CD163位点特异性编辑的猪肺泡巨噬细胞可以降低对猪繁殖与呼吸综合征病毒感染的易感性

doi:10.1016/j.jia.2022.11.010

Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (7): 2188-2199.DOI: 10.1016/j.jia.2022.11.010

CD163位点特异性编辑的猪肺泡巨噬细胞可以降低对猪繁殖与呼吸综合征病毒感染的易感性

收稿日期:2022-09-16 接受日期:2022-11-10 出版日期:2023-07-20 发布日期:2022-11-10

Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus

XU Kui^{1, 2}^*, ZHOU Yan-rong³^*, SHANG Hai-tao^{2, 4}^*, XU Chang-jiang¹, TAO Ran³, HAO Wan-jun³, LIU Sha-sha¹, MU Yu-lian^1#, XIAO Shao-bo^3#, LI Kui^1#

¹State Key Laboratory of Animal Nutrition and Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
²Shenzhen Kingsino Technology Co., Ltd., Shenzhen 518106, P.R.China
³State Key Laboratory of Agricultural Microbiology and Key Laboratory of Preventive Veterinary Medicine in Hubei Province, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, P.R.China
⁴Precision Medicine Institute, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, P.R.China

Received:2022-09-16 Accepted:2022-11-10 Online:2023-07-20 Published:2022-11-10
About author:XU Kui, E-mail: xukui2018xukui@163.com; ZHOU Yan-rong, E-mail: virus@mail.hzau.edu.cn; SHANG Hai-tao, E-mail: shanght@foxmail.com; #Correspondence MU Yu-lian, E-mail: mouyulian@caas.cn; XIAO Shao-bo, E-mail: vet@mail.hzau.edu. cn; LI Kui, E-mail: likui@caas.cn * These authors contributed equally to this study.
Supported by:
This work was supported by the Major Scientific Research Tasks for Scientific and Technological Innovation Projects of the Chinese Academy of Agricultural Sciences (CAAS-ZDRW202006), the National Transgenic Breeding Project (2018ZX08009-26B), the Shenzhen Science and Technology Plan Project (CJGJZD20210408092402006), and the Shenzhen Key Technology Projects (JSGG20180507182028625).

摘要/Abstract

摘要：

猪繁殖与呼吸综合征（Porcine reproductive and respiratory syndrome, PRRS）是猪传染性最强和致死性最高的病毒性疾病之一。尽管分化抗原簇163 (Cluster of differentiation 163, CD163) 蛋白被确定为介导PRRS病毒（Porcine reproductive and respiratory syndrome virus, PRRSV）感染的必需受体，但CD163参与感染的重要氨基酸残基仍不清楚。鉴定这些关键残基对于研究PRRSV的感染机制和制备抗PRRSV的基因编辑猪具有重要意义。对CD163结构的分析表明，CD163 SRCR5结构域内的配体结合口袋(ligand-binding pocket, LBP)(位于CD163的第487位到499位氨基酸)和loop 5–6（位于CD163的第544位到570位氨基酸）可能参与了PRRSV的感染。CD163 LBP位点特异性编辑猪可以完全抵抗PRRSV感染，但loop 5-6的破坏或缺失是否能抑制PRRSV的感染尚未报道。R561（第561位的精氨酸（R））位于loop 5-6内，R561A突变的PK-15细胞可以显著增强对PRRSV的抗感染能力，但定点编辑CD163 R561的猪肺泡巨噬细胞（porcine alveolar macrophages, PAMs）（PRRSV感染的靶细胞）或CD163 R561编辑猪能否抑制PRRSV的感染仍不清楚。这项研究中，我们首先利用CRISPR/Cas9介导的片段删除技术制备了CD163缺失40个氨基酸残基的永生化猪肺泡巨噬细胞（immortalized porcine alveolar macrophage, IPAM）细胞系，这40个残基位于CD163的第523位到562位氨基酸，这其中包含了R561和部分loop 5–6结构，该细胞系被命名为IPAM-CD163^△^523-562。病毒感染实验表明，IPAM-CD163^△^523-562可以完全抵抗 PRRSV的感染。同时，我们利用CRISPR/Cas9介导同源重组技术制备了携带CD163-R561A（CD163的561位精氨酸（R）被替换为丙氨酸（A））的基因编辑克隆猪，并分离得到原代CD163-R561A PAMs。PRRSV攻毒实验结果表明，与野生型的CD163-R561 PAMs相比，CD163-R561A PAMs 对PRRSV的易感性显著降低。以上研究结果表明，CD163的第523位到562位氨基酸中含有介导PRRSV感染的必需氨基酸残基，CD163 R561参与了PRRSV的感染过程，但它不是感染所必需的。这些位点可以作为了解PRRSV感染机制的新靶点，CD163-R561A猪也可作为培育抗PRRSV猪群体的育种材料。

Abstract: Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most infectious viral diseases of swine. Although Cluster of differentiation 163 (CD163) is identified as an essential receptor for mediating PRRS virus (PRRSV) infection, the important residues involved in infection on CD163 are still unclear. Therefore, it is very important to identify these key residues to study the mechanism of PRRSV infection and to generate anti-PRRSV pigs. In this study, we first generated immortalized porcine alveolar macrophage (IPAM) cell lines harboring 40-residues (residues 523–562, including R561 (arginine (R) at position 561)) deletion of CD163. PRRSV infection experiments showed that these IPAM cell lines were completely resistant to PRRSV infection. We then generated cloned pigs carrying CD163- R561A (an arginine (R) to alanine (A) substitution at position 561 of CD163). PRRSV challenge experiments in porcine alveolar macrophages (PAMs) isolated from the CD163-R561A pigs showed significantly lower susceptibility to PRRSV than that of CD163-R561 PAMs. Through this study, we show that CD163 523–562 contains essential residues for mediating PRRSV infection, and that CD163 R561 significantly contributes to PRRSV infection but is not essential for infection. These functional sites can therefore serve as new targets for understanding the mechanism of PRRSV infection. Furthermore, CD163-R561A pigs can be used as an important model for improving pig germplasm with resistance against PRRSV.

Key words: pigs , porcine alveolar macrophages , dual-sgRNA , homology-directed repair , PRRSV , CD163

XU Kui, ZHOU Yan-rong, SHANG Hai-tao, XU Chang-jiang, TAO Ran, HAO Wan-jun, LIU Sha-sha, MU Yu-lian, XIAO Shao-bo, LI Kui. CD163位点特异性编辑的猪肺泡巨噬细胞可以降低对猪繁殖与呼吸综合征病毒感染的易感性[J]. Journal of Integrative Agriculture, 2023, 22(7): 2188-2199.

XU Kui, ZHOU Yan-rong, SHANG Hai-tao, XU Chang-jiang, TAO Ran, HAO Wan-jun, LIU Sha-sha, MU Yu-lian, XIAO Shao-bo, LI Kui. Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus[J]. Journal of Integrative Agriculture, 2023, 22(7): 2188-2199.

参考文献

An T Q, Li J N, Su C M, Yoo D. 2020. Molecular and cellular mechanisms for PRRSV pathogenesis and host response to infection. Virus Research, 286, 197980.

Breedam W V, Delputte P L, Gorp H V, Misinzo G, Vanderheijden N, Duan X B, Nauwynck H J. 2010. Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage. Journal of General Virology, 91, 1659–1667.

Burkard C, Lillico S G, Reid E, Jackson B, Mileham A J, Ait-Ali T, Whitelaw C B, Archibald A L. 2017. Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLoS Pathogens, 13, e1006206.

Calvert J G, Slade D E, Shields S L, Jolie R, Mannan R M, Ankenbauer R G, Welch S K. 2007. CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses. Journal of Virology, 81, 7371–7379.

Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science, 339, 819–823.

Crocker P R, Gordon S. 1986. Properties and distribution of a lectin-like hemagglutinin differentially expressed by murine stromal tissue macrophages. Journal of Experimental Medicine, 164, 1862–1875.

Dhakal S, Renukaradhya G J. 2019. Nanoparticle-based vaccine development and evaluation against viral infections in pigs. Veterinary Research, 50, 90.

Gorp H V, Breedam W V, Doorsselaere J V, Delputte P L, Nauwynck H J. 2010a. Identification of the CD163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus. Journal of Virology, 84, 3101–3105.

Gorp H V, Delputte P L, Nauwynck H J. 2010b. Scavenger receptor CD163, a Jack-of-all-trades and potential target for cell-directed therapy. Molecular Immunology, 47, 1650–1660.

Graversen J H, Madsen M, Moestrup S K. 2002. CD163: A signal receptor scavenging haptoglobin-hemoglobin complexes from plasma. International Journal of Biochemistry & Cell Biology, 34, 309–314.

Guo C H, Wang M, Zhu Z B, He S, Liu H B, Liu X F, Shi X, Tang T, Yu P, Zeng J H, Yang L F, Cao Y C, Chen Y S, Liu X H, He Z Y. 2019. Highly efficient generation of pigs harboring a partial deletion of the CD163 SRCR5 domain, which are fully resistant to porcine reproductive and respiratory syndrome virus 2 infection. Frontiers in Immunology, 10, 1846.

Jiang Y B, Fang L R, Luo R, Xiao S B, Chen H C. 2010. N-acetylpenicillamine inhibits the replication of porcine reproductive and respiratory syndrome virus in vitro. Veterinary Research Communications, 34, 607–617.

Jusa E R, Inaba Y, Kouno M, Hirose O. 1997. Effect of heparin on infection of cells by porcine reproductive and respiratory syndrome virus. American Journal of Veterinary Research, 58, 488–491.

Kappes M A, Faaberg K S. 2015. PRRSV structure, replication and recombination: Origin of phenotype and genotype diversity. Virology, 479–480, 475–486.

Kowal K, Silver R, Slawinska E, Bielecki M, Chyczewski L, Kowal-Bielecka O. 2011. CD163 and its role in inflammation. Folia Histochemica et Cytobiologica, 49, 365–374.

Li B, Fang L R, Xu Z F, Liu S Y, Gao J F, Jiang Y B, Chen H C, Xiao S B. 2009. Recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses. Emerging Infectious Diseases, 15, 2032–2035.

Li X P, Yang Y, Bu L, Guo X G, Tang C C, Song J, Fan N N, Zhao B T, Ouyang Z, Liu Z M, Zhao Y, Yi X L, Quan L Q, Liu S C, Yang Z G, Ouyang H S, Chen Y E, Wang Z, Lai L X. 2014. Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Research, 24, 501–504.

Lunney J K, Fang Y, Ladinig A, Chen N, Li Y, Rowland B, Renukaradhya G J. 2016. Porcine reproductive and respiratory syndrome virus (PRRSV): Pathogenesis and interaction with the immune system. Annual Review of Animal Biosciences, 4, 129–154.

Ma H F, Jiang L G, Qiao S L, Zhi Y B, Chen X X, Yang Y Y, Huang X J, Huang M D, Li R, Zhang G P. 2017. The crystal structure of the fifth scavenger receptor cysteine-rich domain of porcine CD163 reveals an important residue involved in porcine reproductive and respiratory syndrome virus infection. Journal of Virology, 91, e01897–e01913.

Martin-Valls G E, Kvisgaard L K, Tello M, Darwich L, Cortey M, Burgara-Estrella A J, Hernandez J, Larsen L E, Mateu E. 2014. Analysis of ORF5 and full-length genome sequences of porcine reproductive and respiratory syndrome virus isolates of genotypes 1 and 2 retrieved worldwide provides evidence that recombination is a common phenomenon and may produce mosaic isolates. Journal of Virology, 88, 3170–3181.

Onofre G, Kolackova M, Jankovicova K, Krejsek J. 2009. Scavenger receptor CD163 and its biological functions. Acta Medica (Hradec Kralove), 52, 57–61.

Patton J B, Rowland R R, Yoo D, Chang K O. 2009. Modulation of CD163 receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages. Virus Research, 140, 161–171.

Resendiz M, Valenzuela O, Hernandez J. 2018. Response of the cDC1 and cDC2 subtypes of tracheal dendritic cells to porcine reproductive and respiratory syndrome virus. Veterinary Microbiology, 223, 27–33.

Shi C X, Liu Y L, Ding Y Z, Zhang Y G, Zhang J. 2015. PRRSV receptors and their roles in virus infection. Archives of Microbiology, 197, 503–512.

Stoian A M, Rowland R R, Brandariz-Nuñez A. 2022. Identification of CD163 regions that are required for porcine reproductive and respiratory syndrome virus (PRRSV) infection but not for binding to viral envelope glycoproteins. Virology, 574, 71–83.

Tian K G, Yu X L, Zhao T Z, Feng Y J, Cao Z, Wang C B, Hu Y, Chen X Z, Hu D M, Tian X S, Liu D, Zhang S, Deng X Y, Ding Y Q, Yang L, Zhang Y X, Xiao H X, Qiao M M, Wang B, Hou L L, et al. 2007. Emergence of fatal PRRSV variants: Unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS ONE, 2, e526.

Wang D, Cao L, Xu Z, Fang L R, Zhong Y, Chen Q G, Luo R, Chen H C, Li K, Xiao S B. 2013. MiR-125b reduces porcine reproductive and respiratory syndrome virus replication by negatively regulating the NF-kappaB pathway. PLoS ONE, 8, e55838.

Wang T Y, Liu Y G, Li L, Wang G, Wang H M, Zhang H L, Zhao S F, Gao J C, An T Q, Tian Z J, Tang Y D, Cai X H. 2018. Porcine alveolar macrophage CD163 abundance is a pivotal switch for porcine reproductive and respiratory syndrome virus infection. Oncotarget, 9, 12174–12185.

Wasilk A, Callahan J D, Christopher-Hennings J, Gay T A, Fang Y, Dammen M, Reos M E, Torremorell M, Polson D, Mellencamp M, Nelson E, Nelson W M. 2004. Detection of U.S., Lelystad, and European-like porcine reproductive and respiratory syndrome viruses and relative quantitation in boar semen and serum samples by real-time PCR. Journal of Clinical Microbiology, 42, 4453–4461.

Welch S K, Calvert J G. 2010. A brief review of CD163 and its role in PRRSV infection. Virus Research, 154, 98–103.

Wells K D, Bardot R, Whitworth K M, Trible B R, Fang Y, Mileham A, Kerrigan M A, Samuel M S, Prather R S, Rowland R R R. 2017. Replacement of porcine CD163 scavenger receptor cysteine-rich domain 5 with a CD163-like homolog confers resistance of pigs to genotype 1 but not genotype 2 porcine reproductive and respiratory syndrome virus. Virus Research, 91, e01521–e01516.

Wensvoort G, Terpstra C, Pol J M, ter Laak E A, Bloemraad M, de Kluyver E P, Kragten C, van Buiten L, den Besten A, Wagenaar F, Broekhuijsen J M, Moonen P L, Zetstra T, de Boer E A, Tibben H J, de Jong M F, van’t Veld P, Greenland G J, van Gennep J A, Voets M T, et al. 1991. Mystery swine disease in the Netherlands: The isolation of Lelystad virus. Veterinary Quarterly, 13, 121–130.

Whitworth K M, Rowland R R, Ewen C L, Trible B R, Kerrigan M A, Cino-Ozuna A G, Samuel M S, Lightner J E, McLaren D G, Mileham A J, Wells K D, Prather R S. 2016. Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nature Biotechnolgy, 34, 20–22.

Xu K, Zhou Y R, Mu Y L, Liu Z G, Hou S H, Xiong Y J, Fang L R, Ge C L, Wei Y H, Zhang X L, Xu C J, Che J J, Fan Z Y, Xiang G M, Guo J K, Shang H T, Li H, Xiao S B, Li J L, Li K. 2020. CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance. eLife, 9, e57132.

Yang H Q, Zhang J, Zhang X W, Shi J S, Pan Y F, Zhou R, Li G L, Li Z C, Cai G Y, Wu Z F. 2018. CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus. Antiviral Research, 151, 63–70.

Zhou L, Yang H C. 2010. Porcine reproductive and respiratory syndrome in China. Virus Research, 154, 31–37.

Zhou Y R, Fang L R, Wang D, Cai K M, Chen H C, Xiao S B. 2017. Porcine reproductive and respiratory syndrome virus infection induces stress granule formation depending on protein kinase R-like endoplasmic reticulum kinase (PERK) in MARC-145 cells. Frontiers in Cellular and Infection Microbiology, 7, 111.

CD163位点特异性编辑的猪肺泡巨噬细胞可以降低对猪繁殖与呼吸综合征病毒感染的易感性

Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics