Identification of specific amino acid residues in the border disease virus glycoprotein E2 that modify virus growth in pig cells but not in sheep cells

Abstract

Border disease virus (BDV) envelope glycoprotein E2 is required for entry into cells and is a determinant of host tropism for sheep and pig cells. Here, we describe adaptive changes in the BDV E2 protein that modify virus replication in pig cells. To achieve this, two BDV isolates, initially collected from a pig and a sheep on the same farm, were passaged in primary sheep and pig cells in parallel with a rescued variant of the pig virus derived from a cloned full-length BDV cDNA. The pig isolate and the rescued virus shared the same amino acid sequence, but the sheep isolate differed at ten residues, including two substitutions in E2 (K771E and Y925H). During serial passage in cells, the viruses displayed clear selectivity for growth in sheep cells; only the cDNA-derived virus adapted to grow in pig cells. Sequencing revealed an amino acid substitution (Q739R) in the E2 domain DA of this rescued virus. Adaptation at the same residue (Q739K/Q739R) was also observed after passaging of the pig isolate in sheep cells. Use of reverse genetics confirmed that changing residue Q739 to R or K (each positively charged) was sufficient to achieve adaptation to pig cells. Furthermore, this change in host tropism was suppressed if Q739R was combined with K771E. Another substitution (Q728R), conferring an additional positive charge, acquired during passaging, restored the growth of the Q739R/K771E variant. Overall, this study provided evidence that specific, positively charged, residues in the E2 domain DA are crucial for pig-cell tropism of BDV.

Identification of a Common Conformational Epitope on the Glycoprotein E2 of Classical Swine Fever Virus and Border Disease Virus

“…The amino acid sequences and protein structure of glycoprotein E2 are involved in determining the host tropism of different pestiviruses, as the inhibition of binding to permissive and non-permissive cells by E2 proteins is higher when using E2 derived from the homologous pestivirus [ 18 ]. According to a recent study, the residue at position 739 of BDV E2 (corresponding to the residue at position 738 of CSFV E2) is required for viral entry into cells and determines the host tropism for sheep and pig cells, as the substitution of residue Q739 of BDV E2 to either of the positively charged residues R or K was crucial for BDV to adapt to pig cells [ 19 ]. It was assumed that the amino acid substitutions to positively charged amino acids might affect BDV attachment and entry and thus enhance virus replication in pig cells [ 19 ].…”

“…According to a recent study, the residue at position 739 of BDV E2 (corresponding to the residue at position 738 of CSFV E2) is required for viral entry into cells and determines the host tropism for sheep and pig cells, as the substitution of residue Q739 of BDV E2 to either of the positively charged residues R or K was crucial for BDV to adapt to pig cells [ 19 ]. It was assumed that the amino acid substitutions to positively charged amino acids might affect BDV attachment and entry and thus enhance virus replication in pig cells [ 19 ]. However, as the residues at the same position of most CSFV strains are V, I, or T ( Figure 3 ), which are hydrophobic or polar but not positively charged amino acids, the role of the residues at positions 738 in CSFV and 739 in BDV in virus attachment, cell entry, and host adaption to pigs needs further investigation.…”

“…The E2 of pestiviruses plays several important roles during the viral life cycle [ 13 ], such as interacting with cellular receptors to facilitate virus attachment and mediating membrane fusion to allow entry into host cells [ 14 , 15 ], and thereby determines the cell tropism and host specificity of pestiviruses [ 16 , 17 ]. The sequences and structures of E2 proteins are presumed to be responsible for the host specificity with regard to cell entry [ 18 , 19 ] through receptor-mediated endocytosis [ 20 , 21 , 22 ]. Bovine CD46 is the cell surface receptor for BVDV [ 23 ], and inhibition of BVDV infection by CSFV E2 suggests that CSFV E2 and BVDV E2 share an identical receptor [ 14 ].…”