鸟苷酸结合蛋白GBP1和GBP2抑制猪轮状病毒体外复制

doi:10.11843/j.issn.0366-6964.2024.01.023

Abstract

Abstract: The aim of this study was to investigate the effects of the full length, fragmentation and GTPase activity of guanylate-binding protein-1 (GBP1) and guanylate-binding protein-2 (GBP2) on the replication of porcine rotavirus (PoRV). The full-length and truncations (G, M, E, ME regions) of GBP1 and GBP2 genes were amplified by PCR using porcine kidney cell cDNA and GBP1 and GBP2 plasmids as templates and cloned into pCDNA3.1(+) eukaryotic expression vector. Overexpression or silencing of GBP1 and GBP2 genes on rhesus monkey cells (MA104) cells was performed to explore the effect on PoRV replication. Different time variables were set to screen the time points at which GBP1 and GBP2 exerted their effects on PoRV replication. Apply pan-GTPase enzyme inhibitor (CID-1067700) to probe the effect of GTPase enzyme activity of GBP1 and GBP2 on PoRV. The expression of GBP1 and GBP2 proteins were detected by Western blot, quantitative PCR and viral TCID₅₀ assays, and their antiviral functions were investigated. The results showed that pCDNA3.1 (+)-GBP1-full-length-HIS and pCDNA3.1(+)-GBP2(47aa-592aa), (131aa-592aa)-HIS recombinant plasmids were successfully constructed and verified to be expressd. PoRV significantly promoted up-regulations of GBP1 and GBP2 at protein and mRNA expressions. Overexpression of GBP1 and GBP2 significantly inhibited PoRV replication, and silencing the expression of GBP1 and GBP2 genes significantly promoted PoRV replication. The pCDNA3.1 (+)-GBP1/GBP2-G, M, E, ME-HIS structural domain truncations of GBP1 and GBP2 proteins were successfully constructed and verified to be expressed. Overexpression of the truncator gene revealed that the G region of GBP1 and GBP2 exhibited an inhibitory effect on PoRV replication. It was verified by pan-GTPase inhibitory enzyme activity that GTPase enzyme activity is required for GBP1 and GBP2 to inhibit PoRV replication. The results suggest that the GBP1 and GBP2 proteins are able to inhibit PoRV replication at replication phase of the virus, and the inhibitory effect dependents on its G region containing the GTPase enzyme active sites. The results of the study provide a theoretical basis for finding new drug targets for PoRV and developing novel vaccines.

Key words: porcine rotavirus, GBP1, GBP2, antiviral, structural domain

CLC Number:

S852.659.4

CHEN Shuyu, ZHU Xuejiao, ZHOU Jinzhu, TAO Ran, ZHANG Xuehan, Suolangsizhu, Gongga, LI Bin. Inhibition of Porcine Rotavirus in vitro Replication by Guanylate-binding Proteins GBP1 and GBP2[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 245-257.

References 22

[1]	HULL J J A, QI M P, MONTMAYEUR A M, et al. Metagenomic sequencing generates the whole genomes of porcine rotavirus A, C, and H from the United States[J]. PLoS One, 2020, 15(12):e0244498, doi:10. 1371/journal. pone. 0244498.
[2]	LI Y, WANG F X, KAN R C, et al. Genetic and immunological characterization of G9 group A porcine rotaviruses in China[J]. Zoonoses Public Health, 2022, 69(6):694-703, doi:10. 1111/zph. 12958.
[3]	XUE R, TIAN Y, ZHANG Y, et al. Diversity of group A?rotavirus of porcine rotavirus in Shandong province China[J]. Acta Virol, 2018, 62(3):229-234, doi:10. 4149/av_2018_216.
[4]	CUI T T, THEUNS S, XIE J X, et al. Porcine rotavirus mainly infects primary porcine enterocytes at the basolateral surface[J]. Vet Res, 2019, 50(1):110, doi:10. 1186/s13567-019-0728-x.
[5]	SONG D S, KANG B K, OH J S, et al. Multiplex reverse transcription-PCR for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group A rotavirus[J]. J Vet Diagn Invest, 2006, 18(3):278-281, doi:10. 1177/104063870601800309.
[6]	MEMON A M, CHEN F Z, KHAN S B, et al. Development and evaluation of polyclonal antibodies based antigen capture ELISA for detection of porcine rotavirus[J]. Anim Biotechnol, 2023, 34(5):1807-1814, doi:10. 1080/10495398. 2022. 2052304.
[7]	LI L F, YU J H, LI Y F, et al. Guanylate-binding protein 1, an interferon-induced GTPase, exerts an antiviral activity against classical swine fever virus depending on its GTPase activity[J]. J Virol, 2016, 90(9):4412-4426, doi:10. 1128/JVI. 02718-15.
[8]	CUI W, BRAUN E, WANG W, et al. Structural basis for GTP-induced dimerization and antiviral function of guanylate-binding proteins[J]. Proc Natl Acad Sci U S A, 2021, 118(15):e2022269118, doi:10. 1073/pnas. 2022269118.
[9]	DEGRANDI D, KONERMANN C, BEUTER-GUNIA C, et al. Extensive characterization of IFN-induced GTPases mGBP1 to mGBP10 involved in host defense[J]. J Immunol, 2007, 179(11):7729-7740, doi:10. 4049/jimmunol. 179. 11. 7729.
[10]	DEGRANDI D, KRAVETS E, KONERMANN C, et al. Murine guanylate binding protein 2 (mGBP2) controls Toxoplasma gondii replication[J]. Proc Natl Acad Sci U S A, 2013, 110(1):294-299.
[11]	陈梦阁. 小鼠巨噬细胞GBP2和GBP5在抗新孢子虫感染中的作用[D]. 长春:吉林大学, 2021, doi:10. 27162/d. cnki. gjlin. 2021. 006159.CHEN M G. The roles of GBP2 and GBP5 in resistance to Neospora caninum infection in mouse macrophages[D]. Changchun:Jilin University, 2021, doi:10. 27162/d. cnki. gjlin. 2021. 006159. (in Chinese)
[12]	DUAN H, DONG H X, WU S Y, et al. Porcine reproductive and respiratory syndrome virus non-structural protein 4 cleaves guanylate-binding protein 1 via its cysteine proteinase activity to antagonize GBP1 antiviral effect[J]. Vet Res, 2022, 53(1):55, doi:10. 1186/s13567-022-01071-8.
[13]	晏星. 猪GBP1、GBP2基因的克隆及其对PRRSV和PRV增殖的影响[D]. 武汉:华中农业大学, 2014.YAN X. Cloning of porcine GBP1 and GBP2 genes and investigation of its anti-viral effect on PRRSV and PRV proliferation[D]. Wuhan:Huazhong Agricultural University, 2014. (in Chinese)
[14]	牛颖颖. 宿主蛋白GBP1在猪伪狂犬病病毒感染过程中的作用[D]. 杨凌:西北农林科技大学, 2020, doi:10. 27409/d. cnki. gxbnu. 2020. 001681.NIU Y Y. The role of host protein GBP1 in pseudorabies virus infection[D]. Yangling:Northwest A&F University, 2020, doi:10. 27409/d. cnki. gxbnu. 2020. 001681. (in Chinese)
[15]	杨黎. 口蹄疫病毒VP1蛋白经GBP1和TP53调控病毒复制与细胞凋亡研究[D]. 南昌:江西农业大学, 2022, doi:10. 27177/d. cnki. gjxnu. 2022. 000443.YANG L. Study on the regulation of VP1 protein of foot-and-mouth disease virus on virus replication and apoptosis by GBP1 and TP53[D]. Nanchang:Jiangxi Agricultural University, 2022, doi:10. 27177/d. cnki. gjxnu. 2022. 000443. (in Chinese)
[16]	ANDERSON S L, CARTON J M, LOU J, et al. Interferon-induced guanylate binding protein-1 (GBP-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus[J]. Virology, 1999, 256(1):8-14.
[17]	常新见, 周金柱, 范宝超, 等. 猪轮状病毒NJ2012株VP8基因序列分析及原核表达[J]. 畜牧与兽医, 2020, 52(10):68-72.CHANG X J, ZHOU J Z, FAN B C, et al. Sequence analysis and prokaryotic expression of the VP8 gene of porcine rotavirus strain NJ2012[J]. Animal Husbandry & Veterinary Medicine, 2020, 52(10):68-72. (in Chinese)
[18]	朱紫祥, 曹阳春, 曹伟军, 等. 干扰素诱导蛋白鸟苷酸结合蛋白1研究进展[J]. 病毒学报, 2014, 30(4):456-462, doi:10. 13242/j. cnki. bingduxuebao. 002532.ZHU Z X, CAO Y C, CAO W J, et al. Recent progress in interferon induced protein GBP1 research[J]. Chinese Journal of Virology, 2014, 30(4):456-462, doi:10. 13242/j. cnki. bingduxuebao. 002532. (in Chinese)
[19]	张家祺, 甲呷格西, 周群, 等. 宏基因组学技术分析四川省集约化猪场中仔猪腹泻粪便的病毒种群[J]. 畜牧兽医学报, 2023, 54(6):2478-2486.ZHANG J Q, JIA G G X, ZHOU Q, et al. Metagenomics analysis of virus populations in piglet diarrheal feces from intensive pig farms in Sichuan province[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6):2478-2486. (in Chinese)
[20]	ZHOU J Z, HUANG S M, FAN B C, et al. iTRAQ-based proteome analysis of porcine group A rotavirus-infected porcine IPEC-J2 intestinal epithelial cells[J]. J Proteomics, 2021, 248:104354, doi:10. 1016/j. jprot. 2021. 104354.
[21]	PLACE D E, BRIARD B, SAMIR P, et al. Interferon inducible GBPs restrict Burkholderia thailandensis motility induced cell-cell fusion[J]. PLoS Pathog, 2020, 16(3):e1008364, doi:10. 1371/journal. ppat. 1008364.
[22]	NORDMANN A, WIXLER L, BOERGELING Y, et al. A new splice variant of the human guanylate-binding protein 3 mediates anti-influenza activity through inhibition of viral transcription and replication[J]. FASEB J, 2012, 26(3):1290-1300, doi:10. 1096/fj. 11-189886.

Inhibition of Porcine Rotavirus in vitro Replication by Guanylate-binding Proteins GBP1 and GBP2

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