Human papillomaviruses (HPVs) are medically important pathogens that normally induce benign hyperproliferation of epithelia. However, the sexually transmitted high-risk viral genotypes can cause cervical and penile dysplasias and cancer. HPVs establish persistent, often subclinical, infections in cycling basal and parabasal keratinocytes, where the double-stranded, circular DNA genome replicates as extra-chromosomal plasmids at low copy number. Productive amplification takes place upon squamous differentiation. Immunosuppression or surgery/wound healing can reactivate latent infections. There is no effective pharmacologic treatment. Characterization of the mechanisms of viral DNA replication and persistence is of paramount importance to the development of strategies for antiviral therapies and also because HPV provides a simple but elegant model system for replication and chromosome dynamics in eukaryotes. We developed both transient transfection and cell-free systems to study HPV DNA synthesis, which requires the viral origin (Ori) sequences, the Ori recognition protein E2, the DNA helicase E1 and the cellular DNA replication machinery, as well as chaperones and cyclin E/cdk2, a kinase critical for S phase entry. To determine how HPV DNA segregates when the host cells divide, we constructed replication-competent GFP-tagged HPV-11 E1 and E2 proteins and discovered that GFP-11E2 protein associates with mitotic spindles.
Six Specific Aims are: (1) To develop and validate a set of living color fluorescent proteins (FP) fused to E1 and E2, as well as to the Gal4 DNA binding domain, which enables tracing of a modified Ori plasmid containing 40 copies of the Gal4 binding site. We propose to verify that Ori DNA segregates by association with mitotic spindles mediated by E2. (2) To characterize any other sequence elements in HPV necessary for spindle-associated Ori DNA segregation. (3) To identify cellular proteins that mediate the E2-microtubule interaction, using mass spectrometry. (4) To confirm the interactions between E2 and cellular proteins by in vivo colocalization and in vitro interaction. (5) To identify peptide motifs in E2 responsible for these interactions and to examine site-directed mutations in E2 for their abilities to associate with the mitotic spindles and to mediate DNA segregation. (6) To identify the subcellular addresses of FP-tagged E1 and E2 and Ori DNA under nonreplicating and replicating conditions relative to known nuclear domains such as ND10 (PML) bodies and Cajal bodies. Their trafficking will be captured in live cells as time-lapse movies using multi-photon laser confocal scanning microscopy. These investigations will provide a detailed molecular portrait of HPV DNA replication in vivo. The anticipated outcome is a clear definition of the mechanisms of extrachromosomal plasmid segregation and maintenance. ? ? ?
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