Classical swine fever virus glycoprotein E rns is an endoribonuclease with an unusual base specificity.
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).
Hausmann
Yvonne
Y
Institut für Virologie, Justus-Liebig-Universität, Frankfurter Strasse 107, D-35392 Giessen, Germany.
Roman-Sosa
Gleyder
G
Thiel
Heinz-Jürgen
HJ
Rümenapf
Till
T
D006692:Classical Swine Fever Virus / Q000737:chemistry*; D004722:Endoribonucleases / Q000502:physiology*; D006023:Glycoproteins / Q000502:physiology*; D007700:Kinetics; D012313:RNA / Q000378:metabolism*; D011995:Recombination, Genetic; D013379:Substrate Specificity; D014764:Viral Proteins / Q000502:physiology*
D006023:Glycoproteins; D014764:Viral Proteins; D012313:RNA; D004722:Endoribonucleases