The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association

The E2 protein of papillomaviruses is a site-specific DNA binding nuclear p rotein, It functions as the primary replication origin recognition protein and assists in the assembly of the preinitiation complex. It also helps reg ulate transcription from the native viral promoter. The E2 protein consists of an amino-terminal (N) trans-acting domain, a central hinge (H) domain, and a carboxyl-terminal (C) protein dimerization and DNA binding domain. Th e hinge is highly divergent among papillomaviruses, and little is known abo ut its functions. We fused the enhanced green fluorescent protein (GFP) wit h the full-length human papillomavirus type 11 (HPV-11) E2 protein and show ed that the resultant fusion, called gfpE2, maintained transcription and re plication functions of the wild-type protein and formed similar subnuclear foci. Using a series of GFP fusion proteins, we showed that the hinge confe rred strong nuclear localization, whereas the N or C domain was present in both cytoplasm and nucleus. Biochemical fractionation demonstrated that the N domain and hinge, but not the C domain, independently associated with th e nuclear matrix. Mutational analyses showed that a cluster of basic amino acid residues, which is conserved among many mucosotropic papillomaviruses, was required for efficient nuclear localization and nuclear matrix associa tion. This mutation no longer repressed the HPV-11 upstream regulatory regi on-controlled reporter expression. However, a very small fraction of this m utant colocalized with E1 in the nucleus, perhaps by a piggyback mechanism, and was able to support transient replication. We propose that the hinge i s critical for the diverse regulatory functions of the HPV-11 E2 protein du ring mRNA transcription and viral DNA replication.