Abstract
Bovine viral diarrhea virus (BVDV) entry into a host cell is mediated by the interaction of the viral glycoprotein E2 with the cellular transmembrane CD46 receptor. In this study, we generated a stable Madin–Darby Bovine Kidney (MDBK) CD46-knockout cell line to study the ability of different pestivirus A and B species (BVDV-1 and -2) to escape CD46-dependent cell entry. Four different BVDV-1/2 isolates showed a clearly reduced infection rate after inoculation of the knockout cells. However, after further passaging starting from the remaining virus foci on the knockout cell line, all tested virus isolates were able to escape CD46-dependency and grew despite the lack of the entry receptor. Whole-genome sequencing of the escape-isolates suggests that the genetic basis for the observed shift in infectivity is an amino acid substitution of an uncharged (glycine/asparagine) for a charged amino acid (arginine/lysine) at position 479 in the ERNS in three of the four isolates tested. In the fourth isolate, the exchange of a cysteine at position 441 in the ERNS resulted in a loss of ERNS dimerization that is likely to influence viral cell-to-cell spread. In general, the CD46-knockout cell line is a useful tool to analyze the role of CD46 for pestivirus replication and the virus–receptor interaction.
Highlights
The genus Pestivirus belongs to the family Flaviviridae and contains several veterinary-relevant virus species with major animal welfare and economic importance like pestivirus A and B, pestivirus D and pestivirus C
The genome encodes eight nonstructural and four structural proteins in a single open reading frame (ORF) [10], of which the glycoproteins ERNS, E1 and E2 play an important role in the initiation of Bovine viral diarrhea virus (BVDV) uptake by the host cell [11,12]
It has been shown that ERNS interacts with the cell surface heparan sulfate and E1–E2 heterodimers bind the cellular receptor CD46 and mediate clathrin-dependent endocytosis [11,13,14]
Summary
The genus Pestivirus belongs to the family Flaviviridae and contains several veterinary-relevant virus species with major animal welfare and economic importance like pestivirus A and B (bovine viral diarrhea virus types 1 and 2, BVDV-1 and -2), pestivirus D (border disease virus, BDV) and pestivirus C (classical swine fever virus, CSFV) [1,2,3,4]. The genome encodes eight nonstructural and four structural proteins in a single open reading frame (ORF) [10], of which the glycoproteins ERNS , E1 and E2 play an important role in the initiation of BVDV uptake by the host cell [11,12]. CSFV ERNS interacts with the heparan sulfate of porcine cells and both CD46 and heparan sulfate, are major factors for the attachment of CSFV in vitro [15,16]
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