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

Abstract

The genus Pestivirus, family Flaviviridae, includes four historically accepted species, i.e., bovine viral diarrhea virus (BVDV)-1 and -2, classical swine fever virus (CSFV), and border disease virus (BDV). A large number of new pestivirus species were identified in recent years. A common feature of most members is the presence of two unique proteins, N^pro and E^rns, that pestiviruses evolved to regulate the host's innate immune response. In addition to its function as a structural envelope glycoprotein, E^rns is also released in the extracellular space, where it is endocytosed by neighboring cells. As an endoribonuclease, E^rns is able to cleave viral ss- and dsRNAs, thus preventing the stimulation of the host's interferon (IFN) response. Here, we characterize the basic features of soluble E^rns of a large variety of classified and unassigned pestiviruses that have not yet been described. Its ability to form homodimers, its RNase activity, and the ability to inhibit dsRNA-induced IFN synthesis were investigated. Overall, we found large differences between the various E^rns proteins that cannot be predicted solely based on their primary amino acid sequences, and that might be the consequence of different virus-host co-evolution histories. This provides valuable information to delineate the structure-function relationship of pestiviral endoribonucleases.

Keywords: atypical pestiviruses; bovine viral diarrhea virus (BVDV); innate immune evasion; interferon type-I; pestivirus; viral RNase; viral endonuclease.

Figure 1

Amino acid alignment of the sequences of the E^rns analyzed in this study. Sequences carrying the asterisk were not published elsewhere. The corresponding references and, where available, the GenBank numbers are reported in Table 1. To prevent the cleavage of the Strep-tag, the last four amino acids at the C-terminus of APPV-, Bat-, Rat77-, Rat99- and Pangolin-E^rns, were modified in “GFYA”. In yellow are highlighted the cysteine (C) residues important for intra- and inter-molecular disulfide bridges. 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.

Figure 2

Coomassie staining of the various E^rns proteins analyzed. (A) Profile of the wild-type (wt) proteins. The absence of a cysteine in a position analogous to BVDV-Ncp7-171 is reflected in the absence of dimer formation (APPV-, Rat-77-, Rat-99-, and Bat-pestiviruses). (B) Profile of wt and mutant E^rns proteins. Mutant proteins were prevented from dimerization by mutating the corresponding cysteine residue into arginine (Ncp7-mon, BuPV-mon, and PhoPV-mon). The introduction of a ninth cysteine residue on either position 182 or 190 of APPV-E^rns did not induce an appreciable dimerization. Next to the pre-stained size ladder, approximate heights of monomers (M), dimers (D), and polymers (P) are indicated. Uncropped gels are available in Supplementary Material Figure S1.

Figure 3

RNase activity of the E^rns proteins of different pestivirus species analyzed. (A) Agarose gel of BuPV-E^rns is shown as an example for the enzymatic assay, with decreasing signal for the 300 bp dsRNA with increasing concentrations of E^rns. The sizes of the 100, 300, and 500 bp fragments within the 100 bp ladder (L) as size marker are indicated. The uncropped gel is available in Supplementary Material Figure S2. (B) Quantification of the gels as shown in panel A, estimating the amount of the various E^rns proteins required to reduce the signal of dsRNA by 50% (n = 3 ± SD). Proteins that did not display any detectable activity (i.e., E^rns of Gir-PG2, Gir-H138 and Gir-ΔCt), or for which the 50% value could not be calculated (i.e., the chimeric E^rns proteins H138-Ncp7-1 and H138-Ncp7-2), are not shown. The activities relative to E^rns of BVDV-Ncp7 are shown in Table 1.

Figure 4

Representative examples of the immunofluorescence (IF) labeling of E^rns proteins. E^rns was detected by IF microscopy as described using a primary antibody against the Strep-tag present at the C-terminus of each construct and a secondary antibody conjugated with Alexa 488 (green). Cells not treated with E^rns are labelled as “mock”, whereas cells incubated with BVDV-Ncp7-E^rns but treated only with the secondary antibody are labelled with “control”. Nuclei were stained with DAPI (blue) present in the mounting medium. The same image processing settings were used for mock and all constructs. For each microphoto, obtained with a 40× air objective, an area was selected (white square), magnified five times, and displayed to its right-hand side. One representative experiment out of three is shown. As most of the E^rns proteins could not be detected by IF labeling, only BVDV-Ncp7-E^rns and BuPV-E^rns that gave a clear signal are shown, in addition to one example (APPV-E^rns) that could not be visualized. By contrast, substituting the C-terminus of APPV-E^rns with the one of BVDV-Ncp7-E^rns allowed for efficient detection of the chimeric protein.

Figure 5

Inhibition of interferon expression was analyzed using the expression of Mx proteins as a proxy. (A): Example of a Western blot used to quantify the expression of Mx and the housekeeping protein β-actin in cells incubated with Bat-E^rns and subsequently stimulated with poly(IC). The size (kDa) of the pre-stained protein ladder is indicated on the left. The uncropped gel is available in Supplementary Material Figure S3. (B): The amount of each E^rns protein required to reduce the Mx signal by 50% is reported for the proteins that yielded a signal reduction sufficiently strong to be analyzed (n = 3 ± SD). As BoGPV- and BuPV-E^rns were considerably stronger than the other proteins, their results are reported in the inset with a different scale on the y-axis and displaying the amount of these E^rns proteins to reduce the Mx signal by 95%. Proteins that did not display any detectable activity (i.e., E^rns of Gir-PG2, Gir-H138 and pronghorn antelope) are not shown. The activities of the various E^rns proteins in relation to the one of BVDV-Ncp7 is shown in Table 1.