and double-stranded (ds) RNA act as potent IFN inducing signals and we previously showed that E rns 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 E rns against dsRNA was postulated to depend on homodimer formation mediated by disulfide bonds involving residue Cys171. Here, we show that monomeric E rns 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 E rns acts as 'nicking endoribonuclease' degrading ssRNA within doublestranded substrates. This efficiently prevents the activation of IFN and helps to maintain a state of innate immunotolerance in persistently infected animals. Bovine viral diarrhea virus (BVDV), a pestivirus within the family Flaviviridae, occurs throughout the world with far-reaching consequences for animal health and the agricultural economy. Infection of pregnant cows within the first ∼120 days of gestation with a noncytopathic (ncp), but not cytopathic (cp), biotype of BVDV may result in the birth of persistently infected (PI) calves 1-6 . The early time point of fetal infection prior to the development of adaptive immunity and the distinct epitheliochorial placenta of ruminants that is impermeable to antibodies, however, cannot fully account for the successful establishment of persistent infection, because the innate immune defense is operative from the earliest time of fetal development. Therefore, in addition to the establishment of virus-specific B-and T-cell immunotolerance during ontogeny and to the lack of transfer of maternal antibodies to the fetus, the interplay of BVDV with the innate immune response of its host animals might be the most important aspect for the long-term survival of this virus in its host population (for review, see refs 1-6 ). Various pattern recognition receptors (PRR), such as the membrane-bound Toll-like receptors (TLR) or the cytoplasmic RIG-I-like receptors (RLR), activate the synthesis of interferon (IFN) type-I (IFN-α/β) upon detection of viral pathogen-associated molecular patterns (PAMP) 7-9 . IFN is a cytokine involved in the most important innate defense mechanism against virus infection, and most viruses, including the pestiviruses, have developed an enormous variety of strategies to bypass these antiviral effects 5,10-14 . The genome of the enveloped pestiviruses consists of a single-stranded (ss) RNA of positive polarity encoding for a single large polyprotein. Cap-independent translation is started by an internal ribosomal entry site (IRES), and the polyprotein is then further processed by cellular and viral proteases into four structural and at least eight non-structural viral proteins [15] [16] [17] . The N-terminal autoprotease N pro and the structural protein E rns are exclusively present in viruses of the pestivirus genus within the flaviviridae family, and both were implicated to be involved in the evasion of the host's type-I IFN defense 2,17,18 . N pro activates the proteasomal degradation of the interferon regulatory factor (IRF)-3 [19] [20] [21] [22] , and possibly also IRF-7 23 , thus preventing the transcriptional activation of the IFN genes. In this manner, the non-structural protein N pro is able in infected cells to prevent IFN synthesis that is induced by the activation Published: xx xx xxxx OPEN www.nature.com/scientificreports/
Output: Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease OPEN