A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication

• DOI: 10.1128/jvi.01829-10
• Publication Date: 2011-03-31T01:59:24Z
• Authors: Geneviève Morin; Amélie Fradet-Tur...; Paola Di Lello; Fanny Bergeron-La...; James G. Omichinski; Jacques Archambault
• Keywords: 0301 basic medicine; Sequence Homology, Amino Acid; DNA Mutational Analysis; DNA Helicases; Epithelial Cells; Saccharomyces cerevisiae; Calorimetry; Virus Replication; Protein Structure, Secondary; 3. Good health; Viral Proteins; 03 medical and health sciences; DNA, Viral; Mutagenesis, Site-Directed; Trans-Activators; Humans; Nuclear Magnetic Resonance, Biomolecular; Papillomaviridae; Protein Binding; Sequence Deletion

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.