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

Abstract

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.

Recent advances in inhibition of porcine reproductive and respiratory syndrome virus through targeting CD163

“…However, the exact amino acid sequences within SRCR5 domain involved in PRRSV infection remain unclear. To address it, mutational research within CD163 SRCR5 was performed and all four disulfide bonds in SRCR5 are critical and required to support PRRSV infection and proliferation (Stoian et al, 2022). Further, replacement of cysteine at positions 10, 39, 70, and 90 with alanine in SRCR5 region confers resistance of cells to PRRSV infection (Stoian et al, 2022), which suggests that these amino acids are necessary for virus infection (Figure 2).…”

“…To address it, mutational research within CD163 SRCR5 was performed and all four disulfide bonds in SRCR5 are critical and required to support PRRSV infection and proliferation (Stoian et al, 2022). Further, replacement of cysteine at positions 10, 39, 70, and 90 with alanine in SRCR5 region confers resistance of cells to PRRSV infection (Stoian et al, 2022), which suggests that these amino acids are necessary for virus infection (Figure 2). This research also provides an explanation for our study that pigs lacking 41 amino acids which includes three disulfide bonds within SRCR5 domain are not susceptible to infection by PRRSV.…”

“…Therefore, it is promising to develop molecular drugs targeting virus glycoproteins or host receptors. Previous studies have shown that PRRSV glycoproteins such as GP2, GP3, GP4, and GP5 interact with host receptor CD163 to mediate virus entry and uncoating (Stoian et al, 2022). There are various strategies to block virus uncoating.…”

Role of N-linked glycosylation in porcine reproductive and respiratory syndrome virus (PRRSV) infection

“…The construction of the recombinant plasmids, which express the viral GP2, GP3, GP4 and GP5 from P129 strain [39] fused to a HA epitope, have been described previously [33]. The plasmids expressing the different viral glycoproteins, a Q5 site-directed mutagenesis kit (New England Biolabs) and specific primers (Table S2) were used according to the manufacturer's specifications…”

Reappraising host cellular factors involved in attachment and entry to develop antiviral strategies against porcine reproductive and respiratory syndrome virus

“…The crystal structure of the CD163 SRCR5 domain determined by us greatly facilitates precise control and prevention of PRRS from the receptor perspective ( Ma et al, 2017 , 2021 ). Site-directed mutagenesis of the CD163 SRCR5 domain will be beneficial for breeding gene-edited pigs resistant to PRRSV while maintaining CD163 biological functions ( Stoian et al, 2022 ). Based on the crystal structure, a set of small molecule compounds targeting CD163 SRCR5 have been screened through artificial intelligence molecular screening and validated against PRRSV infection in vitro ( Huang et al, 2020 ).…”