Understanding the CD163 - PRRS virus interaction to improve genetic engineering for resistance

猪繁殖与呼吸综合征(PRRS)是造成重大经济损失的猪的一种流行性病毒性疾病。PRRS病毒(PRRS Virus，PRRSV)是一种快速进化的小囊膜RNA病毒。虽然随着畜牧业和疫苗接种的改变而有所改善，但猪繁殖与呼吸综合征仍然对猪的健康和福利产生重大影响。在美国养猪业每年6.5亿美元的传染病成本中，PRRS约占三分之一。PRRS是欧洲、中国和北美养猪业代价最高的疾病，新的PRRSV变种可能具有更大的破坏性，最近在中国和东南亚爆发的高致病变种病毒就证明了这一点。PRRSV具有狭隘的宿主细胞趋向性，仅限于单核/巨噬细胞系的细胞。像所有病毒一样，PRRSV依靠宿主细胞机制进行自我复制并产生新的病毒颗粒。其中一种宿主蛋白CD163被发现对病毒在膜融合进入过程中逃离摄取小泡是必不可少的。CD163表达于巨噬细胞表面，其胞外部分形成一个由九个清道夫受体富含半胱氨酸(SRCR)结构域组成的串珠结构。SRCR结构域5(SRCR5)对PRRSV感染宿主细胞的能力是必不可少的。除了介导PRRSV感染外，CD163在宿主细胞内还具有多种重要功能，如从血液中清除血红蛋白-结合珠蛋白复合体，从而防止可能损害其他细胞的活性氧物种的形成。我最近与其他人的研究表明，基因组编辑是对抗PRRSV感染的一种可行的工具，并证实了CD163在这一过程中的重要性。另一个研究小组的研究表明，CD163基因被敲除的猪对PRRSV感染具有抵抗力。然而，CD163在这些动物中的表达和功能被完全取消，因此它们从血液中清除血红蛋白/结合珠蛋白复合体的能力、缺血修复和炎症反应可能会受到损害。我的工作是通过从猪基因组中删除编码外显子(DeltaSRCR5)来删除SRCR5。我发现这些动物的巨噬细胞不仅对PRRSV感染有抵抗力，而且在巨噬细胞表面表达截短的SRCR5 CD163，同时保持血红蛋白-结合珠蛋白的清除功能。预计SRCR5的移除从而破坏了PRRSV与其受体的钥匙-锁相互作用。因此，它阻止了PRRSV与宿主细胞膜的融合。然而，我们对CD163-PRRSV相互作用的了解还不够，无法排除这种高度突变的RNA病毒适应DeltaSRCR5细胞或猪的可能性。因此，我们需要进一步了解CD163-PRRSV的相互作用，并深入了解PRRSV适应DeltaSRCR5修饰的能力。我对deltaSRCR5的研究表明，从CD163中移除单个外显子可以消除PRRSV感染，强调了基因组编辑技术诱导微妙遗传变化的强大力量。这个项目的目的是进一步评估CD163-PRRSV的相互作用和PRRSV适应deltaSRCR5猪的潜力，以及产生比目前deltaSRCR5猪存在的更微妙的猪基因组变化。我提出了以下策略来解决这些问题：确定CD163的结构域和氨基酸与PRRSV的相互作用；评估PRRSV-CD163的信号和相互作用组；在DeltaSRCR5猪的细胞上连续传代PRRSV以模仿病毒的进化；通过外显子跳过而不是外显子缺失来培育功能强大的DeltaSRCR5猪。

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic viral disease of pigs that causes major economic losses. The causative agent of PRRS, PRRS virus (PRRSV) is a rapidly evolving small enveloped RNA virus. Whilst improvements have been effected with changes in husbandry and vaccination, PRRS still has major impacts on pig health and welfare. PRRS accounts for about a third of the cost of infectious disease to the US pig industry amounting to $650M per year. PRRS is the most costly disease to pig industries of Europe, China and North America and new PRRSV variants have the potential to be even more devastating as demonstrated by recent outbreaks of highly pathogenic variants of the virus in China and Southeast Asia. PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. Like all viruses PRRSV relies on the host cell machinery to replicate itself and generate new viral particles. One of these host proteins, CD163, has been found to be essential for the virus' escape from the uptake vesicle during the entry process by membrane fusion. CD163 is expressed on the surface of macrophages and the extracellular portion of CD163 forms a pearl-on-a-string structure of nine scavenger receptor cysteine-rich (SRCR) domains. SRCR domain 5 (SRCR5) was found to be essential for PRRSV's ability to infect a host cell. In addition to mediating PRRSV infection, CD163 has a variety of important functions within the host cell, such as removing haemoglobin-haptoglobin complexes from the blood, thereby preventing the formation of reactive oxygen species that could damage other cells.My recent research together with others demonstrated that genome editing is a viable tool to combat PRRSV infection and confirmed the importance of CD163 in in this process. Work by another research group has shown that pigs in which the CD163 gene was knocked-out are resistant to PRRSV infection. However, the expression and function of CD163 in these animals is completely abolished and thus their ability to remove haemoglobin/haptoglobin complexes from blood, ischaemic repair and inflammation response may be impaired. My work removed SRCR5 by deletion (deltaSRCR5) of the encoding exon from the pig genome. I found that macrophage cells from these animals were not only resistant to PRRSV infection but also expressed the truncated deltaSRCR5 CD163 on the macrophage surface whilst maintaining haemoglobin-haptoglobin scavenger function.It is expected that removal of SRCR5 and thereby destruction of the "lock" abrogates the key-lock interaction of PRRSV with its receptor. Thereby, it prevents fusion of PRRSV with the host cell membrane. However, we do not understand enough of the CD163-PRRSV interaction to exclude the potential for this highly mutagenic RNA virus to adapt to a deltaSRCR5 cell or pig. Therefore, we need further understanding of the CD163 - PRRSV interaction and insight into PRRSV's ability to adapt to the deltaSRCR5 modification. My research on deltaSRCR5 shows that removal of a single exon from CD163 can abolish PRRSV infection, emphasising the great power of genome editing technology to induce subtle genetic changes.The aim of this project is to further assess the CD163 - PRRSV interaction and the potential of the PRRSV to adapt to a deltaSRCR5 pig as well as to generate even more subtle changes to the pig genome than exist in the current deltaSRCR5 pigs. I propose the following strategies to address these questions: to identify domains and amino acids of CD163 interacting with PRRSV; to assess the PRRSV-CD163 signalling and interactome; to serially passage PRRSV on cells from deltaSRCR5 pigs to mimic virus evolution; to produce functional deltaSRCR5 pigs by exon skipping rather than exon deletion.