title: A conserved amphipathic helix in the N-terminal regulatory region of the papillomavirus E1 helicase is required for efficient viral DNA replication.
abstract: The papillomavirus E1 helicase, with the help of E2, assembles at the viral origin into a double hexamer that orchestrates replication of the viral genome. The N-terminal region (NTR) of E1 is essential for DNA replication in vivo but dispensable in vitro, suggesting that it has a regulatory function. By deletion analysis, we identified a conserved region of the E1 NTR needed for efficient replication of viral DNA. This region is predicted to form an amphipathic α-helix (AH) and shows sequence similarity to portions of the p53 and herpes simplex virus (HSV) VP16 transactivation domains known as transactivation domain 2 (TAD2) and VP16C, which fold into α-helices upon binding their target proteins, including the Tfb1/p62 (Saccharomyces cerevisiae/human) subunit of general transcription factor TFIIH. By nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we found that a peptide spanning the E1 AH binds Tfb1 on the same surface as TAD2/VP16C and with a comparable affinity, suggesting that it does bind as an α-helix. Furthermore, the E1 NTRs from several human papillomavirus (HPV) types could activate transcription in yeast, and to a lesser extent in mammalian cells, when fused to a heterologous DNA-binding domain. Mutation of the three conserved hydrophobic residues in the E1 AH, analogous to those in TAD2/VP16C that directly contact their target proteins, decreased transactivation activity and, importantly, also reduced by 50% the ability of E1 to support transient replication of DNA in C33A cells, at a step following assembly of the E1-E2-ori preinitiation complex. These results demonstrate the existence of a conserved TAD2/VP16C-like AH in E1 that is required for efficient replication of viral DNA.
authors: Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montre´al (IRCM), Canada.
authors: Geneviève
authors: G
authors: Morin
authors: Amélie
authors: A
authors: Fradet-Turcotte
authors: Paola
authors: P
authors: Di Lello
authors: Fanny
authors: F
authors: Bergeron-Labrecque
authors: James G
authors: JG
authors: Omichinski
authors: Jacques
authors: J
authors: Archambault
mesh_terms: D002151:Calorimetry; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000378:metabolism*; D004847:Epithelial Cells / Q000821:virology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D027383:Papillomaviridae / Q000502:physiology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D015534:Trans-Activators; D014764:Viral Proteins; D004265:DNA Helicases