Matrix protein mediated shutdown of host cell metabolism limits vesicular stomatitis virus-induced interferon-alpha responses to plasmacytoid dendritic cells

Upon infection with many different viruses, plasmacytoid dendritic cells (pDC) produce large amounts of type I interferon (IFN-alpha/beta). To address why upon vesicular stomatitis virus (VSV) infection pDC, but not conventional myeloid DC (mDC), are induced to produce IFN-alpha, pDC and mDC were differentiated from bone marrow cells (BM-DC). Upon VSV infection BM-pDC produced IFN-alpha, whereas BM-mDC did not. Notably, upon infection with VSV-M2, a VSV variant expressing a M51R mutant matrix (M) protein that showed a reduced sequestration of host cell metabolism, BM-pDC and BM-mDC mounted massive IFN-alpha responses. Both DC subsets showed comparable RNA levels of retinoic acid inducible gene-I (RIG-I) and Toll-like receptor (TLR) 7 and were able to respond upon triggering with double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) analogs. Moreover, upon VSV-M2 infection IFN-alpha production by both DC subsets was largely dependent on viral replication. Interestingly, upon virus infection BM-pDC, but not BM-mDC, up-regulated mRNA levels of nuclear export factors Nup96/98, probably reflecting cellular mechanisms to circumvent viral escape strategies. Collectively, these results indicated that cell types induced to produce IFN-alpha upon viral infection are not primarily defined by cellular receptor configurations but rather by complex virus/host cell interactions.     

Modulation of PKR activity in cells infected by bovine viral diarrhea virus

Bovine viral diarrhea virus is an important animal pathogen. The cytopathic and noncytopathic biotypes of the virus are associated with distinct pathologic entities. A striking difference between the two biotypes is viral RNA accumulation in infected cells. Viral dsRNA is thought to activate protein kinase PKR; an important mediator of innate immunity. In this study, we investigated PKR activation and its consequences in BVDV-infected cells. Infection with cp BVDV was found to induce PKR activation, eIF2alpha phosphorylation, translation inhibition and NF-kappaB activation. In contrast, PKR activity and eIF2alpha phosphorylation were not induced during infection with the ncp BVDV. In addition, cells infected with ncp BVDV showed no PKR phosphorylation in response to infection with the unrelated poliovirus whereas uninfected ncp BVDV cells when infected with poliovirus showed high levels of phosphorylated PKR. Cells infected with ncp BVDV failed to respond to synthetic dsRNA (poly I:C) treatment with NF-kappaB activation. However, the NF-kappaB response to bacterial lipopolysaccarides (LPS) was normal in these cells, suggesting a specific suppression of antiviral response signaling in ncp BVDV infected cells. These results indicate that ncp BVDV has evolved specific mechanisms to prevent activation of PKR and its antiviral effectors, most likely to facilitate the establishment and maintenance of persistent infection.     

In vitro-generated viral double-stranded RNA in contrast to polyinosinic:polycytidylic acid induces interferon-alpha in human plasmacytoid dendritic cells

Double-stranded RNA (dsRNA) arises in the cytoplasm during viral replication and was shown to participate in the interferon (IFN)-alpha induction process. Besides the intracellular recognition, released dsRNA from dying, infected cells can function as a pathogen-associated molecular pattern (PAMP) for the innate immune system. In the present study, in vitro-generated dsRNA fragments of genomic sequences of Newcastle disease virus were used to induce IFN-alpha release in human peripheral blood mononuclear cells (PBMC), in immature myeloid dendritic cells (mDC) and in immature plasmacytoid DC (pDC). The extracellular administration of dsRNA fragments but not the application of the corresponding single-stranded RNA (ssRNA) strands led to an IFN-alpha production in PBMC. The synthetic dsRNA analogue polyinosinic acid : polycytidylic acid [Poly(I : C)] could only stimulate IFN-alpha production in enriched mDC but not in pDC. In contrast, dsRNA fragments induced IFN-alpha only in pDC. Complexation of dsRNA fragments with transfection reagents increased the efficiency of IFN-alpha induction and commuted ssRNA molecules into IFN-alpha inducers. However, stimulation of in vitro-generated murine Toll-like receptor 7 (TLR7) knockout DC and human TLR-transfected HEK293 cells with dsRNA fragments gave no evidences for the involvement of pDC-specific TLR7 or TLR9 in the observed IFN-alpha induction.     

N of classical swine fever virus is an antagonist of double-stranded RNA-mediated apoptosis and IFN-α/β induction

Classical swine fever virus (CSFV) protects cells from double-stranded (ds) RNA-mediated apoptosis and IFN-alpha/beta induction. This phenotype is lost when CSFV lacks N(pro) (DeltaN(pro) CSFV). In the present study, we demonstrate that N(pro) counteracts dsRNA-mediated apoptosis and IFN-alpha/beta induction independently of other CSFV elements. For this purpose, we generated porcine SK-6 and PK-15 cell lines constitutively expressing N(pro) fused to the enhanced green fluorescent protein (EGFP). The survival of the SK6-EGFP-N(pro) cell line after polyinosinic polycytidylic acid [poly(IC)] treatment was comparable to that of CSFV-infected SK-6 cells and was significantly higher than the survival of the parent cell line. In PK-15 cells, the presence of EGFP-N(pro) prevented the DeltaN(pro) CSFV- and poly(IC)-mediated IFN-alpha/beta production. Importantly, N(pro) also inhibited IFN-alpha and IFN-beta promoter-driven luciferase expression in human cells and blocked IFN-alpha/beta induction mediated by Newcastle disease virus. This establishes a novel function for N(pro) in counteraction of the IFN-alpha/beta induction pathway.     

Analysis of the bovine viral diarrhea virus genome for possible cellular insertions.

Mucosal disease is the most severe disease resulting from bovine viral diarrhea virus (BVDV) infection in cattle. Two biotypes of BVDV may be isolated from animals with mucosal disease: cytopathic (cp) and noncytopathic (ncp). These "pairs" of cp/ncp viruses are often closely related and it has been suggested that the cp virus arises from a ncp virus by insertion of cellular RNA in the p125 region of the BVDV genome. We have used four pairs of cp/ncp BVDV isolated from cattle with mucosal disease, to examine the genomic sequence of the region of the genome coding for the nonstructural protein p125 (processed to p54/p80 in cp viruses) by PCR analysis and sequencing. We did not detect any cellular gene insertions in any of the four ncp viruses; however, we found a large duplication of the p80 gene and a ubiquitin gene insertion in three of the four cp isolates. Our results suggest that cellular RNA insertions in the p125 region may contribute significantly to the cytopathogenicity of BVDV. However, this does not appear to be the only mechanism of cytopathogenicity as we did not detect any insertions or duplications in one of the cp viruses. Comparison of the DNA sequence in the p80 region revealed greater homology within the "pairs" than to NADL, which lend further support to the hypothesis that a cp virus is originated from a ncp virus.     

Subset-dependent modulation of dendritic cell activity by circovirus type 2

Viral interactions with dendritic cells (DCs) have important consequences for immune defence function. Certain single-stranded DNA viruses that associate with a number of species, including humans and pigs, exhibit interesting characteristics in this context. Porcine circovirus type 2 (PCV2) can persist within myeloid DCs in the absence of virus replication. Internalization was observed with both conventional blood DCs and plasmacytoid DCs [natural interferon-producing cells (NIPCs)], as well as DC precursors. This PCV2-DC interaction neither induced nor inhibited DC differentiation. The maturation of myeloid DCs induced by a cocktail of interferon-alpha/tumour necrosis factor-alpha (IFN-alpha/TNF-alpha), and the ability to process and present antigen to T lymphocytes, remained intact in the presence of PCV2. The virus was clearly internalized by the DCs, a process noted with both mature and immature cells. This suggested a non-macropinocytic uptake, confirmed by an insensitivity to wortmannin but sensitivity to cytochalasin D, chlorpromazine and bafilomycin. Nevertheless, PCV2 was immunomodulatory, being effected through the reaction of NIPC to danger signals. When NIPCs responded to the CpG-oligonucleotide (CpG-ODN), their costimulatory function which induces myeloid DC maturation was clearly impaired by the presence of PCV2. This was caused by a PCV2-induced inhibition of the IFN-alpha and TNF-alpha normally produced following interaction with CpG-ODN. Thus, the immunomodulatory activity of PCV2 is mediated through the disruption of NIPC function. This would impair the maturation of associated myeloid DC and have major implications for the efficient recognition of viral and bacterial danger signals, favouring the establishment of infections additional to that of PCV2.     

Characterization of RNA Synthesis and Translation of Bovine Viral Diarrhea Virus (BVDV)

Full length and replicon genomes of various strains of bovine viral diarrhea virus (BVDV) have been characterized. Analysis of growth kinetics for a pair of cytopathogenic (cp) and noncytopathogenic (ncp) strains revealed that ncp strain synthesized viral RNA at much reduced level compared to the cp strain. Kinetics of translation and replication, the effects of bi-cistronic versus mono-cistronic genomes, and cis requirements for viral replication were also examined in a BVDV replicon DI9c. Importantly, our results suggest a tight regulation and a switch from translation to replication, and demonstrated the cis requirements of NS4B and NS5A in replication.     

Matrix protein mediated shutdown of host cell metabolism limits vesicular stomatitis virus-induced interferon-alpha responses to plasmacytoid dendritic cells

Upon infection with many different viruses, plasmacytoid dendritic cells (pDC) produce large amounts of type I interferon (IFN-/β). To address why upon vesicular stomatitis virus (VSV) infection pDC, but not conventional myeloid DC (mDC), are induced to produce IFN-, pDC and mDC were differentiated from bone marrow cells (BM-DC). Upon VSV infection BM-pDC produced IFN-, whereas BM-mDC did not. Notably, upon infection with VSV-M2, a VSV variant expressing a M51R mutant matrix (M) protein that showed a reduced sequestration of host cell metabolism, BM-pDC and BM-mDC mounted massive IFN- responses. Both DC subsets showed comparable RNA levels of retinoic acid inducible gene-I (RIG-I) and Toll-like receptor (TLR) 7 and were able to respond upon triggering with double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) analogs. Moreover, upon VSV-M2 infection IFN- production by both DC subsets was largely dependent on viral replication. Interestingly, upon virus infection BM-pDC, but not BM-mDC, up-regulated mRNA levels of nuclear export factors Nup96/98, probably reflecting cellular mechanisms to circumvent viral escape strategies. Collectively, these results indicated that cell types induced to produce IFN- upon viral infection are not primarily defined by cellular receptor configurations but rather by complex virus/host cell interactions.     

Modulation of double-stranded RNA-mediated gene induction by interferon in human umbilical vein endothelial cells.

Endothelial cells respond to double-stranded RNA (dsRNA) with expression of a number of important immunomodulatory and inflammatory response genes, including adhesion molecules, cytokines, and antiviral genes. Considerable differences are seen when genes are induced by dsRNA compared with cytokines. Much higher levels of mRNA for interleukin-6 (IL-6), 2',5'-oligoadenylate synthetase (2',5'-OAS), protein kinase (PKR), and interferon (IFN) regulatory factor-1 (IRF-1) result from incubation with dsRNA than with IL-1beta, tumor necrosis factor-alpha (TNF-alpha), or IFN-alpha, whereas the differences in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin mRNA expression in response to dsRNA, IL-1beta, and TNF-alpha are relatively minor. IFN-alpha priming enhances responsiveness of some, but not all, genes to dsRNA but not to IL-1beta, but the optimal time for pretreatment varies considerably among different dsRNA-responsive genes. Protein translation is reduced in human umbilical vein endothelial cells (HUVEC) in response to incubation with dsRNA, and this decrease is accentuated if cells are primed with IFN-alpha. Despite this decrease, IFN-alpha priming causes very high levels of IL-6 protein expression in response to dsRNA but not in response to IL-1beta or TNF-alpha. These studies demonstrate that priming with class I IFN can enhance the response to dsRNA through the heightened expression of genes that contribute to both the cellular response to viral infection and the host immunologic response.     

Use of double-stranded RNA templates by the tombusvirus replicase in vitro: Implications for the mechanism of plus-strand initiation

Plus-stranded RNA viruses replicate efficiently in infected hosts producing numerous copies of the viral RNA. One of the long-standing mysteries in RNA virus replication is the occurrence and possible role of the double-stranded (ds)RNA formed between minus- and plus-strands. Using the partially purified Cucumber necrosis virus (CNV) replicase from plants and the recombinant RNA-dependent RNA polymerase (RdRp) of Turnip crinkle virus (TCV), in this paper, we demonstrate that both CNV replicase and the related TCV RdRp can utilize dsRNA templates to produce viral plus-stranded RNA in vitro. Sequence and structure of the dsRNA around the plus-strand initiation site had a significant effect on initiation, suggesting that initiation on dsRNA templates is a rate-limiting step. In contrast, the CNV replicase could efficiently synthesize plus-strand RNA on partial dsRNAs that had the plus-strand initiation promoter "exposed", suggesting that the polymerase activity of CNV replicase is strong enough to unwind extended dsRNA regions in the template during RNA synthesis. Based on the in vitro data, we propose that dsRNA forms might have functional roles during tombus- and carmovirus replication and the AU-rich nature of the terminus could be important for opening the dsRNA structure around the plus-strand initiation promoter for tombus- and carmoviruses and possibly many other positive-strand RNA viruses.     

The interferon response circuit: induction and suppression by pathogenic viruses

Type I interferons (IFN-alpha/beta) are potent antiviral cytokines and modulators of the adaptive immune system. They are induced by viral infection or by double-stranded RNA (dsRNA), a by-product of viral replication, and lead to the production of a broad range of antiviral proteins and immunoactive cytokines. Viruses, in turn, have evolved multiple strategies to counter the IFN system which would otherwise stop virus growth early in infection. Here we discuss the current view on the balancing act between virus-induced IFN responses and the viral counterplayers.     

N(pro) of classical swine fever virus is an antagonist of double-stranded RNA-mediated apoptosis and IFN-alpha/beta induction

Classical swine fever virus (CSFV) protects cells from double-stranded (ds) RNA-mediated apoptosis and IFN-alpha/beta induction. This phenotype is lost when CSFV lacks N(pro) (DeltaN(pro) CSFV). In the present study, we demonstrate that N(pro) counteracts dsRNA-mediated apoptosis and IFN-alpha/beta induction independently of other CSFV elements. For this purpose, we generated porcine SK-6 and PK-15 cell lines constitutively expressing N(pro) fused to the enhanced green fluorescent protein (EGFP). The survival of the SK6-EGFP-N(pro) cell line after polyinosinic polycytidylic acid [poly(IC)] treatment was comparable to that of CSFV-infected SK-6 cells and was significantly higher than the survival of the parent cell line. In PK-15 cells, the presence of EGFP-N(pro) prevented the DeltaN(pro) CSFV- and poly(IC)-mediated IFN-alpha/beta production. Importantly, N(pro) also inhibited IFN-alpha and IFN-beta promoter-driven luciferase expression in human cells and blocked IFN-alpha/beta induction mediated by Newcastle disease virus. This establishes a novel function for N(pro) in counteraction of the IFN-alpha/beta induction pathway.     

The effect of different bovine viral diarrhea virus genotypes and biotypes on the metabolic activity and activation status of bovine peripheral blood mononuclear cells

The effects of cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhea virus (BVDV) on the cellular metabolic activity and activation status of bovine peripheral blood mononuclear cells (PBMC) were investigated. Cellular DNA and protein synthesis was determined by [3H]thymidine and [3H]valine incorporation, respectively, in phytohemagglutinin (PHA)-stimulated PBMC. All cp strains and most ncp BVDV strains significantly inhibited DNA synthesis in PHA-stimulated PBMC; however, only cp BVDV strains inhibited protein synthesis. A plaque assay and immunofluorescence test confirmed productive BVDV infection of PBMC. In addition, viral RNA synthesis was demonstrated in BVDV-infected PBMC by RT-PCR. The interleukin-2 receptor (IL-2R) was used as a marker for the activation status of BVDV-infected PBMC. The expression of IL-2R was preserved in virus-infected cells, even though DNA and protein synthesis was suppressed. These findings suggest a novel mechanism of virus-induced immune suppression in which BVDV inhibits basic metabolic activities of bovine PBMC. The activation signals, however, are maintained.     

Genetic recombination at different points in the Npro-coding region of bovine viral diarrhea viruses and the potentials to change their antigenicities and pathogenicities

Cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain KS86-1 cp was isolated from a cow persistently infected with non-cytopathogenic (ncp) BVDV strain KS86-ncp after development of mucosal disease by superinfection with cp BVDV strain Nose. cp BVDV strains 799cp and 839cp were also isolated from independent cattle that developed mucosal disease by superinfection with cp BVDV KS86-1cp. In the present study, genetic analysis revealed that the genes of cp BVDV strains 799cp and 839cp were chimeras between the genes of the persisting ncp BVDVs and that of superinfecting KS86-1cp. The genetic recombination that generates 799cp occurred between the identical points in the N(pro) gene region, whereas genetic recombination that generates 839cp occurred between different points in the N(pro) gene region. Both 799cp and 839cp were inherited Jiv gene of KS86-1cp strain and envelope protein genes of the persisting viruses. In addition, neutralization test disclosed that antigenicities of 799cp, 839cp, and KS86-1cp were also similar to each persisting virus. These findings indicate that exogenous cp BVDV containing insertion of Jiv gene in the 5 terminal region can induce genetic recombination with the original ncp BVDV at different points in the N(pro) gene region, and those viruses have high potential to change those antigenicities and pathogenicities by RNA recombination.     

Monocyte-derived human macrophages and peripheral blood mononuclear cells infected with ebola virus secrete MIP-1alpha and TNF-alpha and inhibit poly-IC-induced IFN-alpha in vitro

Ebola virus infection of humans is associated with high levels of circulating inflammatory chemokines and cytokines. We demonstrate that direct infection of human PBMC results in the induction of MCP-1, MIP-1alpha, RANTES, and TNF-alpha as early as 24 h p.i. in response to live virus. Monocyte-derived macrophages infected with live Ebola-virus secreted MIP-1alpha and TNF-alpha specifically while RANTES and MCP-1 were secreted by with both live or inactivated virus stimulation and do not require viral replication. Type I interferons (IFN-alpha and -beta), IL-1beta and IL-10, were not induced by Ebola virus. Furthermore, live virus infection of both PBMCs and monocytes-derived macrophages inhibited IFN-alpha induced by double-stranded RNA in vitro. These data provide the first direct evidence of a role for macrophages in the pathogenesis to Ebola virus and suggest that Ebola virus can inhibit cellular antiviral mechanisms mediated by type I interferons.     

Activation of cell signaling pathways is dependant on the biotype of bovine viral diarrhea viruses type 2

Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a worldwide distribution. Besides the segregation into two distinct species (BVDV1/BVDV2) two different biotypes, a cytopathic (cp) and a noncytopathic (ncp) biotype, are distinguished based on their behavior in epithelial cell cultures. One of the most serious forms of BVDV infection affecting immunocompetent animals of all ages is severe acute BVD (sa BVD) which is caused by highly virulent ncp BVDV2 strains. Previous studies revealed that these highly virulent ncp viruses cause cell death in a lymphoid cell line (BL3) which is not clearly associated with typical apoptotic changes (e.g. PARP cleavage) observed after infection with cp BVDV. To further characterize the underlying molecular mechanisms, we first analyzed the role of the mitochondria and caspases as key mediators of apoptosis. Compared to infection with cp BVDV2, infection with highly virulent ncp BVDV2 resulted in a delayed and less pronounced disruption of the mitochondrial transmembrane potential (DeltaPsi(m)) and a weaker activation of the caspase cascade. In contrast, infection with low virulence ncp BVDV2 showed no significant differences from the uninfected control cells. Since different pro- and anti-apoptotic cellular signaling pathways may become activated upon virus infection, we compared the effect of different BVDV2 strains on cellular signaling pathways in BL3 cells. Stress-mediated p38 MAPK phosphorylation was detected only in cells infected with cp BVDV2. Interestingly, infection with highly virulent ncp BVDV2 was found to influence the phosphoinositide 3-kinase (PI3K)-Akt pathway. This indicates that BL3 cells respond differently to infection with BVDV depending on virulence and biotype.     

Experimental induction of mucosal disease: consequences of superinfection of persistently infected cattle with different strains of cytopathogenic bovine viral diarrhea virus

Summary.  Mucosal disease (MD) can be induced in cattle persistently infected with noncytopathogenic bovine viral diarrhea virus (ncp BVD virus) by superinfecting them with antigenically related cytopathogenic (cp) BVD virus strains. While some of these animals succumb to early onset MD after 2 to 3 weeks post infectionem (p.i.), others only react by producing neutralizing antibodies against the cp BVD virus strain and may develop late onset MD after longer incubation periods. The aim of this study was to determine if an increasing degree of antigenic homology between the ncp and the superinfecting cp BVD virus strains as determined by their comparative reactivity with E2 glycoprotein specific monoclonal antibodies (mabs) increases the probability of inducing early or late onset MD, respectively. For this, each two of eight clinically healthy animals from the same herd and persistently infected with the same ncp BVD viruses were superinfected with four different cp BVD virus strains. As only two of these animals developed late onset MD, one animal from a different herd that developed early onset MD was included in the study. Besides clinical observation and testing for antibody production, virus isolation and characterization of the cp BVD virus isolates were performed. The results indicate that antigenic similarity as determined by comparative mab analysis alone is not sufficient to allow prediction of the outcome of the disease. Accepted November 28, 1997 Received July 11, 1997     

A 45-Nucleotide Insertion in the NS2 Gene is Responsible for the Cytopathogenicity of a Bovine Viral Diarrhoea Virus Strain

Cytopathogenicity (cp) markers have recently been investigated in the genomes of field isolates of bovine viral diarrhoea virus (BVDV). Most of the isolates originated from mucosal disease (MD) cases observed after vaccination with a live attenuated vaccine, termed here BVDV-X. The NS2-3 genes of these isolates and of the vaccine proved to be identical, including a 45-nucleotide (nt) viral insertion at nt position 4355. The insertion originated from the NS4B/5A junction region of the BVDV genome. Interestingly, in BVDV strain CP7 a 27-nt insertion originating from the NS2 is located exactly at the same position. Complete genome analysis of BVDV-X did not reveal further potential cp markers. Furthermore, expression studies indicated that the insertion promotes NS2-3 cleavage. In order to examine the possible role of the 45-nt insertion in viral cytopathogenicity in details, a full-length infectious cDNA clone of BVDV-X was generated, and bovine turbinate (BT) cells were transfected with RNA transcribed from the clone. The recovered virus, termed BVDV-XR, showed slight retardation in growth in comparison with the original BVDV-X, and induced cytopathogenic effect (CPE). Since the natural non-cytopathogenic (ncp) counterpart of the vaccine virus was not available, an insertion-negative mutant cDNA clone was generated from BVDV-XR by PCR-directed mutagenesis. The recovered virus, termed BVDV-XR-INS-, showed the same growth characteristics as its cp counterpart BVDV-XR, but caused no CPE. These findings provide a direct proof that the 45-nt insertion at position 4355 has a basic role in the cytopathogenic character of this BVDV strain.