State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China.
J Virol. 2022 Dec 21;96(24):e0162622. doi: 10.1128/jvi.01626-22. Epub 2022 Dec 1.
Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, not only causes diarrhea in piglets but also possesses the potential to infect humans. To better understand host-virus genetic dependencies and find potential therapeutic targets for PDCoV, we used a porcine single-guide RNA (sgRNA) lentivirus library to screen host factors related to PDCoV infection in LLC-PK1 cells. The solute carrier family 35 member A1 (SLC35A1), a key molecule in the sialic acid (SA) synthesis pathway, was identified as a host factor required for PDCoV infection. A knockout of SLC35A1 caused decreases in the amounts of cell surface sialic acid (SA) and viral adsorption; meanwhile, trypsin promoted the use of SA in PDCoV infection. By constructing and assessing a series of recombinant PDCoV strains with the deletion or mutation of possible critical domain or amino acid residues for SA binding in the S1 N-terminal domain, we found that S T182 might be a PDCoV SA-binding site. However, the double knockout of SLC35A1 and amino peptidase N (APN) could not block PDCoV infection completely. Additionally, we found that different swine enteric coronaviruses, including transmissible gastroenteritis coronavirus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome coronavirus, are differentially dependent on SA. Overall, our study uncovered a collection of host factors that can be exploited as drug targets against PDCoV infection and deepened our understanding of the relationship between PDCoV and SA. Identifying the host factors required for replication will be helpful to uncover the pathogenesis mechanisms and develop antivirals against the emerging coronavirus porcine deltacoronavirus (PDCoV). Herein, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout screen, the results of which revealed that the solute carrier family 35 member A1 (SLC35A1) is a host factor required for PDCoV infection that acts by regulating cell surface sialic acid (SA). We also identified the T182 site in the N-terminal domain of PDCoV S1 subunit as being associated with the SA-binding site and found that trypsin promotes the use of cell surface SA by PDCoV. Furthermore, different swine enteric coronaviruses use SLC35A1 differently for infection. This is the first study to screen host factors required for PDCoV replication using a genome-wide CRISPR-Cas9 functional knockout, thereby providing clues for developing antiviral drugs against PDCoV infection.
猪德尔塔冠状病毒（PDCoV）是一种新兴的肠道致病性冠状病毒，不仅会导致仔猪腹泻，还具有感染人类的潜力。为了更好地了解宿主-病毒的遗传依赖性，并为 PDCoV 找到潜在的治疗靶点，我们使用猪的单引导 RNA（sgRNA）慢病毒文库在 LLC-PK1 细胞中筛选与 PDCoV 感染相关的宿主因子。溶质载体家族 35 成员 A1（SLC35A1）是唾液酸（SA）合成途径中的关键分子，被鉴定为 PDCoV 感染所需的宿主因子。SLC35A1 的敲除导致细胞表面唾液酸（SA）的量减少和病毒吸附减少；同时，胰蛋白酶促进了 SA 在 PDCoV 感染中的使用。通过构建和评估一系列重组 PDCoV 菌株，这些菌株在 S1 N 端结构域中可能的关键结构域或氨基酸残基缺失或突变以用于 SA 结合，我们发现 S T182 可能是 PDCoV 的 SA 结合位点。然而，SLC35A1 和氨基肽酶 N（APN）的双敲除并不能完全阻断 PDCoV 的感染。此外，我们发现不同的猪肠道冠状病毒，包括传染性胃肠炎冠状病毒、猪流行性腹泻病毒和猪急性腹泻综合征冠状病毒，对 SA 的依赖性不同。总的来说，我们的研究揭示了一系列宿主因子，这些因子可以被利用作为针对 PDCoV 感染的药物靶点，并加深了我们对 PDCoV 与 SA 之间关系的理解。确定复制所需的宿主因子将有助于揭示发病机制并开发针对新兴冠状病毒猪德尔塔冠状病毒（PDCoV）的抗病毒药物。在这里，我们进行了全基因组聚类规则间隔短回文重复（CRISPR）-Cas9 敲除筛选，结果表明溶质载体家族 35 成员 A1（SLC35A1）是 PDCoV 感染所需的宿主因子，通过调节细胞表面唾液酸（SA）起作用。我们还确定了 PDCoV S1 亚单位 N 端结构域中的 T182 位点与 SA 结合位点有关，并发现胰蛋白酶促进 PDCoV 利用细胞表面 SA。此外，不同的猪肠道冠状病毒对 SLC35A1 的感染利用方式不同。这是首次使用全基因组 CRISPR-Cas9 功能敲除筛选 PDCoV 复制所需的宿主因子的研究，从而为开发针对 PDCoV 感染的抗病毒药物提供了线索。
