Classical Swine Fever Virus Glycoprotein E ^rns Is an Endoribonuclease with an Unusual Base Specificity

Abstract

The glycoprotein E(rns) of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for E(rns). Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5' of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that E(rns) is a highly active endoribonuclease (k(cat)/K(m) = 2 x 10(6) to 10 x 10(6) M(-1) s(-1)) with a strong affinity to NpU containing single-stranded RNA substrates (K(m) = 85 to 260 nM).