Mutations within scavenger receptor cysteine-rich (SRCR) protein domain 5 of porcine CD163 involved in infection with porcine reproductive and respiratory syndrome virus (PRRS)

CD163, a macrophage-specific membrane scavenger receptor, serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The removal of scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR5) of CD163 is sufficient to make transfected cells or genetically modified pigs resistant to PRRSV-1 and PRRSV-2 genotypes, and substitution of SRCR5 with SRCR8 from human CD163-like protein (hCD163L1) confers resistance to PRRSV-1 but not PRRSV-2 isolates. However, the specific regions within the SRCR5 polypeptide involved in PRRSV infection remain largely unknown. In this report, we performed mutational studies in order to identify which regions or amino acid sequences in the SRCR5 domain are critical for PRRSV infection. The approach used in this study was to make proline-arginine (PR) insertions along the SRCR5 polypeptide. Constructs were transfected into HEK293T cells, and then evaluated for infection with PRRSV-2 or PRRSV-1. For PRRSV-2, four PR insertions located after amino acids 8 (PR-9), 47 (PR-48), 54 (PR-55), and 99 (PR-100) had the greatest impact on infection. For PRRSV-1, insertions after amino acids 57 (PR-58) and 99 (PR-100) were critical. Computer simulations based on the crystal structure of SRCR5 showed that the mutations that affected infection localized to a similar region on the surface of the 3-D structure. Specifically, we found two surface patches that are essential for PRRSV infection. PR-58 and PR-55, which were separated by only three amino acids, had reciprocal effects on PRRSV-1 and PRRSV-2. Substitution of Glu-58 with Lys-58 reduced PRRSV-1 infection without affecting PRRSV-2, which partially explains the resistance to PRRSV-1 caused by the SRCR5 replacement with the homolog human SRCR8 previously observed. Finally, resistance to infection was observed following the disruption of any of the four conserved disulfide bonds within SRCR5. In summary, the results confirm that there are distinct differences between PRRSV-1 and PRRSV-2 on recognition of CD163; however, all mutations that affect infection locate on a similar region on the same face of SRCR5.