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 concentr ations, Using a recombinant 25 kDa L4R (rL4R) protein and a gel mobili ty shift assay with radiolabelled oligonucleotides, the K-d for a 45me r 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 molecul es bound to a single 45mer oligonucleotide, and using oligonucleotides of different lengths it was calculated that one rL4R molecule bound e very 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 radiolabell ed 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 pot ential roles of L4R protein in vaccinia virus early transcription are discussed.