School of Medicine, Department of Medial Microbiology and Immunology, University of California Davis, Davis, CA, USA.
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
J Gen Virol. 2022 May;103(5). doi: 10.1099/jgv.0.001740.
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.
CD163 是一种巨噬细胞特异性膜吞噬受体，可作为猪繁殖与呼吸综合征病毒 (PRRSV) 的细胞进入受体。去除 CD163 的富含半胱氨酸的清道夫受体结构域 5 (SRCR5) 足以使转染细胞或基因修饰的猪对 PRRSV-1 和 PRRSV-2 基因型产生抗性，并且用来自人 CD163 样蛋白 (hCD163L1) 的 SRCR8 替代 SRCR5 可赋予对 PRRSV-1 的抗性，但不能赋予对 PRRSV-2 分离株的抗性。然而，与 PRRSV 感染相关的 SRCR5 多肽中的特定区域在很大程度上仍不清楚。在本报告中，我们进行了突变研究，以确定 SRCR5 结构域中哪些区域或氨基酸序列对于 PRRSV 感染至关重要。本研究中使用的方法是在 SRCR5 多肽上进行脯氨酸-精氨酸 (PR) 插入。将构建体转染到 HEK293T 细胞中，然后评估其对 PRRSV-2 或 PRRSV-1 的感染情况。对于 PRRSV-2，位于氨基酸 8（PR-9），47（PR-48），54（PR-55）和 99（PR-100）之后的四个 PR 插入对感染的影响最大。对于 PRRSV-1，位于氨基酸 57（PR-58）和 99（PR-100）之后的插入是关键的。基于 SRCR5 晶体结构的计算机模拟表明，影响感染的突变定位于 3-D 结构表面的相似区域。具体来说，我们发现了两个对 PRRSV 感染至关重要的表面斑块。仅相隔三个氨基酸的 PR-58 和 PR-55 对 PRRSV-1 和 PRRSV-2 具有相互作用。用赖氨酸 58 替代谷氨酸 58 可降低 PRRSV-1 的感染而不影响 PRRSV-2，这部分解释了先前观察到的用同源物人 SRCR8 替换 SRCR5 引起的对 PRRSV-1 的抗性。最后，破坏 SRCR5 内的四个保守二硫键中的任何一个都会导致感染抵抗。总之，这些结果证实了 PRRSV-1 和 PRRSV-2 在识别 CD163 方面存在明显差异；然而，所有影响感染的突变都位于 SRCR5 的同一面上的相似区域。
