Modulation of ADAM17 Levels by Pestiviruses Is Species-Specific

Abstract

Upon host cell infection, viruses modulate their host cells to better suit their needs, including the downregulation of virus entry receptors. ADAM17, a cell surface sheddase, is an essential factor for infection of bovine cells with several pestiviruses. To assess the effect of pestivirus infection on ADAM17, the amounts of cellular ADAM17 and its presence at the cell surface were determined. Mature ADAM17 levels were reduced upon infection with a cytopathic pestivirus bovis (bovine viral diarrhea virus, cpBVDV), pestivirus suis (classical swine fever virus, CSFV) or pestivirus giraffae (strain giraffe), but not negatively affected by pestivirus L (Linda virus, LindaV). A comparable reduction of ADAM17 surface levels, which represents the bioactive form, could be observed in the presence of E2 of BVDV and CSFV, but not LindaV or atypical porcine pestivirus (pestivirus scrofae) E2. Superinfection exclusion in BVDV infection is caused by at least two proteins, glycoprotein E2 and protease/helicase NS3. To evaluate whether the lowered ADAM17 levels could be involved in superinfection exclusion, persistently CSFV- or LindaV-infected cells were challenged with different pestiviruses. Persistently LindaV-infected cells were significantly more susceptible to cpBVDV infection than persistently CSFV-infected cells, whilst the other pestiviruses tested were not or only hardly able to infect the persistently infected cells. These results provide evidence of a pestivirus species-specific effect on ADAM17 levels and hints at the possibility of its involvement in superinfection exclusion.

Keywords: ADAM17; maturation; pestivirus; receptor downregulation.

Figure 1

Pestivirus infection can change the cellular amounts of mature and immature ADAM17. (A) MDBK cells were infected with cpBVDV-1, BVDV-2, CSFV, strain giraffe or LindaV or mock infected (neg). Cells were lysed 24, 48 or 72 hpi and mature ADAM17 (white arrowhead, apparent molecular weight 95–100 kD), immature ADAM17 (black arrowhead, apparent molecular weight 120 kD) and β-actin (grey arrowhead) were detected by Western blot analysis using fluorescence-based detection. An unspecific band also detected by the employed antibody is indicated by a black arrow. (B) Quantification of the relative amounts (normalized to β-actin) of mature (i) and immature (ii) ADAM17 in comparison with non-infected cells is shown, as well as the ratio of mature to immature ADAM17 expressed in % (iii) as determined by Western blot analysis. Timepoints correspond to the timepoints given in (A). Shown are mean and standard deviation of n = 3 independent experiments. Statistical significance of the results was calculated with a Student’s t-test.

Figure 2

BVDV and CSFV E2 lower ADAM17 surface levels. (A) HEK293 cells 48 h after transfection expressing either BVDV Npro to E2 labeled at its N-terminus with a fluorophore (BVDVNpro-E2_fluo) or the same construct with a deletion of the E2 ectodomain (BVDVNpro-E2_fluo∆E2ectodomain) were analyzed by flow cytometry for the expression of ADAM17 at the cell surface. Relative mean fluorescence intensities were calculated by comparing the levels of surface fluorescence intensities of transfected and non-transfected cells. Shown are mean and standard deviation of n = 5 independent experiments. Statistical significance of the results was calculated with a Student’s t-test. (B) HEK293 Tet-off cells 48 h after transfection expressing either BVDV, APPV, CSFV or LindaV E2 with a C-terminal mCherry tag were analyzed for the surface expression of ADAM17 by flow cytometry. Relative mean fluorescence intensities were calculated by comparing the levels of surface fluorescence intensities of transfected and non-transfected cells. Shown are mean and standard deviation of n = 3 independent experiments. Statistical significance of the results was calculated with a Student’s t-test. (C) Changes in ADAM17 levels upon induction of pestivirus E2 expression in SK6 Tet-on cells are shown. Shown are one representative Western blot and the quantification of the % of mature and immature ADAM17 in doxycycline-induced versus non-induced cells (normalized to β-actin). Mature ADAM17 (white arrowhead, apparent molecular weight 95–100 kD), immature ADAM17 (black arrowhead, apparent molecular weight 120 kD) and β-actin (grey arrowhead) were detected by Western blot analysis using fluorescence-based detection. An unspecific band also detected by the employed antibody is indicated by a black arrow. Mean and standard deviation of n = 3 independent experiments are depicted. Statistical significance of the results was calculated with a Student’s t-test.

Figure 3

Persistently LindaV-infected cells are more susceptible to superinfection by a cpBVDV-1 than persistently CSFV-infected cells. Virus titers (blue bars) observed in MDBK, MDBK_bADAM17 and CRIB_bADAM17 cells persistently infected with CSFV (A) or LindaV (B) upon superinfection with cpBVDV-1 compared with titers in non-infected cells are shown. (C) Comparison of the percent reduction of titer of the superinfecting virus in persistently CSFV- or LindaV-infected cells compared with non-infected cells is shown. Data are presented as the mean and standard deviation of three independent experiments (n = 3). Statistical significance was determined using two-way ANOVA.