The mengovirus leader protein suppresses alpha/beta interferon production by inhibition of the iron/ferritin-mediated activation of NF-kappa B

In our studies on the biological function of the mengovirus leader protein, we identified a casein kinase II (CK-2) phosphorylation site in the protein. Here we report that the mengovirus leader protein can be phosphorylated by CK-2 in vitro. Expression of a recombinant leader protein in which the consensus CK-2 sequence around threonine 47 was disturbed resulted in a mutant protein that could no longer be phosphorylated. The CK-2 consensus sequence was modified by site-directed mutagenesis and subsequently introduced into a mengovirus cDNA clone to investigate the effect of the phosphorylation of the leader protein on virus replication and on the host cell response. Modifications by which the CK-2 consensus sequence was disturbed resulted in mutant viruses with reduced growth kinetics. We demonstrated that the integrity of the CK-2 phosphorylation site of the mengovirus leader protein was specifically related to the suppression of NF-kappa B activation and subsequent suppression of alpha/beta interferon production in infected cells. We also found that the integrity of the CK-2 phosphorylation site of the leader protein coincided with an increase of ferritin expression in the infected cell. These data indicate that the leader protein suppresses the iron-mediated activation of NF-kappa B and thereby inhibits alpha/beta interferon expression in the infected cell.     

The leader peptide of Theiler's murine encephalomyelitis virus is a zinc-binding protein.

The leader (L) peptide is located in the amino-terminal part of the polyprotein of members of the Cardiovirus (which includes Theiler's murine encephalomyelitis virus) and Aphthovirus genera of picornaviruses. Although the function of L is unknown, strain DA of Theiler's murine encephalomyelitis virus with a mutation of L produces a cell-specific restricted infection. We now report that the DA L peptide is a metalloprotein and that zinc binds to a Cys-His motif that is conserved among cardioviruses.     

Human cytomegalovirus immediate-early 2 gene expression blocks virus-induced beta interferon production

The effect of human cytomegalovirus (HCMV) gene expression on beta interferon (IFN-beta) expression was examined. We demonstrate that the HCMV immediate-early 2 (IE2) gene product IE86 can effectively block the induction of IFN-beta during HCMV infection. IE86 also efficiently blocked the induction of IFN-beta following Sendai virus infection, demonstrating that IE86's ability to block induction of IFN-beta is not limited to HCMV infection, identifying IE2 as an IFN-beta antagonist.     

The amino-terminal domain of bovine viral diarrhea virus Npro protein is necessary for alpha/beta interferon antagonism

The alpha/beta interferon (IFN-alpha/beta) system is the first line of defense against viral infection and a critical link between the innate and adaptive immune responses. IFN-alpha/beta secretion is the hallmark of cellular responses to acute RNA virus infections. As part of their survival strategy, many viruses have evolved mechanisms to counteract the host IFN-alpha/beta response. Bovine viral diarrhea virus (BVDV) (genus Pestivirus) was reported to trigger interferon production in infected cultured cells under certain circumstances or to suppress it under others. Our studies with various cultured fibroblasts and epithelial bovine cells indicated that cytopathic (cp) BVDV induces IFN-alpha/beta very inefficiently. Using a set of engineered cp BVDVs expressing mutant Npro and appropriate controls, we found that the IFN-alpha/beta response to infection was dependent on Npro expression and independent of viral replication efficiency. In order to investigate whether the protease activity of Npro is required for IFN-alpha/beta antagonism, we engineered Npro mutants lacking protease activity by replacement of amino acid E22, H49, or C69. We found that E22 and H49 substitutions abolished the ability of Npro to suppress IFN, whereas C69 had no effect, suggesting that the structural integrity of the N terminus of Npro was more important than its catalytic activity for IFN-alpha/beta suppression. A catalytically active mutant with a change at a conserved Npro region near the N terminus (L8P) in both BVDV biotypes did not antagonize IFN-alpha/beta production, confirming its involvement in this process. Taken together, these results not only provide direct evidence for the role of Npro in blocking IFN-alpha/beta induction, but also implicate the amino-terminal domain of the protein in this function.     

Resistance of lymphocytic choriomeningitis virus to alpha/beta interferon and to gamma interferon.

The susceptibility to alpha/beta interferon (IFN-alpha/beta) or to gamma interferon (IFN-gamma) of various lymphocytic choriomeningitis virus (LCMV) strains was evaluated in C57BL/6 mice and in various cell lines. Anti-IFN-gamma treatment in vivo revealed that the LCMV strains Armstrong, Aggressive, and WE were most susceptible to IFN-gamma whereas Traub, Cl 13-Armstrong, and Docile were resistant. The same pattern of susceptibility to recombinant IFN-gamma was observed in vitro. In vivo treatment with anti-IFN-alpha/beta showed a sizeable increase in replication of Aggressive, Armstrong, and WE; effects were less pronounced for Docile, Cl 13-Armstrong, or Traub. Correspondingly, WE, Armstrong, and Aggressive were all relatively sensitive to purified IFN-alpha/beta in vitro, and Cl 13-Armstrong, Docile, and Traub were more resistant. Overall, there was a good correlation between the capacity of LCMV strains to establish a persistent infection in adult immunocompetent mice and their relative resistance to IFN-gamma and IFN-alpha/beta.     

Inhibition of L-deleted foot-and-mouth disease virus replication by alpha/beta interferon involves double-stranded RNA-dependent protein kinase

We previously demonstrated that the ability of foot-and-mouth disease virus (FMDV) to form plaques in cell culture is associated with the suppression of alpha/beta interferon (IFN-alpha/beta). In the present study, we used Escherichia coli-expressed porcine and bovine IFN-alpha or -beta individually to demonstrate that each was equally effective in inhibiting FMDV replication. The block in FMDV replication appeared to be at the level of protein translation, suggesting a role for double-stranded RNA-dependent protein kinase (PKR). In support of these findings, treatment of porcine and bovine cells with 2-aminopurine, an inhibitor of PKR, increased the yield of virus 8.8- and 11.2-fold, respectively, compared to that in untreated infected cells. In addition, results of FMDV infection in mouse embryonic fibroblast cells derived from gene knockout mice lacking the gene for RNase L(-/-) or PKR(-/-) or both indicated an important role for PKR in the inhibition of FMDV replication.     

Cyclosporin A inhibits the production of gamma interferon (IFN gamma), but does not inhibit production of virus-induced IFN alpha/beta

The effect of cyclosporin A (CsA) on the production of gamma interferon (IFN gamma) versus IFN alpha/beta was studied using mouse and human lymphocytes and fibroblasts. Spleen cells from C57Bl/6 mice produced low but significant levels (40-60 U/ml) of IFN gamma after 2 to 3 days of culture with irradiated DBA spleen cells. The addition of CsA at concentrations as low as 0.1 microgram/ml completely inhibited (less than 10 U/ml) IFN gamma production in these cultures. High levels of IFN gamma (170-1200 U/ml) were produced when either C57Bl/6 spleen cells or Ficoll-Hypaque-purified human peripheral blood lymphocytes (PBL) were cultured with the T-cell mitogen staphylococcal enterotoxin A (SEA). The addition of CsA (0.1 microgram/ml) to these cultures also completely inhibited (less than 10 U/ml) IFN gamma production. This inhibition was shown not to be due to a change in the kinetics of IFN gamma production or to a change in the amount of SEA required for stimulation. IFN gamma production in SEA-stimulated mouse spleen cells was inhibited at 3 days of culture even when CsA was added at 24 or 48 hr postculture initiation. Thus, CsA inhibits IFN gamma production even when early events associated with lymphocyte activation have been allowed to take place. In contrast to IFN gamma production, IFN alpha/beta production by Newcastle disease virus (NDV)-infected mouse and human lymphocytes or fibroblasts was not inhibited by the addition of CsA (1 microgram/ml). CsA also did not block the action of IFN gamma or IFN alpha/beta since addition of CsA (1 microgram/ml) to reference IFN standards had no effect on their antiviral activity. Thus, CsA inhibits the production of IFN gamma by T cells but appears to have no effect on the production of IFN alpha/beta by virus-infected cells or on the antiviral action of already produced IFN gamma and IFN alpha/beta.     

Interleukin 2 induces interferon alpha/beta production in mouse bone marrow cells

We have previously reported that mouse bone marrow (BM) cells stimulated with alloantigen produce cytotoxic effector T-cell activity and produce interferon (IFN-)alpha/beta. In this report we show evidence suggesting that interleukin 2 (IL-2) may play a role in this IFN-alpha/beta production by alloantigen-stimulated BM cells. Alloantigen-induced IFN production by bone marrow cells was completely inhibited when cultures were supplemented with antisera to IL-2. Cell-free supernatants obtained at 2 days from cultures containing C57BL/6 BM cells and irradiated DBA/2J spleen cells were also shown to contain low levels of IL-2 activity and induced significant IFN production in fresh BM cells. Different IL-2 preparations were tested for their ability to induce IFN-alpha/beta production in mouse BM cells. Mouse BM cells cultured with recombinant human IL-2 or highly purified mouse IL-2 produced high levels of IFN-alpha/beta activity after 2-3 days of culture with significant IFN activity being detected as early as 24 hr of culture. IL-2-induced IFN-alpha/beta production was partially resistant to irradiation. In contrast, irradiated (2000 rad) bone marrow cells failed to produce any IFN when cultured with alloantigen in the absence of IL-2. T-cell-depleted BM cells or BM cells obtained from C57BL/10 nude mice produced high levels of IFN-alpha/beta following stimulation with IL-2. In addition, bone marrow cells depleted of Ia+, Qa 5+, or Asialo GM+1 cells produced IFN in response to IL-2. Thus, neither T cells nor NK cells are required for IL-2-induced IFN-alpha/beta production by BM cells. The action of IL-2 on bone marrow cells to induce IFN production was mediated by the classical IL-2 receptor, since monoclonal antibodies to the IL-2 receptor present on T cells blocked this response and since bone marrow cells depleted of IL-2 receptor-bearing cells failed to produce IFN when cultured with IL-2. These results suggest that non-T cells resident in the BM have receptors for IL-2 and can produce IFN-alpha/beta upon stimulation by IL-2. Since IFN has been shown to affect different aspects of hematopoiesis, the production of IFN by BM cells stimulated by IL-2 may be important in the control of hematopoiesis. In addition, IL-2-induced IFN production may play a role in graft-versus-host disease.     

Synergistic inhibition of pseudorabies virus replication by porcine alpha/beta interferon and gamma interferon in vitro

Interferon (IFN) is crucial for initiating the innate immune response and for the generation of the adaptive response. IFN, in most species, comprises IFN-alpha (IFN-alpha), IFN-beta (IFN-beta) and IFN-gamma (IFN-gamma). In this study, we compared the capacity of porcine IFN-alpha, -beta and -gamma, or a combination of them, to protect IBRS-2 cells (porcine kidney cells) from infection with pseudorabies virus (PRV). The results demonstrated that porcine IFN-beta (PoIFN-beta) was the most efficient of the three IFNs in conferring resistance PRV infection; 100 U/mL PoIFN-beta inhibited PRV plaque formation 5.3-fold. Compared with PoIFN-beta, porcine IFN-gamma (PoIFN-gamma) was less capable of inhibiting PRV plaque formation (3.3-fold inhibition). Porcine IFN-alpha (PoIFN-alpha) had the least capability of the three PoIFNs, and inhibited PRV plaque formation only 1.26-fold. The inhibitory capacity increased to only 2.3-fold with a treatment of 12,800 U/mL PoIFN-alpha. A combination of PoIFN-gamma and PoIFN-alpha or PoIFN-beta inhibited PRV plaque formation 12.8-fold or 100-fold, respectively. Treatment of IBRS-2 cells with PoIFN-alpha/beta and PoIFN-gamma inhibited PRV replication 29- or 146-fold. Additionally, real-time PCR analyses of the PRV immediate early (IE) gene revealed that IE mRNA expression was profoundly decreased in cells stimulated with PoIFN-alpha/beta and PoIFN-gamma (23.8-133.0-fold) compared with vehicle-treated cells. All the findings indicate that PoIFN-gamma acts synergistically with other PoIFNs (PoIFN-alpha and -beta) to potently inhibit PRV replication in vitro.     

Impaired antiviral response and alpha/beta interferon induction in mice lacking beta interferon

We have generated mice lacking the gene for beta interferon and report that they are highly susceptible to vaccinia virus infection. Furthermore, in cultured embryo fibroblasts, viral induction of alpha interferon and of 2-5A synthetase genes is impaired. We also show that beta interferon does not prime its own expression.