Human Papillomaviruses (HPVs) are major DNA tumor viruses involved in cervical and anogenital cancers. HPV virions consist of a molecule of DNA contained within a capsid composed of L1 major and L2 minor capsid proteins. HPVs replicate in the nucleus of host epithelial cells, and nuclear import and export of HPV proteins and nucleic acids are crucial for their replication cycle. E6 and E7 are the transforming proteins of high risk HPVs, and regulation of their nuclear import may play an important role in the transformation process. Our data suggest that HPV16 E6 oncoprotein is imported into the nucleus through several pathways. The nuclear localization signal of HPV16 E6 is partially conserved in high risk HPV18 E6, and it is not conserved in low risk HPV11 E6. We propose to identify the karyopherins (Kaps) involved and biochemically characterize the nuclear import pathways for high risk HPV16/18 E6 oncoproteins. Our data with wild type and mutants of HPV16 E7 strongly suggest that E7 uses a novel nuclear import pathway(s). We propose to identify the import receptor(s) involved in nuclear import of E7 and characterize this novel transport pathway(s). To accomplish these goals we will use a variety of methods including: nuclear import and binding assays, affinity isolation, mass spectrometry, cloning and mutagenesis. The L1 and L2 capsid proteins enter the nucleus twice during the virus life cycle: immediately after the virions infect the cells, and during the late productive phase when the newly synthesized L1 and L2 proteins co-assemble with the replicated HPV-DNA to form infectious virions. We have characterized the nuclear import pathways used by the L1 major capsid proteins of high and low risk HPVs. Here we propose: 1) to identify the Kaps involved in nuclear import of L2 minor capsid proteins of high risk HPV16/18 and characterize these transport pathways, and 2) to investigate the potential role of L2 proteins in facilitating nuclear import of HPV DNA. The import of viral DNA into the nucleus of infected host cells is a crucial event in the life cycle of HPVs, and these studies are expected to shed light on this process. Moreover, these studies should be helpful for future development of anti-viral drugs and innovative therapies against cervical cancers.
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