Current progress on innate immune evasion mediated by Npro protein of pestiviruses.

Interferon (IFN), the most effective antiviral cytokine, is involved in innate and adaptive immune responses and is essential to the host defense against virus invasion. Once the host was infected by pathogens, the pathogen-associated molecular patterns (PAMPs) were recognized by the host pattern recognition receptors (PRRs), which activates interferon regulatory transcription factors (IRFs) and nuclear factor-kappa B (NF-κB) signal transduction pathway to induce IFN expression. Pathogens have acquired many strategies to escape the IFN-mediated antiviral immune response. Pestiviruses cause massive economic losses in the livestock industry worldwide every year. The immune escape strategies acquired by pestiviruses during evolution are among the major difficulties in its control. Previous experiments indicated that Erns, as an envelope glycoprotein unique to pestiviruses with RNase activity, could cleave viral ss- and dsRNAs, therefore inhibiting the host IFN production induced by viral ss- and dsRNAs. In contrast, Npro, the other envelope glycoprotein unique to pestiviruses, mainly stimulates the degradation of transcription factor IRF-3 to confront the IFN response. This review mainly summarized the current progress on mechanisms mediated by Npro of pestiviruses to antagonize IFN production.

Pestiviruses infection: Interferon-virus mutual regulation.

Pestiviruses are a class of viruses that in some cases can cause persistent infection of the host, thus posing a threat to the livestock industry. Interferons (IFNs) are a group of secreted proteins that play a crucial role in antiviral defense. In this review, on the one hand, we elaborate on how pestiviruses are recognized by the host retinoic acid-inducible gene-I (RIG-I), melanoma-differentiation-associated protein 5 (MDA5), and Toll-like receptor 3 (TLR3) proteins to induce the synthesis of IFNs. On the other hand, we focus on reviewing how pestiviruses antagonize the production of IFNs utilizing various strategies mediated by self-encoded proteins, such as the structural envelope protein (Erns) and non-structural protein (Npro). Hence, the IFN signal transduction pathway induced by pestiviruses infection and the process of pestiviruses blockade on the production of IFNs intertwines into an intricate regulatory network. By reviewing the interaction between IFN and pestiviruses (based on studies on BVDV and CSFV), we expect to provide a theoretical basis and reference for a better understanding of the mechanisms of induction and evasion of the innate immune response during infection with these viruses.

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

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, Npro and Erns, that pestiviruses evolved to regulate the host's innate immune response. In addition to its function as a structural envelope glycoprotein, Erns is also released in the extracellular space, where it is endocytosed by neighboring cells. As an endoribonuclease, Erns 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 Erns 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 Erns 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.

ADAM17 Is an Essential Factor for the Infection of Bovine Cells with Pestiviruses.

The entry of BVDV into bovine cells was studied using CRIB cells (cells resistant to infection with bovine viral diarrhea virus [BVDV]) that have evolved from MDBK cells by a spontaneous loss of susceptibility to BVDV. Recently, larger genetic deletions were reported but no correlation of the affected genes and the resistance to BVDV infection could be established. The metalloprotease ADAM17 was reported as an essential attachment factor for the related classical swine fever virus (CSFV). To assess whether ADAM17 might be involved in the resistance of CRIB-1 cells to pestiviruses, we analyzed its expression in CRIB-1 and MDBK cells. While ADAM17 protein was detectable in MBDK cells, it was absent from CRIB-1 cells. No functional full-length ADAM17 mRNA could be detected in CRIB cells and genetic analysis revealed the presence of two defective alleles. Transcomplementation of functional ADAM17 derived from MDBK cells in CRIB-1 cells resulted in a nearly complete reversion of their resistance to pestiviral infection. Our results demonstrate that ADAM17 is a key cellular factor for the pestivirus resistance of CRIB-1 cells and establishes its essential role for a broader range of pestiviruses.

A novel NS3/4A protease dependent cleavage site within pestiviral NS2.

Pestiviruses like bovine viral diarrhoea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae. A special feature of the Flaviviridae is the importance of nonstructural (NS) proteins for both genome replication and virion morphogenesis. The NS2-3-4A region and its regulated processing by the NS2 autoprotease and the NS3/4A protease plays a central role in the pestiviral life cycle. We report the identification and characterization of a novel internal cleavage in BVDV NS2, which is mediated by the NS3/4A protease. Further mapping using the NS2 of BVDV-1 strain NCP7 showed that cleavage occurs between L188 and G189. This cleavage site represents a novel sequence motif recognized by the NS3/4A protease and is conserved between the pestivirus species A, B and D. Inhibition of this internal NS2 cleavage by mutating the cleavage site did not cause obvious effects on RNA replication or virion morphogenesis in cultured cell lines. Accordingly, this novel internal NS2 cleavage adds an additional layer to the already complex polyprotein processing of Pestiviruses and might further extend the repertoires of the multifunctional NS2. However, unravelling of the functional relevance of this novel processing event in NS2, therefore, awaits future in vivo studies.

The Erns Carboxyterminus: Much More Than a Membrane Anchor.

Pestiviruses express the unique essential envelope protein Erns, which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of Erns is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host's innate immune response to pestivirus infection. The unusual membrane anchor of Erns raises questions with regard to proteolytic processing of the viral polyprotein at the Erns carboxy-terminus. Moreover, the membrane anchor is crucial for establishing the critical equilibrium between retention and secretion and ensures intracellular accumulation of the protein at the site of virus budding so that it is available to serve both as structural component of the virion and factor controlling host immune reactions. In the present manuscript, we summarize published as well as new data on the molecular features of Erns including aspects of its interplay with the other two envelope proteins with a special focus on the biochemistry of the Erns membrane anchor.

Charged Residues in the Membrane Anchor of the Pestiviral Erns Protein Are Important for Processing and Secretion of Erns and Recovery of Infectious Viruses.

The pestivirus envelope protein Erns is anchored in membranes via a long amphipathic helix. Despite the unusual membrane topology of the Erns membrane anchor, it is cleaved from the following glycoprotein E1 by cellular signal peptidase. This was proposed to be enabled by a salt bridge-stabilized hairpin structure (so-called charge zipper) formed by conserved charged residues in the membrane anchor. We show here that the exchange of one or several of these charged residues reduces processing at the Erns carboxy-terminus to a variable extend, but reciprocal mutations restoring the possibility to form salt bridges did not necessarily restore processing efficiency. When introduced into an Erns-only expression construct, these mutations enhanced the naturally occurring Erns secretion significantly, but again to varying extents that did not correlate with the number of possible salt bridges. Equivalent effects on both processing and secretion were also observed when the proteins were expressed in avian cells, which points at phylogenetic conservation of the underlying principles. In the viral genome, some of the mutations prevented recovery of infectious viruses or immediately (pseudo)reverted, while others were stable and neutral with regard to virus growth.

Downstream Sequences Control the Processing of the Pestivirus Erns-E1 Precursor.

Like other enveloped viruses, pestiviruses employ cellular proteases for processing of their structural proteins. While typical signal peptidase cleavage motifs are present at the carboxy terminus of the signal sequence preceding Erns and the E1/E2 and E2/P7 sites, the Erns-E1 precursor is cleaved by signal peptidase at a highly unusual structure, in which the transmembrane sequence upstream of the cleavage site is replaced by an amphipathic helix. As shown before, the integrity of the amphipathic helix is crucial for efficient processing. The data presented here demonstrate that the E1 sequence downstream of this cleavage site is also important for the cleavage. Carboxy-terminal truncation of the E1 moiety as well as internal deletions in E1 reduced the cleavage efficiency to less than 30% of the wild-type (wt) level. Moreover, the C-terminal truncation by more than 30 amino acids resulted in strong secretion of the uncleaved fusion proteins. The reduced processing and increased secretion were even observed when 10 to 5 amino-terminal residues of E1 were left, whereas extensions by 1 or 3 E1 residues resulted in reduced processing but no significantly increased secretion. In contrast to the E1 sequences, a 10-amino-acid c-myc tag fused to the Erns C terminus had only marginal effect on secretion but was also not processed efficiently. Mutation of the von Heijne sequence upstream of E2 not only blocked the cleavage between E1 and E2 but also prevented the processing between Erns and E2. Thus, processing at the Erns-E1 site is a highly regulated process.IMPORTANCE Cellular signal peptidase (SPase) cleavage represents an important step in maturation of viral envelope proteins. Fine tuning of this system allows for establishment of concerted folding and processing processes in different enveloped viruses. We report here on SPase processing of the Erns-E1-E2 glycoprotein precursor of pestiviruses. Erns-E1 cleavage is delayed and only executed efficiently when the complete E1 sequence is present. C-terminal truncation of the Erns-E1 precursor impairs processing and leads to significant secretion of the protein. The latter is not detected when internal deletions preserving the E1 carboxy terminus are introduced, but also these constructs show impaired processing. Moreover, Erns-E1 is only processed after cleavage at the E1/E2 site. Thus, processing of the pestiviral glycoprotein precursor by SPase is done in an ordered way and depends on the integrity of the proteins for efficient cleavage. The functional importance of this processing scheme is discussed in the paper.

Delayed by Design: Role of Suboptimal Signal Peptidase Processing of Viral Structural Protein Precursors in Flaviviridae Virus Assembly.

The Flaviviridae virus family is classified into four different genera, including flavivirus, hepacivirus, pegivirus, and pestivirus, which cause significant morbidity and mortality in humans and other mammals, including ruminants and pigs. These are enveloped, single-stranded RNA viruses sharing a similar genome organization and replication scheme with certain unique features that differentiate them. All viruses in this family express a single polyprotein that encodes structural and nonstructural proteins at the N- and C-terminal regions, respectively. In general, the host signal peptidase cleaves the structural protein junction sites, while virus-encoded proteases process the nonstructural polyprotein region. It is known that signal peptidase processing is a rapid, co-translational event. Interestingly, certain signal peptidase processing site(s) in different Flaviviridae viral structural protein precursors display suboptimal cleavage kinetics. This review focuses on the recent progress regarding the Flaviviridae virus genus-specific mechanisms to downregulate signal peptidase-mediated processing at particular viral polyprotein junction sites and the role of delayed processing at these sites in infectious virus particle assembly.

Determination of Critical Requirements for Classical Swine Fever Virus NS2-3-Independent Virion Formation.

For members of the Flaviviridae, it is known that, besides the structural proteins, nonstructural (NS) proteins also play a critical role in virion formation. Pestiviruses, such as bovine viral diarrhea virus (BVDV), rely on uncleaved NS2-3 for virion formation, while its cleavage product, NS3, is selectively active in RNA replication. This dogma was recently challenged by the selection of gain-of-function mutations in NS2 and NS3 which allowed virion formation in the absence of uncleaved NS2-3 in BVDV type 1 (BVDV-1) variants encoding either a ubiquitin (Ubi) (NS2-Ubi-NS3) or an internal ribosome entry site (IRES) (NS2-IRES-NS3) between NS2 and NS3. To determine whether the ability to adapt to NS2-3-independent virion morphogenesis is conserved among pestiviruses, we studied the corresponding NS2 and NS3 mutations (2/T444-V and 3/M132-A) in classical swine fever virus (CSFV). We observed that these mutations were capable of restoring low-level NS2-3-independent virion formation only for CSFV NS2-Ubi-NS3. Interestingly, a second NS2 mutation (V439-D), identified by selection, was essential for high-titer virion production. Similar to previous findings for BVDV-1, these mutations in NS2 and NS3 allowed for low-titer virion production only in CSFV NS2-IRES-NS3. For efficient virion morphogenesis, additional exchanges in NS4A (A48-T) and NS5B (D280-G) were required, indicating that these proteins cooperate in NS2-3-independent virion formation. Interestingly, both NS5B mutations, selected independently for NS2-IRES-NS3 variants of BVDV-1 and CSFV, are located in the fingertip region of the viral RNA-dependent RNA polymerase, classifying this structural element as a novel determinant for pestiviral NS2-3-independent virion formation. Together, these findings will stimulate further mechanistic studies on the genome packaging of pestiviruses.IMPORTANCE For Flaviviridae members, the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses, the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain of function. However, so far, the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant has not been clarified. To unravel the course of genome packaging, the additional sets of mutations obtained for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.

Novel Pestivirus Species in Pigs, Austria, 2015.

A novel pestivirus species was discovered in a piglet-producing farm in Austria during virologic examinations of congenital tremor cases. The emergence of this novel pestivirus species, provisionally termed Linda virus, in domestic pigs may have implications for classical swine fever virus surveillance and porcine health management.

Lipid Binding of the Amphipathic Helix Serving as Membrane Anchor of Pestivirus Glycoprotein Erns.

Pestiviruses express a peculiar protein named Erns representing envelope glycoprotein and RNase, which is important for control of the innate immune response and persistent infection. The latter functions are connected with secretion of a certain amount of Erns from the infected cell. Retention/secretion of Erns is most likely controlled by its unusual membrane anchor, a long amphipathic helix attached in plane to the membrane. Here we present results of experiments conducted with a lipid vesicle sedimentation assay able to separate lipid-bound from unbound protein dissolved in the water phase. Using this technique we show that a protein composed of tag sequences and the carboxyterminal 65 residues of Erns binds specifically to membrane vesicles with a clear preference for compositions containing negatively charged lipids. Mutations disturbing the helical folding and/or amphipathic character of the anchor as well as diverse truncations and exchange of amino acids important for intracellular retention of Erns had no or only small effects on the proteins membrane binding. This result contrasts the dramatically increased secretion rates observed for Erns proteins with equivalent mutations within cells. Accordingly, the ratio of secreted versus cell retained Erns is not determined by the lipid affinity of the membrane anchor.

Structures and Functions of Pestivirus Glycoproteins: Not Simply Surface Matters.

Pestiviruses, which include economically important animal pathogens such as bovine viral diarrhea virus and classical swine fever virus, possess three envelope glycoproteins, namely Erns, E1, and E2. This article discusses the structures and functions of these glycoproteins and their effects on viral pathogenicity in cells in culture and in animal hosts. E2 is the most important structural protein as it interacts with cell surface receptors that determine cell tropism and induces neutralizing antibody and cytotoxic T-lymphocyte responses. All three glycoproteins are involved in virus attachment and entry into target cells. E1-E2 heterodimers are essential for viral entry and infectivity. Erns is unique because it possesses intrinsic ribonuclease (RNase) activity that can inhibit the production of type I interferons and assist in the development of persistent infections. These glycoproteins are localized to the virion surface; however, variations in amino acids and antigenic structures, disulfide bond formation, glycosylation, and RNase activity can ultimately affect the virulence of pestiviruses in animals. Along with mutations that are driven by selection pressure, antigenic differences in glycoproteins influence the efficacy of vaccines and determine the appropriateness of the vaccines that are currently being used in the field.

Crystal structure of the pestivirus envelope glycoprotein E(rns) and mechanistic analysis of its ribonuclease activity.

Pestiviruses, which belong to the Flaviviridae family of RNA viruses, are important agents of veterinary diseases causing substantial economical losses in animal farming worldwide. Pestivirus particles display three envelope glycoproteins at their surface: E(rns), E1, and E2. We report here the crystal structure of the catalytic domain of E(rns), the ribonucleolytic activity of which is believed to counteract the innate immunity of the host. The structure reveals a three-dimensional fold corresponding to T2 ribonucleases from plants and fungi. Cocrystallization experiments with mono- and oligonucleotides revealed the structural basis for substrate recognition at two binding sites previously identified for T2 RNases. A detailed analysis of poly-U cleavage products using (31)P-NMR and size exclusion chromatography, together with molecular docking studies, provides a comprehensive mechanistic picture of E(rns) activity on its substrates and reveals the presence of at least one additional nucleotide binding site.

Complementation studies with the novel "Bungowannah" virus provide new insights in the compatibility of pestivirus proteins.

In recent years several atypical pestiviruses have been described. Bungowannah virus is the most divergent virus in this group. Therefore, heterologous complementation was used to clarify the phylogenetic relationship and to analyze the exchangeability of genome regions encoding structural proteins. Using a BVDV type 1 backbone, chimeric constructs with substituted envelope proteins E(rns), E1 and E2, were investigated. While all constructs replicated autonomously, infectious high titer chimeric virus could only be observed after exchanging the complete E1-E2 encoding region. The complementation of E1 and E2 alone resulted only in replicons. Complementation of BVDV-E(rns) was only efficient if Bungowannah virus-E(rns) was expressed from a bicistronic construct. Our data provide new insights in the compatibility of pestivirus proteins and demonstrate that heterologous complementation could be useful to characterize new pestiviruses.

The pestivirus glycoprotein Erns is anchored in plane in the membrane via an amphipathic helix.

E(rns) is a structural glycoprotein of pestiviruses found to be attached to the virion and to membranes within infected cells via its COOH terminus, although it lacks a hydrophobic anchor sequence. The COOH-terminal sequence was hypothesized to fold into an amphipathic alpha-helix. Alanine insertion scanning revealed that the ability of the E(rns) COOH terminus to bind membranes is considerably reduced by the insertion of a single amino acid at a wide variety of positions. Mutations decreasing the hydrophobicity of the apolar face of the putative helix led to reduction of membrane association. Proteinase K protection assays showed that E(rns) translated in vitro in the presence of microsomal membranes was protected, whereas a mutant with an artificial transmembrane region and a short cytosolic tag was shortened by the protease treatment. A tag fused to the COOH terminus of wild type E(rns) was not accessible for antibodies within digitonin-permeabilized cells, but the variant with the tag located downstream of the artificial transmembrane region was detected under the same conditions. These results are in accordance with the model that the COOH-terminal membrane anchor of E(rns) represents an amphipathic helix embedded in plane into the membrane. The integrity of the membrane anchor was found to be important for recovery of infectious virus.

Method for detection of extraneous active bovine viral diarrhoea virus and classical swine fever virus in animal viral vaccines by RT-PCR, which amplify negative-strand viral RNA in infected cells.

An oligonucleotide sense primer, Pst324alpha, was designed and used for synthesizing cDNA from negative-strand viral RNA in infected cells and used for rapid detection of active extraneous bovine viral diarrhoea virus (BVDV) and classical swine fever virus (CSFV) in animal viral vaccines by culturing a sample in cells followed by reverse transcriptase-polymerase chain reaction (RT-PCR). Active and inactivated viruses of BVDV No. 12-43 strain and CSFV GPE(-)strain were inoculated to bovine testicle and swine testicle cells for incubation. After the complete extraction of RNA from these cells, cDNA was synthesized using Pst324alpha, and PCR was carried out using primers 324 and 326 (novel RT-PCR). Amplification of novel RT-PCR products was observed in cells infected with active viruses but not in cells inoculated with inactivated viruses, inoculums and cultured media after incubation. This novel RT-PCR was able to amplify viral sequences from cells infected with only a small number of infectious particles (less than 10 TCID50) at three days postinoculation and was as sensitive as the general RT-PCR using a random primer and the interference and immunofluorescent antibody (FA) methods. The results of experiments on detection of BVDV RNA from vaccines contaminated with active and inactivated BVDV showed that the sensitivity of the novel RT-PCR was almost the same as the sensitivities of the interference and FA methods. These results suggest that the novel RT-PCR is easier and more rapid than the interference method for detection of active BVDV and that the novel RT-PCR is a reliable means for detection of active extraneous BVDV for quality control of animal vaccines.

Translocation activity of C-terminal domain of pestivirus Erns and ribotoxin L3 loop.

The pestivirus envelope glycoprotein E(rns) has RNase activity and therefore was suspected to enter cells to cleave RNA. The protein contains an RNase domain with a C-terminal extension, which shows homology with a membrane-active peptide. The modular architecture and the C-terminal homology suggested that the C terminus could be responsible for the presumed translocation. Peptides corresponding to the C-terminal domain of E(rns) and also the homologous L3 loop of ribotoxin II were indeed able to translocate across the eukaryotic cell membrane and were targeted to the nucleoli. The entire E(rns) protein was also able to translocate into the cell. Furthermore, other labeled proteins and even active enzymes could be transported inside the cell when they were attached to the C-terminal E(rns) peptide. Translocation was energy-independent and not mediated by a protein receptor. The peptides showed no specificity for cell type or species.

Imino sugars inhibit the formation and secretion of bovine viral diarrhea virus, a pestivirus model of hepatitis C virus: implications for the development of broad spectrum anti-hepatitis virus agents.

One function of N-linked glycans is to assist in the folding of glycoproteins by mediating interactions of the lectin-like chaperone proteins calnexin and calreticulin with nascent glycoproteins. These interactions can be prevented by inhibitors of the alpha-glucosidases, such as N-butyl-deoxynojirimycin (NB-DNJ) and N-nonyl-DNJ (NN-DNJ), and this causes some proteins to be misfolded and retained within the endoplasmic reticulum (ER). We have shown previously that the NN-DNJ-induced misfolding of one of the hepatitis B virus (HBV) envelope glycoproteins prevents the formation and secretion of virus in vitro and that this inhibitor alters glycosylation and reduces the viral levels in an animal model of chronic HBV infection. This led us to investigate the effect of glucosidase inhibitors on another ER-budding virus, bovine viral diarrhea virus, a tissue culture surrogate of human hepatitis C virus (HCV). Here we show that in MDBK cells alpha-glucosidase inhibitors prevented the formation and secretion of infectious bovine viral diarrhea virus. Data also are presented showing that NN-DNJ, compared with NB-DNJ, exhibits a prolonged retention in liver in vivo. Because viral secretion is selectively hypersensitive to glucosidase inhibition relative to the secretion of cellular proteins, the possibility that glucosidase inhibitors could be used as broad-based antiviral hepatitis agents is discussed. A single drug against HBV, HCV, and, possibly, HDV, which together chronically infect more than 400 million people worldwide, would be of great therapeutic value.