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. 2006 Jul;80(14):6926-35.
doi: 10.1128/JVI.02443-05.

"Self" and "nonself" manipulation of interferon defense during persistent infection: bovine viral diarrhea virus resists alpha/beta interferon without blocking antiviral activity against unrelated viruses replicating in its host cells

Matthias Schweizer  1 , Philippe Mätzener, Gabriela Pfaffen, Hanspeter Stalder, Ernst Peterhans
Affiliations

"Self" and "nonself" manipulation of interferon defense during persistent infection: bovine viral diarrhea virus resists alpha/beta interferon without blocking antiviral activity against unrelated viruses replicating in its host cells

Matthias Schweizer et al. J Virol. 2006 Jul.

Abstract

Bovine viral diarrhea virus (BVDV), together with Classical swine fever virus (CSFV) and Border disease virus (BDV) of sheep, belongs to the genus Pestivirus of the Flaviviridae. BVDV is either cytopathic (cp) or noncytopathic (ncp), as defined by its effect on cultured cells. Infection of pregnant animals with the ncp biotype may lead to the birth of persistently infected calves that are immunotolerant to the infecting viral strain. In addition to evading the adaptive immune system, BVDV evades key mechanisms of innate immunity. Previously, we showed that ncp BVDV inhibits the induction of apoptosis and alpha/beta interferon (IFN-alpha/beta) synthesis by double-stranded RNA (dsRNA). Here, we report that (i) both ncp and cp BVDV block the induction by dsRNA of the Mx protein (which can also be induced in the absence of IFN signaling); (ii) neither biotype blocks the activity of IFN; and (iii) once infection is established, BVDV is largely resistant to the activity of IFN-alpha/beta but (iv) does not interfere with the establishment of an antiviral state induced by IFN-alpha/beta against unrelated viruses. The results of our study suggest that, in persistent infection, BVDV is able to evade a central element of innate immunity directed against itself without generally compromising its activity against unrelated viruses ("nonself") that may replicate in cells infected with ncp BVDV. This highly selective "self" and "nonself" model of evasion of the interferon defense system may be a key element in the success of persistent infection in addition to immunotolerance initiated by the early time point of fetal infection.

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Figures

FIG. 1.
FIG. 1.
ncp as well as cp BVDV inhibits the synthesis of Mx induced by poly(IC). MDBK cells were either mock infected or infected with the SuwaNcp (A) or the R1935 cp (B) BVDV strain and incubated for 18 h at 37°C. Thereafter, poly(IC) was added at concentrations of 0 to 100 μg/ml as indicated. Cytosolic extracts were prepared 24 h later, and the expression of Mx protein was analyzed by Western blotting as described in Materials and Methods. Simultaneous staining of β-actin on the same membrane was used as a control for the protein loading of the individual lanes.
FIG. 2.
FIG. 2.
BVDV does not inhibit the induction of Mx induced by IFN-α. MDBK cells were either mock infected or infected with the SuwaNcp (A) or the R1935 cp (B and C) BVDV strain and incubated for 18 h at 37°C. Thereafter, rbo IFN-αI.1 was added at 0 to 10 ng/ml as indicated. Cytosolic extracts were prepared 24 h later, and the expression of cellular Mx protein (A and B) and that of viral NS3 and NS23 proteins (C) were analyzed by Western blotting as described in Materials and Methods. Simultaneous staining of β-actin on the same membranes was used as a control for the protein loading of the individual lanes.
FIG. 3.
FIG. 3.
Expression of Mx protein in IFN-responsive (MDBK and calf testicle cells) and nonresponsive (MDBK-V7) cells. (A) MDBK cells (upper panel) or MDBK-V7 cells (lower panel) were treated with poly(IC) at concentrations of 0 to 100 μg/ml and with rbo IFN-αI.1 at concentrations of 0.01 to 30 ng/ml, respectively, as indicated. Cytosolic extracts were prepared 24 h later, and the expression of Mx protein was analyzed by Western blotting as described in Materials and Methods. Simultaneous staining of β-actin on the same membrane was used as a control for the protein loading of the individual lanes. The film was exposed for 10 seconds or 4 min as indicated. (B) Calf testicle cells were either mock infected or infected with the ncp BVDV strains SuwaNcp (S-Ncp), Pe515-Ncp (515Ncp), TGAN, and 890 and the cp BVDV strains SuwaCp (S-Cp), Pe515-Cp (515Cp), TGAC, R1935, and NADL, respectively, at an MOI of 5 and incubated for 1 or 2 days at 37°C. Twenty micrograms of poly(IC) (pIC) per milliliter or 20 ng/ml rbo IFN-αI.1 (IFN) was used as a positive control for the expression of Mx protein. Cytosolic extracts were prepared at the time points indicated, and the expression of Mx protein and β-actin was analyzed by Western blotting as described in Materials and Methods. p.i., postinfection.
FIG. 4.
FIG. 4.
BVDV replication in BT cells or monocyte-derived Mφ is inhibited only when IFN-α/β is added prior to virus infection. BT cells (A and B) or Mφ (C and D) in six-well plates were infected with the SuwaNcp BVDV strain (○) at an MOI of 0.01, or uninfected cells were incubated with rbo IFN-αI.1 (▪) at various concentrations from 0 to 100 ng/ml as indicated in the figure. Twenty-four hours later, the IFN-treated cells (▪) were infected with the SuwaNcp BVDV strain and the previously infected cells (○) were treated with IFN as described above and incubated for another 24 h. (A and C) The BVDV titer in the supernatant was analyzed as described in Materials and Methods. (B and D) Total RNA was isolated, and intracellular viral RNA was quantified by real-time TaqMan RT-PCR as described in Materials and Methods. The amount of viral RNA is expressed relative to the corresponding amount in cells treated without IFN as calculated by the ΔΔCt method, and representative experiments are shown. TCID50, 50% tissue culture infective dose.
FIG. 5.
FIG. 5.
Passaging of ncp BVDV-infected cells in the presence of rbo IFN-α is unable to eliminate the virus. MDBK cells in 25-cm2 tissue culture flasks were infected with the SuwaNcp BVDV strain as described in Materials and Methods. At 24 h postinfection, the virus titer in the supernatant was analyzed (P1′), and the medium was replaced with fresh medium without (▴) or with 1 (⧫), 10 (□), or 100 (○) ng/ml rbo IFN-αI.1. Thereafter, cells were passaged twice a week for 10 times as indicated (P1 to P10) with or without IFN as described for the first passage. For the last passage (P11), all samples were incubated in medium without IFN. After every passage, the BVDV titer in the supernatant (A) and the amount of intracellular viral RNA (B) were analyzed as described for Fig. 4 (representative experiments are shown). In order to exclude the selection of IFN-resistant cells, parallel cultures of MDBK cells at passages 1, 7, and 10 were infected with VSV at an MOI of 0.01, and the amount of VSV in the supernatant was titrated 24 h postinfection (C). TCID50, 50% tissue culture infective dose.
FIG. 6.
FIG. 6.
ncp BVDV does not inhibit or only slightly inhibits the activity of rbo IFN-α against unrelated viruses in BT cells. BT cells were mock infected (▪) or infected with the TGAN (A), 890 (B), or Ncp7 (C) ncp BVDV strains (○), respectively, at an MOI of 3 at 24 h prior to the addition of rbo IFN-αI.1 at various concentrations as indicated (w/o, without IFN). Six hours (A and B) or 24 h (C) after the addition of IFN, the cells were infected with VSV (A and B) or EMCV (C) at an MOI of 0.01 and incubated for another 20 to 24 h. (A) DNA fragmentation was quantified by propidium iodide staining of nuclei followed by fluorescence-activated cell sorting analysis as described in Materials and Methods. The percentages on the y axis indicate the relative number of nuclei with subgenomic DNA compared to the corresponding sample in the absence of IFN, which was set to 100%. For clarity, the mean ± 95% confidence interval (n = 3 to 8) is shown only for cells treated with more than 0.01 ng/ml IFN. (B) The titer of the challenge virus VSV was analyzed as described in Materials and Methods. For clarity, the mean ± 95% confidence interval (n = 2 to 6) is shown only for cells treated with at least 0.01 ng/ml IFN. TCID50, 50% tissue culture infective dose. (C) Viable, adherent cells were stained with crystal violet (CV) and the intensity measured at 590 nm as described in Materials and Methods (average of triplicates ± standard deviation). OD590, optical density at 590 nm.
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