Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease

Abstract

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.

Classical Swine Fever Virus NproAntagonizes IRF3 To Prevent Interferon-Independent TLR3- and RIG-I-Mediated Apoptosis

“…6A and B ). CSFV was not assessed for its capacity to antagonize poly(I·C)-mediated apoptosis as the ΔN pro virus still encodes the soluble and secretable endoribonuclease E rns , which acts as a scavenger receptor for dsRNA ( 44 , 45 ); a double mutant ΔN pro ΔE rns virus was unavailable.…”

“…The SK6 cells that were infected with either CSFV Alfort, CSFV Brescia or rCSFV Alfort prior to SeV treatment displayed significantly reduced Bax localisation to the mitochondria (p<.001), however those infected with ΔN pro rCSFV Alfort displayed levels of localisation comparable to uninfected control cells (FIG 6A, 6B). CSFV was not assessed for its capacity to antagonise poly(I:C)-mediated apoptosis as the ΔN pro virus still encodes the soluble and secretable endoribonuclease E rns which acts as a scavenger receptor for dsRNA (45,46); a double mutant ΔN pro ΔE rns virus was unavailable.…”

The Erns Carboxyterminus: Much More Than a Membrane Anchor

“…The E rns protein of pestiviruses represents a very special product of viral evolution, a kind of a swiss army knife that integrates a role as structural protein essential for generation of infectious viral particles and a virulence factor function important for controlling the host’s innate immune response to pestivirus infection. With regard to both of these functions, our knowledge of the detailed molecular mechanisms is very poor, despite considerable and successful efforts taken to characterize this protein even on the structural level [ 5 , 6 , 9 , 11 , 12 , 16 , 20 , 21 , 23 , 123 , 124 , 125 , 126 ]. Deletion of the E rns coding sequence from the viral genome prevents the generation of infectious viruses (this report and [ 6 , 95 , 96 , 119 ]).…”

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

“…Wild-type E rns of the BVDV stain Ncp7 was subcloned into the pCI mammalian expression vector (Promega; kindly provided by P. Plattet) by Eurofins Genomics GmbH (Ebersberg, Germany). All mutants and GFP fusion constructs used in this study were subcloned using the “In-Fusion HD cloning kit” from Clontech (Takara Bio Europe SAS, Saint-Germain-en-Laye, France) as described for H30F and C171R [ 25 ]. Primers are available upon request.…”