The glycoprotein Erns plays a central role in the biology of the pestivirus bovine viral diarrhea virus (BVDV). This soluble endonuclease mediates the escape from an interferon (IFN) response in the infected fetus, thereby permitting the establishment of persistent infection. Viral single-stranded (ss) and double-stranded (ds) RNA act as potent IFN inducing signals and we previously showed that Erns efficiently cleaves these substrates, thereby inhibiting an IFN response that is crucial for successful fetal infection. Considering that a large variety of RNases and DNases require dimerisation to cleave double-stranded substrates, the activity of Erns against dsRNA was postulated to depend on homodimer formation mediated by disulfide bonds involving residue Cys171. Here, we show that monomeric Erns is equally able to cleave dsRNA and to inhibit dsRNA-induced IFN synthesis as the wild-type form. Furthermore, both forms were able to degrade RNA within a DNA/RNA- as well as within a methylated RNA/RNA-hybrid, with the DNA and the methylated RNA strand being resistant to degradation. These results support our model that Erns acts as ‘nicking endoribonuclease’ degrading ssRNA within double-stranded substrates. This efficiently prevents the activation of IFN and helps to maintain a state of innate immunotolerance in persistently infected animals.

Pestiviruses including bovine viral diarrhea virus (BVDV), border disease virus (BDV) and classical swine fever virus (CSFV), occur worldwide and are important pathogens of livestock. A large part of their success can be attributed to the induction of central immunotolerance including B- and T-cells upon fetal infection leading to the generation of persistently infected (PI) animals. In the past few years, it became evident that evasion of innate immunity is a central element to induce and maintain persistent infection. Hence, the viral non-structural protease N(pro) heads the transcription factor IRF-3 for proteasomal degradation, whereas an extracellularly secreted, soluble form of the envelope glycoprotein E(rns) degrades immunostimulatory viral single- and double-stranded RNA, which makes this RNase unique among viral endoribonucleases. We propose that these pestiviral interferon (IFN) antagonists maintain a state of innate immunotolerance mainly pertaining its viral nucleic acids, in contrast to the well-established immunotolerance of the adaptive immune system, which is mainly targeted at proteins. In particular, the unique extension of 'self' to include the viral genome by degrading immunostimulatory viral RNA by E(rns) is reminiscent of various host nucleases that are important to prevent inappropriate IFN activation by the host's own nucleic acids in autoimmune diseases such as Aicardi-Goutières syndrome or systemic lupus erythematosus. This mechanism of "innate tolerance" might thus provide a new facet to the role of extracellular RNases in the sustained prevention of the body's own immunostimulatory RNA to act as a danger-associated molecular pattern that is relevant across various species.

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.

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