ADAM17 Is an Essential Factor for the Infection of Bovine Cells with Pestiviruses

Abstract

The entry of BVDV into bovine cells was studied using CRIB cells (cells resistant to infection with bovine viral diarrhea virus [BVDV]) that have evolved from MDBK cells by a spontaneous loss of susceptibility to BVDV. Recently, larger genetic deletions were reported but no correlation of the affected genes and the resistance to BVDV infection could be established. The metalloprotease ADAM17 was reported as an essential attachment factor for the related classical swine fever virus (CSFV). To assess whether ADAM17 might be involved in the resistance of CRIB-1 cells to pestiviruses, we analyzed its expression in CRIB-1 and MDBK cells. While ADAM17 protein was detectable in MBDK cells, it was absent from CRIB-1 cells. No functional full-length ADAM17 mRNA could be detected in CRIB cells and genetic analysis revealed the presence of two defective alleles. Transcomplementation of functional ADAM17 derived from MDBK cells in CRIB-1 cells resulted in a nearly complete reversion of their resistance to pestiviral infection. Our results demonstrate that ADAM17 is a key cellular factor for the pestivirus resistance of CRIB-1 cells and establishes its essential role for a broader range of pestiviruses.

Keywords: ADAM17; bovine viral diarrhea virus; cells resistant to BVDV infection; pestivirus.

Figure 1

Glycosylated forms of ADAM17 are absent in CRIB-1 cells. (A) Western blot analysis of ADAM17 in the cell lysate of MDBK and CRIB-1 cells and in the cellular glycoprotein fraction of MDBK and CRIB-1 cells generated by precipitation with Concanavalin A (ConA) beads. The different bands are indicated by arrow heads. In CRIB-1 cells, only the band designated as unspecific, as its origin is unknown, can be discerned in cell lysate, whilst no band can be detected when glycoproteins are precipitated with ConA beads. (B) Detection of ADAM17 and β-actin by Western blot analysis of cell lysates of MDBK, CRIB-1 and CRIB-1 cells expressing ADAM17 from a lentiviral vector (+ADAM17). Upon transcomplementation, the glycosylated pro- and mature form of ADAM17 can be detected in CRIB-1 cells, indicating that the pathways responsible for glycosylation and processing of ADAM17 are intact in CRIB-1 cells.

Figure 2

Genetic alterations of the ADAM17 gene in CRIB-1 cells. Nucleotide positions relative to the ARS-UCD1.2 reference bovine genome. Alignments of reads in the modified regions are shown in Supplementary Figure S1.

Figure 3

Transcomplementation of bovine ADAM17 renders CRIB-1 cells susceptible to pestiviruses. Susceptibility of MDBK cells (blue) or CRIB-1 (green) cells with (+) or without (−) transcomplementation of ADAM17 or SH3BGRL to different pestiviruses in % of the susceptibility of MDBK cells. Shown are mean and standard deviation of four independent experiments.

Figure 4

Effect of ADAM17 transcomplementation on BVDV spread. (A) Analysis of BVDV spread by flow cytometry. The amount of infected MDBK or CRIB-1 cells with (+) or without (−) transcomplementation of ADAM17 or SH3BGRL was quantified by flow cytometry 72 h after infection with BVDV C87 (E2-mClover) with an MOI of 0.001. Shown are mean and standard deviation of three independent experiments. (B) BVDV plaque morphology on MDBK or CRIB-1 cells with (ADAM17) or without (neg) transcomplementation of bovine ADAM17 after staining with the anti-E2 antibody 6A5. Cell nuclei were stained with DAPI. Scale bar = 200 µm.