Positively Charged Amino Acids in the Pestiviral Erns Control Cell Entry, Endoribonuclease Activity and Innate Immune Evasion

Abstract

The genus Pestivirus, family Flaviviridae, includes four economically important viruses of livestock, i.e., bovine viral diarrhea virus-1 (BVDV-1) and -2 (BVDV-2), border disease virus (BDV) and classical swine fever virus (CSFV). Erns and Npro, both expressed uniquely by pestiviruses, counteract the host’s innate immune defense by interfering with the induction of interferon (IFN) synthesis. The structural envelope protein Erns also exists in a soluble form and, by its endoribonuclease activity, degrades immunostimulatory RNA prior to their activation of pattern recognition receptors. Here, we show that at least three out of four positively-charged residues in the C-terminal glycosaminoglycan (GAG)-binding site of BVDV-Erns are required for efficient cell entry, and that a positively charged region more upstream is not involved in cell entry but rather in RNA-binding. Moreover, the C-terminal domain on its own determines intracellular targeting, as GFP fused to the C-terminal amino acids of Erns was found at the same compartments as wt Erns. In summary, RNase activity and uptake into cells are both required for Erns to act as an IFN antagonist, and the C-terminal amphipathic helix containing the GAG-binding site determines the efficiency of cell entry and its intracellular localization.

The Molecular Basis for Erns Dimerization in Classical Swine Fever Virus

“…We have shown recently that mutation of a variety of amino acids (especially charged residues) in the membrane anchor region of E rns prevents the recovery of infectious viruses, and that at least one defect resulting from these tested exchanges concerns the assembly and budding step of pestiviruses [10,60]. Another recent publication reported that exchanges of positively charged residues in the anchor region can block virus entry and also transport of secreted E rns into target cells [61]. With this knowledge in mind, it is rather likely that isolation of S209C as the only pseudorevertant is not necessarily a consequence of structural aspects but could simply be due to impairment of functions of the E rns membrane anchor by other mutations leading to non-viable viruses.…”

Fifty Shades of Erns: Innate Immune Evasion by the Viral Endonucleases of All Pestivirus Species

“…The orange boxes are focusing on the two RNase active sites conserved among most pestiviruses (except for APPV-and Bat-pestivirus), whereas the black box highlights the residue important for RNase activity as identified in this study using E rns of giraffe pestiviruses. The proposed "positive-region" and "heparin-binding-domain" [24] are boxed in blue.…”

“…For staining of cellular Mx proteins, a primary mouse monoclonal antibody against MxA was used [24]. As a house-keeping protein, β-actin was detected with a mouse monoclonal anti-β-actin antibody (Sigma, Buchs, Switzerland).…”

“…To evaluate the molecular weight of the various E rns proteins, 2 µg of each were analyzed under non-reducing conditions as previously described [24]. Briefly, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by standard staining with Coomassie Brilliant Blue G250 (Bio-Rad Laboratories, Cressier, Switzerland).…”

“…In addition, the C-terminal end of E rns contains a positively charged domain that enables the protein to interact with cell surface glycosaminoglycans (GAG) [21][22][23]. The presence of this GAG-binding site is required for cell attachment followed by its uptake via clathrin-mediated endocytosis [9], leading most likely to the localization of E rns in an endolysosomal compartment [7,24].…”