Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response - PubMed (original) (raw)

Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response

J Schlender et al. J Virol. 2000 Sep.

Abstract

The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-alpha) receptor. Treatment of cells with recombinant universal IFN-alpha A/D or IFN-beta revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-alpha/beta mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.

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Figures

FIG. 1

(A) Diagram of the genomes of recombinant BRSV. The locations of transcripts (shaded bars) and protein-encoding frames (open bars) are shown relative to the viral genome (vRNA) (solid bars). In the enlargement, the organizations of full-length and NS deletion mutants are compared. Leader RNA is marked by vertical stripes, and the relative positions of the corresponding nucleotides and restriction sites used for cloning are indicated. (B) Organization of rRV containing tagged BRSV NS1 or BRSV NS2 open reading frames between the RV G and L genes.

FIG. 2

Lack of NS1 and NS2 transcripts in recombinant BRSVs. Total RNA from BSR cells infected with the indicated viruses was isolated 2 to 4 days postinfection and analyzed by Northern hybridization with probes spanning the NS1, NS2, NS1 to NS2, and N genes, respectively. NS1, NS2, and N mRNAs are indicated.

FIG. 3

NS deletion mutants are more attenuated in MDBK cells than in BSR cells. Nearly confluent BSR T7-5 (A) and MDBK (B) cell monolayers were infected at an MOI of 0.1 with BRSV, BRSV ΔNS1, BRSV ΔNS2, or BRSV ΔNS1/2. Infectious virus titers were determined every 2 days as described in Materials and Methods. From day 6 onward, replication of all mutants in Vero cells and of wt BRSV in MDBK cells leads to massive cell destruction. Values are from two independent experiments, each performed in triplicate. Bars show standard deviations.

FIG. 4

Supernatants from virus-infected MDBK cells or infected macrophages inhibit the growth of BRSV NS deletion mutants in cocultured Vero cells. (A) Schematic of the design of cocultivation experiments. (B) MDBK cells or LPS-stimulated bovine macrophage effector cells (EC) were infected with BRSV at a MOI of 1, seeded into Nunc Anopore membrane cell culture inlays, and cocultured with Vero responder cells (RC) infected with wt BRSV, BRSV ΔNS1, BRSV ΔNS2, or BRSV ΔNS1/2. After 3 days, the inlays were removed and infectious virus titers in Vero cultures were determined. The results are shown as percent inhibition, including standard deviation (+/−), and fold reduction (of the mean) relative to controls using noninfected MDBK or noninfected, nonstimulated macrophages. Values were obtained from six (MDBK) and four (macrophages) cocultivation experiments.

FIG. 5

An IFNAR2 monoclonal antibody neutralizes the effect of the inhibitory factor produced by MDBK or macrophage supernatants. Vero responder cells infected at an MOI of 0.1 with rBRSV ΔNS1, rBRSV ΔNS2, rBRSV ΔNS1/2, or wt rBRSV were incubated for 3 h with 5 μg each of a monoclonal antibody against IFNAR2 (lanes 2), MHC class I (lanes 3), or TNFR1 (lanes 4) or in the absence of antibodies (lanes 1). Cocultivation with infected MDBK cells (see Fig. 4 for the design of the experiment) was done in the presence of 1 μg of the respective antibody per ml. Titers were determined in six (lanes 1 and 2) or two experiments (lanes 3 and 4). Bars show standard deviation.

FIG. 6

All BRSV NS deletion mutants are IFN-α/β sensitive. Vero cells infected at an MOI of 0.1 with BRSV, BRSV ΔNS1, BRSV ΔNS2, or BRSV ΔNS1/2 were incubated with the indicated amounts of recombinant IFN-α A/D (A) or IFN-β (B). Infectious virus titers from four independent experiments were determined 4 days postinfection. Bars show standard deviation.

FIG. 7

IFN resistance of BRSV in MDBK and Vero cells. MDBK cells (solid columns) or Vero cells (open columns) were infected at an MOI of 1 with rBRSV and treated with the indicated amounts of recombinant IFN-α A/D. Infectious virus titers were determined 3 days postinfection. Bars represent standard deviations.

FIG. 8

IFN resistance of RV in cells coinfected with RVs expressing NS1 and NS2. Vero cells (A) or MDBK cells (B) were infected with wt RV SAD VB, SAD VB-NS1, or SAD VB-NS2 or coinfected with SAD VB-NS1 and SAD VB-NS2. Immediately after infection, cultures were treated with the indicated amounts of IFN-α A/D. Infectious virus titers were determined 2 days postinfection. Results represent the mean values of at least four independent experiments, with error bars indicating standard deviation.

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Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response - PubMed (original) (raw)