Evaluating Large Spontaneous Deletions in a Bovine Cell Line Selected for Bovine Viral Diarrhea Virus Resistance

Abstract

Bovine viral diarrhea virus's (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Keywords: BVDV; CRIB; CRISPR; GRID2; MDBK; PTPN12; RABGAP1L.

Figure 1

Physical maps of the three large homozygous deleted regions in the CRIB cell line. Genome map of ARS-UCD1.2 in the region of the three large homozygous deleted regions. Blue arrows, genes (open reading frames); black lines, overlapping heterozygous deletion alleles; red box, homozygous deleted region. Panel (A) heterozygous deletion alleles of PTPN12 on chromosome 4. Panel (B) heterozygous deletion alleles of GRID2 on chromosome 6. Panel (C) heterozygous deletion alleles of RABGAP1L on chromosome 16. Shown on top of the bovine map are the transcripts annotated for RABGAP1L in the Hereford ARS-UCD1.2 reference genome. Below the map are IsoSeq transcripts from multiple cattle breeds (see Methods). Panel (D), genome map with the full-length bovine RABGAP1L gene as determined using IsoSeq transcripts.

Figure 2

Knockout of PTPN12 does not significantly impact BVDV infection in MDBK cells. Panel (A), map of bovine PTPN12 and surrounding genes: blue arrows, genes (open reading frames); vertical blue lines, coding regions (exons); black lines, heterozygous deletions in two alleles of PTPN12 on chromosome 4; red square, homozygous deleted region (33,808 bp); red arrow, exon target of PTPN12 gRNA. Panel (B), RNASeq analyses of relative RNA transcript abundance in MDBK and CRIB cells (trimmed mean of M (TMM) normalization value). Panel (C), western blot of SDS-PAGE for PTPN12 and GAPDH (loading control). Panel (D), Multistep virus growth curve. Cells were infected with BVDV strain NADL (MOI 0.01). Cells were collected and processed 0–96 h post-infection for quantitation of viral RNA using RT-qPCR. Results represent the mean ± standard deviation (n = 3).

Figure 3

Knockout of GRID2 does not significantly impact BVDV infection in MDBK cells. Panel (A) map of bovine GRID2 and surrounding genes: blue arrows, genes (open reading frames); vertical blue lines, coding regions (exons); black lines, heterozygous deletions in two alleles of GRID2 on chromosome 6; red square, homozygous deleted region (63,200 bp); red arrow, exon target of GRID2 gRNA. Panel (B) Relative RNA expression levels in MDBK and CRIB cells (TMM normalization value). Pane (C) western blot of SDS-PAGE for GRID2 and GAPDH (loading control). Bovine cerebellum was used as a positive control for GRID2 expression. Panel (D) Multistep virus growth curve. Cells were infected with BVDV strain NADL (MOI 0.01). Cells were collected and processed 0–96 h post-infection for quantitation of viral RNA using RT-qPCR. Results represent the mean ± standard deviation (n = 3).

Figure 4

Disruption of the RABGAP1L gene does not significantly impact BVDV infection in MDBK cells. Panel (A), map of bovine RABGAP1L and surrounding genes: blue arrows, genes (open reading frames); black lines, heterozygous deletions in two alleles of RABGAP1L on chromosome 16; light red deletion block, homozygous deleted region (69,384 bp); red block, CRISPR/Cas9-mediated homozygous deletion. Shown is the corrected annotation for RABGAP1L based on IsoSeq data (see methods and Figure 1C). Panel (B), Relative RNA expression levels in MDBK and CRIB cells (TMM normalization value). Panel (C), Multistep virus growth curve. Cells were infected with BVDV strain NADL (MOI 0.01). Cells were collected and processed 0–96 h post-infection for quantitation of viral RNA using RT-qPCR. Results represent the mean ± standard deviation (n = 3).

Figure 5

Triple KO of PTPN12, GRID2, and RABGAP1L genes does not impact BVDV infection in MDBK cells. Panel (A) western blot of SDS-PAGE for PTPN12, GRID2, and GAPDH (loading control). Panel (B) MDBK and MDBK-TKO cell morphology (10x). Panel (C) Cell growth in T-25 flasks. Panel (D) Multistep virus growth curve. Cells were infected with BVDV strain NADL (MOI 0.01). Cells were collected and processed 0–96 h post-infection for quantitation of viral RNA using RT-qPCR. Results represent the mean ± standard deviation (n = 3). Panel (E) Cells were infected with various cytopathic (cp) and non-cytopathic (ncp) BVDV isolates (MOI = 0.01) and collected at 0 and 96 h post-infection for quantitation of viral RNA using RT-qPCR. Results represent means at 96 h ± standard deviation (n = 3).