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To study the activity of the expressed BRSV NS proteins, Vero cells were infected at an MOI of 5 with parental RV (VB wt) or with each of the recombinants, or were co- infected with both recombinants, VB bNS1 and VB bNS2, at an MOI of 2.5 each. Directly after infection, cells were treated with increasing amounts of recombinant IFN-α A/D and the production of infectious RV titers was analysed 2 days post infection. Growth of the parental RV and of NS1 or NS2 protein-expressing viruses from single infections was similarly affected (Fig. 10A). On application of 50 IU of IFN-α titers dropped by approximately 1 log unit and then further decreased very slowly with increasing IFN amounts, indicating either a weak IFN response of Vero cells or a high intrinsic resistance of RV to IFN-mediated responses in Vero cells. In cells co- infected with NS1 and NS2-expressing viruses, however, protection of virus replication could be shown. Virus titers remained significantly higher than in single infections and decreased slowly in a dose-dependent manner. Similar results were also obtained when viral protein expression was monitored. Only in cells coinfected with both recombinants, viral protein synthesis was maintained in the presence of up to 150 IU of IFN-α (Fig. 11A).

Fig. 10: IFN resistance in cells coinfected with RVs expressing BRSV NS1 and NS2. Vero cells (A) or MDBK cells (B) were infected with wild-type VB, VB bNS1, or VB bNS2, or coinfected with VB bNS1 and VB bNS2. Immediately after infection, cells were treated with the indicated amounts of IFN-α A/D. Infectious virus titers were determined 2 days post infection. Results represent the mean values of at least four independent experiments, with error bars indicating standard deviations.

In parallel experiments performed in MDBK cells, parental RV and the NS-expressing recombinants replicated to slightly lower titers in untreated MDBK cells than in Vero cells. In contrast to Vero cells, treatment with IFN more dramatically reduced infectious titers of wt RV and of NS-expressing viruses from single infections. An immediate reduction in infectious titers by 3 log units indicated a highly effective IFN-mediated cell response. However, in cells co- infected with viruses expressing NS1 and NS2 proteins, virus replication was completely protected against IFN until more than 150 IU of IFN was applied (Fig. 10B). These results were reflected by the analysis of RV protein synthesis. In untreated cells, all recombinants produced substantial and comparable amounts of RV proteins whereas in IFN- treated cells, only coinfections with VB bNS1 and VB bNS2 led to substantial protein synthesis until more than 150 to 200 IU was applied (Fig. 11B). These results confirm that the two BRSV NS proteins are required and sufficient for exerting the observed IFN antagonist activity. In addition, they show that both NS proteins are able to confer resistance to IFN- mediated antiviral response not only to BRSV but also to another, unrelated rhabdovirus.

Fig. 11: RV protein synthesis in IFN challenge experiments. Vero (A) or MDBK cells (B) were infected as described in Fig. 8, and cultures were treated with 0, 50, 150, 500 and 2,000 IU (A) or 0, 50, 150, 175, 500 IU of IFN-α (B). Cells were harvested 2 days post infection and extracts were analysed by western blot. RV N protein was detected using a serum raised against RV (S50). (C) Western blot analysis of BRSV NS protein expression in Vero cells infected with VB bNS1 and VB bNS2. Cells were treated with 0, 50 or 150 IU of IFN-α directly after seeding, and harvested 2 days post infection. NS1 protein was detected using an antibody recognizing the HA-tag (Y-11). For detection of NS2, an antibody recognizing the FLAG-tag (M2) was used.

To rule out the possibility that the addition of the HA- or FLAG-tag to NS1 and NS2, respectively, influences their function(s) in the viral life cycle, recombinant BRSVs expressing either NS1ha or NS2fl were generated (BRSV ha and BRSV fl, respectively; Fig. 12A). Both recombinant BRSVs were recovered by cotransfection of BSR T7/5 cells with BRSV full- length cDNA with support plasmids encoding BRSV N, P, L, and M2 genes as described in 2-4, and synthesis of the tagged NS proteins was monitored by Western blotting (Fig. 12B).

Fig. 12: IFN resistance of rBRSVs expressing tagged BRSV NS genes is not altered. (A) Genomes of rBRSV expressing NS1ha (BRSV ha) or NS2fl (BRSV fl). (B) Western blot analysis of cell lysates from Vero cells infected with BRSV ha and BRSV fl. NS1ha was detected using an antibody recognizing the HA-tag (Y- 11). NS2fl was recognized by an antibody raised against the synthetic FLAG epitope (M2). (C) Vero or MDBK cells infected at an MOI of 0.1 with BRSV wt, BRSV ha, or BRSV fl were incubated with the indicated amounts of recombinant IFN-α directly after seeding. Infectious titers were determined 3 days post infection. Results are shown as fold reduction in viral titer and represent the mean values of two independent experiments.

IFN challenge experiments were performed in Vero and MDBK cells to determine the protective capacity of the modified NS proteins towards exogenous IFN. As expected, no difference in IFN resistance was observed for BRSV ha or BRSV fl when compared with wt BRSV (Fig. 13). We therefore conclude that the addition of a protein tag to either NS protein does not compromise its IFN antagonist function in the context of a viral infection.