Vaccinia virion protein VP8, the 25 kDa product of the L4R gene, binds single-stranded DNA and RNA with similar affinity

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 K-d for a 45mer oligonucleotide was determined to be 2 nM, The K-d 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 K-d 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.