Crystal Structure of the Pestivirus Envelope Glycoprotein Erns and Mechanistic Analysis of Its Ribonuclease Activity

Abstract

Pestiviruses, which belong to the Flaviviridae family of RNA viruses, are important agents of veterinary diseases causing substantial economical losses in animal farming worldwide. Pestivirus particles display three envelope glycoproteins at their surface: E rns , E1, and E2. We report here the crystal structure of the catalytic domain of E rns , the ribonucleolytic activity of which is believed to counteract the innate immunity of the host. The structure reveals a threedimensional fold corresponding to T2 ribonucleases from plants and fungi. Cocrystallization experiments with mono-and oligonucleotides revealed the structural basis for substrate recognition at two binding sites previously identified for T2 RNases. A detailed analysis of poly-U cleavage products using 31 P-NMR and size exclusion chromatography, together with molecular docking studies, provides a comprehensive mechanistic picture of E rns activity on its substrates and reveals the presence of at least one additional nucleotide binding site.

The Molecular Biology of Pestiviruses

“…A survey of the location of N-glycosylation sites in the E rns sequences from different pestiviruses revealed that the majority is located on the convex side, so that it seems likely that glycosylation on the concave side would interfere with E rns dimer formation or enzymatic activity. Structural analysis confirmed the T2 fold of the protein and showed that the Zn 2+ ion bound to histidine 81 in the active center (Krey et al, 2012), which explains the inhibition of RNase activity by this ion. The highest structural similarity was observed for the plant T2 RNase MC1 that also shows a preference for NpU bonds (Krey et al, 2012).…”

“…Structural analysis confirmed the T2 fold of the protein and showed that the Zn 2+ ion bound to histidine 81 in the active center (Krey et al, 2012), which explains the inhibition of RNase activity by this ion. The highest structural similarity was observed for the plant T2 RNase MC1 that also shows a preference for NpU bonds (Krey et al, 2012). The structural data argue in favor of a two-step interaction between the RNase and its substrate.…”

“…In contrast, B1 could interact with any nucleotide. As mentioned above, E rns forms homodimers and in vitro experiments with the purified enzyme have shown an approximately fourfold slower cleavage and higher affinity by the dimer compared to the monomer (Krey et al, 2012). The increased binding affinity is supposed to result from the doubled number of positively charged binding sites in the active site cavity formed when the two monomers anneal at their concave sides.…”

“…RNase activity of E rns has a pH optimum of pH 6.0 and is inhibited by Zn 2+ and Mn 2+ , but insensitive to chelating agents (Schneider et al, 1993;Windisch et al, 1996). The crystal structure of the E rns ectodomain (amino acids 1-165) has been solved (Krey et al, 2012). It contains five α helices and seven β strands (Fig.…”

X-Ray Structure of the Pestivirus NS3 Helicase and Its Conformation in Solution

“…1A (2,5,6). Structural studies are available for nonstructural protein 5B (NS5B) (7), E rns (8), E2 (9,10), and N pro (11,12). N pro and E rns are unique to pestiviruses, while all the others have their counterparts in the other genera.…”