Viral Traits and Cellular Knock-Out Genotype Affect Dependence of BVDV on Bovine CD46

Abstract

The role of bovine CD46 in the host cell entry of BVDV has been established for more than a decade. By generating novel MDBK CD46 knock-out clones, we confirm previously reported data on the CD46 motives important for BVDV binding and the importance of the G479R exchange within BVDV E^rns to gain independence of bovine CD46 during entry. The comparison of different knock-out genotypes revealed a high variability of cellular susceptibility for a BVDV encoding the G479R exchange. These data highlight the effect of clonal selection of knock-outs on virus susceptibility, which should be considered when planning knock-out experiments.

Keywords: CRISPR-CAS9 mediated knock-out; bovine CD46; bovine viral diarrhea virus; pestivirus; susceptibility.

Figure 1

Genotype of generated MDBK CD46 knock-out cell lines and susceptibility to different BVDV-1 strains. (A) (i) Localization of the sgRNAs and the primers used for amplification of the modified region by PCR on the CD46 gene. (ii) Reactivity of the cell lines with anti-CD46 antibodies CA17 and CA26. − indicates no reactivity, + indicates reactivity. (iii) Effect of modifications within the CD46 alleles on the expressed protein. Stop codons are indicated by red asterisk. Residues important for BVDV E2 binding according to [11] are indicated. Wt = unmodified MDBK cells. (B) Susceptibility of the different clones to BVDV-1 strains C87, CP7, and NADL in comparison to the parental MDBK cell line. The average and standard deviation of three independent experiments are depicted. Color code and clone numbering is according to (A-iii). (C) Effect of the susceptibility of clone 7 to C87 and C87 encoding a G at position 479 of the polyprotein (C87R479G).