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

The genus , family , 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). E and N, 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 E 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-E 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 E was found at the same compartments as wt E. In summary, RNase activity and uptake into cells are both required for E 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.