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.

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.

Distinct roles for the IIId2 sub-domain in pestivirus and picornavirus internal ribosome entry sites.

Viral internal ribosomes entry site (IRES) elements coordinate the recruitment of the host translation machinery to direct the initiation of viral protein synthesis. Within hepatitis C virus (HCV)-like IRES elements, the sub-domain IIId(1) is crucial for recruiting the 40S ribosomal subunit. However, some HCV-like IRES elements possess an additional sub-domain, termed IIId2, whose function remains unclear. Herein, we show that IIId2 sub-domains from divergent viruses have different functions. The IIId2 sub-domain present in Seneca valley virus (SVV), a picornavirus, is dispensable for IRES activity, while the IIId2 sub-domains of two pestiviruses, classical swine fever virus (CSFV) and border disease virus (BDV), are required for 80S ribosomes assembly and IRES activity. Unlike in SVV, the deletion of IIId2 from the CSFV and BDV IRES elements impairs initiation of translation by inhibiting the assembly of 80S ribosomes. Consequently, this negatively affects the replication of CSFV and BDV. Finally, we show that the SVV IIId2 sub-domain is required for efficient viral RNA synthesis and growth of SVV, but not for IRES function. This study sheds light on the molecular evolution of viruses by clearly demonstrating that conserved RNA structures, within distantly related RNA viruses, have acquired different roles in the virus life cycles.