding the molecular mechanisms of the nuclear import of E6 oncoproteins. In this study we investigated the nuclear import of the E6 oncoprotein of high-risk HPV16. We found that the HPV16 E6 oncoprotein interacts with Kap α2 and can enter the nuclei of digitonin-permeabilized cells via Kap α2β1 heterodimers. Interestingly, HPV16 E6 also interacted with Kap β1 and Kap β2, and binding of RanGTP to these Kap βs inhibited their interaction with HPV16 E6 NLS. In agreement with these binding data, nuclear import of the HPV16 E6 oncoprotein could be mediated by either Kap β1 or Kap β2. The C terminus of HPV16 E6 (amino acids 121 to 151) interacted with either Kap α2, Kap β1, or Kap β2 and mediated nuclear import of a glutathione S-transferase (GST) reporter via either of these pathways. We found that mutation of R124 to G reduced the nuclear import of the HPV16 E6 oncoprotein. Moreover, an E6 deletion mutant lacking 121KKQR124 was not imported into the nucleus, suggesting that the KKQR sequence within the NLS is essential for nuclear import of HPV16 E6 oncoprotein. Together, these data suggest that the HPV16 E6 oncoprotein interacts via its C-terminal NLS with Kap α2, Kap β1, and Kap β2 and exploits these interactions to enter the nucleus via Kap α2β1-, Kap β1-, and Kap β2-mediated pathways.