title: Recruitment of replication protein A by the papillomavirus E1 protein and modulation by single-stranded DNA.
abstract: With the exception of viral proteins E1 and E2, papillomaviruses depend heavily on host replication machinery for replication of their viral genome. E1 and E2 are known to recruit many of the necessary cellular replication factors to the viral origin of replication. Previously, we reported a physical interaction between E1 and the major human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA). E1 was determined to bind to the 70-kDa subunit of RPA, RPA70. In this study, using E1-affinity coprecipitation and enzyme-linked immunosorbent assay-based interaction assays, we show that E1 interacts with the major ssDNA-binding domain of RPA. Consistent with our previous report, no measurable interaction between E1 and the two smaller subunits of RPA was detected. The interaction of E1 with RPA was substantially inhibited by ssDNA. The extent of this inhibition was dependent on the length of the DNA. A 31-nucleotide (nt) oligonucleotide strongly inhibited the E1-RPA interaction, while a 16-nt oligonucleotide showed an intermediate level of inhibition. In contrast, a 10-nt oligonucleotide showed no observable effect on the E1-RPA interaction. This inhibition was not dependent on the sequence of the DNA. Furthermore, ssDNA also inhibited the interaction of RPA with papillomavirus E2, simian virus 40 T antigen, human polymerase alpha-primase, and p53. Taken together, our results suggest a potential role for ssDNA in modulating RPA-protein interactions, in particular, the RPA-E1 interactions during papillomavirus DNA replication. A model for recruitment of RPA by E1 during papillomavirus DNA replication is proposed.
authors.affiliation: Department of Microbiology, and Witebsky Center for Microbial Pathogenesis and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, USA.
authors.forename: Yueh-Ming
authors.initials: YM
authors.lastname: Loo
authors.forename: Thomas
authors.initials: T
authors.lastname: Melendy
mesh_terms: D001667:Binding, Competitive; D011232:Chemical Precipitation; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry / Q000494:pharmacology; D027383:Papillomaviridae; D051716:Replication Protein A; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C084304:E1 protein, Human papillomavirus type 11; D009841:Oligonucleotides; C497167:RPA1 protein, human; D051716:Replication Protein A; D014764:Viral Proteins