Bayliss C D, Smith G L
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Nucleic Acids Res. 1997 Oct 15;25(20):3984-90. doi: 10.1093/nar/25.20.3984.
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.
痘苗病毒蛋白VP8是L4R基因的一种25 kDa产物，是一种丰富的病毒粒子蛋白，可结合单链（ss）和双链（ds）DNA。在高盐浓度下，ssDNA的结合更受青睐。使用重组25 kDa L4R（rL4R）蛋白和放射性标记寡核苷酸的凝胶迁移率变动分析，确定45聚体寡核苷酸的Kd为2 nM。50 mM KCl对Kd无影响，但100 mM KCl可使其降低35倍。多个rL4R分子与单个45聚体寡核苷酸结合，通过使用不同长度的寡核苷酸计算得出，每17个核苷酸有一个rL4R分子结合。脱氧核糖和核糖多核苷酸均可竞争与ssDNA的结合。使用130 nt放射性标记的ssRNA进行凝胶迁移率变动分析，观察到从病毒粒子中纯化的rL4R和天然VP8均能结合RNA。在无盐条件下，rL4R对该ssRNA底物的Kd为3 nM，且结合呈正协同效应。文中讨论了L4R蛋白在痘苗病毒早期转录中的潜在作用。
