title: Vaccinia virion protein VP8, the 25 kDa product of the L4R gene, binds single-stranded DNA and RNA with similar affinity.
abstract: Vaccinia virus protein VP8 is a 25 kDa product of the L4R gene and is an abundant virion protein that binds single-stranded (ss) and double-stranded (ds) DNA. Binding of ssDNA is preferred at high salt concentrations. Using a recombinant 25 kDa L4R (rL4R) protein and a gel mobility shift assay with radiolabelled oligonucleotides, the Kd for a 45mer oligonucleotide was determined to be 2 nM. The Kd was unaltered by 50 mM KCl but was reduced 35-fold by 100 mM KCl. Multiple rL4R molecules bound to a single 45mer oligonucleotide, and using oligonucleotides of different lengths it was calculated that one rL4R molecule bound every 17 nt. Binding to ssDNA was competed by both deoxyribo- and ribo-polynucleotides. RNA binding was observed for both rL4R and native VP8, purified from virions, using a gel mobility shift with a radiolabelled ssRNA of 130 nt. The Kd of rL4R for this ssRNA substrate was 3 nM in the absence of salt and binding was positively cooperative. The potential roles of L4R protein in vaccinia virus early transcription are discussed.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
authors.forename: C D
authors.initials: CD
authors.lastname: Bayliss
authors.forename: G L
authors.initials: GL
authors.lastname: Smith
mesh_terms: D001667:Binding, Competitive; D004265:DNA Helicases / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014616:Vaccinia virus / Q000737:chemistry / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; C108986:vaccinia virus protein VP8; D012313:RNA; D011189:Potassium Chloride; D004265:DNA Helicases