Bovine Viral Diarrhea Virus Strain- and Cell Type-Specific Inhibition of Type I Interferon Pathways

Abstract

The interaction of noncytopathogenic bovine viral diarrhea virus type 1 (BVDV-1ncp) with antigen-presenting cell (APC) subsets is of great interest due to the recent increase in severe acute BVDV outbreaks attributed to this genotype (1, 12, 13). We report the effect of a BVDV-1ncp strain (Ho916ncp [13]) causing severe acute infection on in vitro type I interferon (alpha/beta interferon [IFN-α/β]) production compared to that of a mild-acute strain (Ky1203ncp) (15). Ho916ncp but not Ky1203ncp induced IFN-α/β in only lineage-negative (LIN−) cells, a recently described enriched IFN-α/β-producing cell population in cattle (A. Gibson, S. Miah, P. Griebel, J. Brownlie, and D. Werling, submitted for publication). In agreement with published data, monocytes and monocyte-derived dendritic cells (moDC) did not produce IFN-α/β in response to either strain (4, 11, 17). Experimental infection of calves with BVDVncp results in the detection of IFN-α/β and the IFN-α/β-inducible gene Mx (5, 20) as well as serum IFN-α/β activity in pregnant dams and the fetus (22). The identification of IFN-α/β-producing cells within the lymph nodes of BVDVncp-infected calves (4) and of enriched circulating IFN-α/β-producing cells (Gibson et al., submitted) points to an APC subset as a cellular source of IFN-α/β during in vivo BVDVncp infections. In the present study, monocytes, moDC, and LIN− cells, generated from the same animal (11, 24-26, 28; Gibson et al., submitted), were exposed to either strain at a multiplicity of infection (MOI) of 0.1 and IFN-α/β was assessed after 48 h, using a previously described Mx-CAT (chloramphenicol acetyltransferase) reporter assay (4-6, 9). LIN− cells, but not monocytes or moDC, produced significant amounts of IFN-α/β in response to Ho916ncp but not to Ky1203ncp or mock-infected control (Fig. ​(Fig.1).1). To assess whether these observed differences in IFN-α/β production were due to differences in viral replication, the presence of BVDV was determined by immunoperoxidase staining using a bovine hyperimmune serum, similar to what was described previously (11). Astonishingly, no clear differences in abilities to infect the different cell types were seen for the two BVDVncp strains (Fig. ​(Fig.2).2). As differences in viral replication did not explain differences in IFN-α/β production, we next assessed the potential effects of both BVDVncp strains on interferon response factors (IRFs). BVDVncp is known to inhibit IFN-α/β production through viral proteins Npro (2, 3, 10, 14) and Erns (16, 18, 19), which block IFN-α/β production by targeting IRF-3 for degradation by polyubiquitination (7, 14) or by degrading viral RNA (19). Translocation of IRF-3 occurs during infection but not binding to DNA (2). In contrast, IRF-7 is neither activated nor translocated to the nucleus (2). FIG. 1. Type I interferon response to BVDVncp. Monocytes (MON), moDC, and LIN− cells (LIN) were isolated from the same animal and stimulated with 50 μg ml−1 of poly(I:C), BVDVncp strains Ho916ncp and Ky1203ncp at an MOI of 0.1, and corresponding ... FIG. 2. Antigen-presenting cell susceptibility to BVDVncp. Monocytes (A), moDC (B), and LIN− cells (C) were infected with BVDVncp strains Ho916ncp and Ky1203ncp for 48 h (MOI, 0.1). Cells were fixed and stained for the presence of BVDV antigen using a ... We assessed the impact of both strains on IFN-α/β signaling components IRF-3 and IRF-7 by Western blotting, which showed that IRF-3 and IRF-7 were increased in all three cell types infected with Ho916ncp compared to mock-infected controls. In contrast, Ky1203ncp induced a reduction of IRF-7 expression in monocytes and moDC and a reduction in IRF-3 expression in all three cell types. IRF-7 remained unchanged in LIN− cells, potentially indicating cell type-specific degradation in monocytes and moDC for Ky1203ncp (Fig. ​(Fig.3).3). Ho916ncp-induced IFN-α/β production by LIN− cells appears to be independent of IRF-3 or IRF-7 modulation; however, LIN− cells, unlike monocytes or moDC, express TLR7 (27; Gibson et al., submitted), which has been recently implicated in the recognition of other pestiviruses, such as West Nile virus and dengue virus (8, 21, 23). Ho916ncp, as a severe acute BVDV-1ncp strain could be more readily accessible to the endosomal compartment within LIN− cells, thus producing cell type-restricted IFN-α/β through TLR7. Our data show for the first time differences in BVDV-1ncp strains with regard to a cell-specific IFN-α/β response and offer some suggestions for this modulation. FIG. 3. Relative expression of IRF-3 and IRF-7 in response to BVDVncp. Monocytes (MON), moDC, and LIN− (LIN) cells were isolated from the same animal and stimulated with Ho916ncp and Ky1203ncp alongside mock-infected controls for 48 h. Protein lysates ...