N蛋白磷酸化位点突变影响猪繁殖与呼吸综合征病毒的复制及亚基因组的转录

doi:10.11843/j.issn.0366-6964.2019.12.011

Abstract

Abstract: To explore whether the N-protein phosphorylation of porcine reproductive and respiratory syndrome virus (PRRSV) affects viral replication, the ser-105 and ser-120 of the PRRSV N-protein were mutated into alanine alone or simultaneously. The rescued viruses were named as A105, A120 and A105-120, and their growth curve and subgenomic transcription level were determined respectively. The viral replication rate and titer of the mutant virus in MARC-145 were significantly lower than that of parental strain PRRSV XH-GD (P<0.01 or P<0.05). While in the primary PAM cells, the replication rate of the mutant virus decreased (P<0.01), but the final virus titer was not affected. The genomic (g) RNA transcription level of the mutant virulence group was significantly decreased (P<0.01 or P<0.05), and the phosphorylation site mutation significantly reduced the transcription of the longer subgenomic mRNA (sgmRNA)2/3 (P<0.01 or P<0.05), but increased the expression levels of the shorter sgmRNA 4/5 with time. Based on the gRNA expression level, the expression of sgmRNAs expression was analyzed. Our current work showed that the N-protein phosphorylation significantly affects the expression of sgmRNA4/5(P<0.01 or P<0.05). Mutation of N protein phosphorylation site affects PRRSV replication and subgenomic transcription.

Key words: porcine reproductive and respiratory syndrome virus, N protein, phosphorylation, subgenome

CLC Number:

S852.659.6

YI Heyou, YU Zhiqing, CHEN Yao, LI Qi, WEI Yingfang, ZHANG Guihong. Mutation of N Protein Phosphorylation Site Affects Replication and Subgenomic Transcription of Porcine Reproductive and Respiratory Syndrome Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(12): 2470-2478.

References 32

[1]	CANELLI E, CATELLA A, BORGHETTI P, et al. Phenotypic characterization of a highly pathogenic Italian porcine reproductive and respiratory syndrome virus (PRRSV) type 1 subtype 1 isolate in experimentally infected pigs[J]. Vet Microbiol, 2017, 210:124-133.
[2]	SPARKS J W, KARRIKER L A, DAY D N, et al. Vaccination mitigates the impact of PRRSv infection on the pharmacokinetics of ceftiofur crystalline-free acid in pigs[J]. J Vet Pharmacol Ther, 2017, 40(4):363-369.
[3]	RAMOS N, MIRAZO S, CASTRO G, et al. First-time detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection in Uruguay[J]. Transbound Emerg Dis, 2018, 65(2):352-356.
[4]	TONG G Z, ZHOU Y J, HAO X F, et al. Highly pathogenic porcine reproductive and respiratory syndrome, China[J]. Emerg Infect Dis, 2007, 13(9):1434-1436.
[5]	LIU J K, ZHOU X, ZHAI J Q, et al. Emergence of a novel highly pathogenic porcine reproductive and respiratory syndrome virus in China[J]. Transbound Emerg Dis, 2017, 64(6):2059-2074.
[6]	SUN Y K, HAN X L, WEI Y F, et al. Phylogeography, phylodynamics and the recent outbreak of lineage 3 porcine reproductive and respiratory syndrome viruses in China[J]. Transbound Emerg Dis, 2019, 66(5):2152-2162.
[7]	MUSIC N, GAGNON C A. The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis[J]. Anim Health Res Rev, 2010, 11(2):135-163.
[8]	YU LX, WANG X, YU H, et al. The emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus with additional 120aa deletion in Nsp2 region in Jiangxi, China[J]. Transbound Emerg Dis, 2018, 65(6):1740-1748.
[9]	XU L, ZHOU L, SUN W F, et al. Nonstructural protein 9 residues 586 and 592 are critical sites in determining the replication efficiency and fatal virulence of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus[J]. Virology, 2018, 517:135-147.
[10]	MEULENBERG J J M, PETERSEN-DEN Besten A, DE KLUYVER E P, et al. Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus[J]. Virology, 1995, 206(1):155-163.
[11]	WANG C B, MENG H, GAO Y J, et al. Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus[J]. Vet Res, 2017, 48(1):54.
[12]	WANG F X, YANG Y, LIU X, et al. Development of monoclonal antibody for differentiating porcine reproductive and respiratory syndrome virus and identification of a novel non-structural protein 2 epitope peptide[J]. Virus Disease, 2017, 28(4):408-415.
[13]	JOURDAN S S, OSORIO F, HISCOX J A. An interactome map of the nucleocapsid protein from a highly pathogenic North American porcine reproductive and respiratory syndrome virus strain generated using SILAC-based quantitative proteomics[J]. Proteomics, 2012, 12(7):1015-1023.
[14]	YOO D, WOOTTON S K, LI G, et al. Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-Associated protein fibrillarin[J]. J Virol, 2003, 77(22):12173-12183.
[15]	DUAN X, NAUWYNCK H J, PENSAERT M B. Virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV)[J]. Vet Microbiol, 1997, 56(1-2):9-19.
[16]	SHABIR N, KHATUN A, NAZKI S, et al. In vitro immune responses of porcine alveolar macrophages reflect host immune responses against porcine reproductive and respiratory syndrome viruses[J]. BMC Vet Res, 2018, 14(1):380.
[17]	SUGAI A, SATO H, HAGIWARA K, et al. Newly identified minor phosphorylation site threonine-279 of measles virus nucleoprotein is a prerequisite for nucleocapsid formation[J]. J Virol, 2014, 88(2):1140-1149.
[18]	ZENGEL J, PICKAR A, XU P, et al. Roles of phosphorylation of the nucleocapsid protein of mumps virus in regulating viral RNA transcription and replication[J]. J Virol, 2015, 89(14):7338-7347.
[19]	WU C H, CHEN P J, YEH S H. Nucleocapsid phosphorylation and RNA helicase DDX1 recruitment enables coronavirus transition from discontinuous to continuous transcription[J]. Cell Host Microbe, 2014, 16(4):462-472.
[20]	ALBATAINEH M T, KADOSH D. Regulatory roles of phosphorylation in model and pathogenic fungi[J]. Med Mycol, 2016, 54(4):333-352.
[21]	CHEN Y, XING X L, LI Q, et al. Serine 105 and 120 are important phosphorylation sites for porcine reproductive and respiratory syndrome virus N protein function[J]. Vet Microbiol, 2018, 219:128-135.
[22]	杨峰,周宏超,许信刚,等. 猪繁殖与呼吸综合征病毒实时荧光定量PCR检测方法的建立及初步应用[J]. 动物医学进展,2018, 39(9):25-29.YANG F,ZHOU H C,XU X G, et al. Establishment and preliminary application of a real-time PCR for PRRSV detection[J]. Progress in Veterinary Medicine, 2018, 39(9):25-29. (in Chinese)
[23]	VENTEO A, REBOLLO B, SARRASECA J, et al. A novel double recognition enzyme-linked immunosorbent assay based on the nucleocapsid protein for early detection of European porcine reproductive and respiratory syndrome virus infection[J]. J Virol Met, 2012,181(1):109-113.
[24]	曹宗喜, 焦培荣,谭树义,等. 基于DNA-launched的猪繁殖与呼吸综合征病毒反向遗传操作系统的建立[J]. 动物医学进展, 2016, 37(11):1-8.CAO Z X, JIAO P R, TAN S Y, et al. Establishment of DNA-launched reverse genetics system of PRRSV[J]. Progress in Veterinary Medicine, 2016, 37(11):1-8. (in Chinese)
[25]	CHEN Y, YU Z Q, YI H Y, et al. The phosphorylation of the N protein could affect PRRSV virulence in vivo[J]. Vet Microbiol, 2019, 231:226-231.
[26]	TAKEUCHI H, SAITO H, NODA T, et al. Phosphorylation of the HIV-1 capsid by MELK triggers uncoating to promote viral cDNA synthesis[J]. PLoS Pathog, 2017, 13(7):e1006441.
[27]	SUÁREZ P, DÍAZ-GUERRA M, PRIETO C, et al. Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis[J]. J Virol, 1996, 70(5):2876-2882.
[28]	SHI C X, LIU Y L, DING Y Z, et al. PRRSV receptors and their roles in virus infection[J]. Arch Microbiol, 2015, 197(4):503-512.
[29]	LUNNEY J K, FANG Y, LADINIG A, et al. Porcine reproductive and respiratory syndrome virus (PRRSV):pathogenesis and interaction with the immune system[J]. Annu Rev Anim Biosci, 2016, 4:129-154.
[30]	DE WILDE A H, SNIJDER E J, KIKKERT M, et al. Host factors in coronavirus replication[M]//TRIPP R A, TOMPKINS S M. Roles of Host Gene and Non-coding RNA Expression in Virus Infection. Cham:Springer, 2018:1-42.
[31]	EMMOTT E, MUNDAY D, BICKERTON E, et al. The cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology[J]. J Virol, 2013, 87(17):9486-9500.
[32]	SPENCER K A, DEE M, BRITTON P, et al. Role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein[J]. Virology, 2008, 370(2):373-381.

Mutation of N Protein Phosphorylation Site Affects Replication and Subgenomic Transcription of Porcine Reproductive and Respiratory Syndrome Virus

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 32

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	SONG Wenyan, ZHANG Hanwen, WU Aodi, ZHANG Liyan, LIU Zhao, YE Tongtong, CHEN Chuangfu, SHENG Jinliang. Screening of Nanobodies against Porcine Reproductive and Respiratory Syndrome Virus GP5 Protein and Exploration of Their Inhibitory Effect on Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 258-270.
[2]	WANG Zhiyuan, LIU Boqi, XU Zhiying, XU Sijia, XING Jiabao, ZHANG Guihong, WANG Heng, SUN Yankuo. Analysis of ORF5 Gene Variation of Porcine Reproductive and Respiratory Syndrome Virus in Local Regions of China from 2021 to 2022 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3812-3823.
[3]	TIAN Shimao, WAN Qianhui, XU Xiaodong, KONG Yingying, TIAN Ke, TANG Yubing, CHEN Jilong, YANG Guihong. Ubiquitinase of NF-κB Signal Pathway Regulated by Neuromedin B and Its Receptor NMBR during Influenza A Virus H9N2 Subtype Infection [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3118-3126.
[4]	LIU Jiankui, XU Ye, LIU Chen, YU Hui, YANG Yuan, HE Le, LI Jiarui, DAN Huijuan, DAI Ailing, YANG Xiaoyan, WEI Chunhua. Genomic Characteristics of Porcine Reproductive and Respiratory Syndrome Virus in Fujian Province from 2017 to 2021 based on Whole Genome [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1579-1589.
[5]	LU Mengqi, YANG Wengjie, LI Ping, YU Peng, DONG Ling, NIU Xiaoyu, YANG Keli, ZOU Weihua, SONG Hui. Changes of PRRSV Infection on Microorganisms in Lungs and Intestines of Piglets based on 16S rRNA Sequencing [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1664-1678.
[6]	LI Zhuowei, WANG Fang, WANG Junjun, CHEN Zhenhan, LI Huanrong, ZHOU Shuanghai, LIU Xuewei. Inhibitory Effect of Dicumarol on Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus in vitro [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1160-1168.
[7]	LI Huawei, WANG Xuying, QIAO Hongxing, LI Xinfeng, JI Xiangbo, GUO Kewei, YANG Zhongyuan. Establishment of a Lentiviral Vector-mediated Stable Expression of eIF5A Cell Line and Its Effect on Porcine Reproductive and Respiratory Syndrome Virus Propagation [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1169-1176.
[8]	ZHONG Lemiao, LIU Binghui, WU Chunlin, WU Yijian. Preparation and Evaluation of Subunit Vaccine based on Adhesin Protein of Mycoplasma gallisepticum [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5171-5183.
[9]	ZHOU Likun, DONG Xinyuan, LI Jiahui, CUI Zhiying, ZHAO Shijie, XU Jie, CHEN Jing, ZHANG Yina, XIA Ping’an. Regulatory Role of MID2 on Porcine Reproductive and Respiratory Syndrome Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4735-4744.
[10]	WANG Junjun, SONG Huimin, LI Zhuowei, JIANG Ping, ZHOU Shuanghai, LI Huanrong, LIU Xuewei. Effect of Interaction between N Protein and Ribosomal Protein S20 of Porcine Reproductive and Respiratory Syndrome Virus on Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 293-303.
[11]	XIAO Ke, CHEN Ting, ZHAO Qiping, ZHU Shunhai, DONG Hui, LIU Manyu, YU Yu, HUANG Bing, HAN Hongyu. Preliminary Study on the Characteristics and Functions of HD Dome-containing Proteins in Eimeria tenella [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2608-2620.
[12]	WAN Ying, ZHOU Gaijing, MA Yuan, SHI Zhengwang, XIAO Shuqi, LUO Juncong, SONG Rui, CAO Liyan, YANG bo, WANG Lijuan, TIAN Hong, ZHENG Haixue. Development and Application of N-protein Blocking ELISA for Detecting Porcine Epidemic Diarrhea Virus Antibodies [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1173-1181.
[13]	JIA Hanxiao, YUAN Honggen, LI Zhen, GUAN Shuaiyin, ZHANG Jinhua, SONG Yunfeng. Screening of Proteins Interacting with PCV2 Replication Initiation Region and Study on the Regulation of Virus Replication by PARP1 Protein [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3511-3521.
[14]	LI Chen, QIU Cunyi, FENG Yixue, HAN Likang, DU Lianzhao, WANG Qianqian, MA Xin, LI Ruixiang, WANG Xinglong. Observation of Anti-porcine Reproductive and Respiratory Syndrome Virus Effect of Shenhu Baidu Granules in vivo [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(8): 2344-2353.
[15]	LUO Chuanzhen, ZHANG Yuchen, LOU Jiangcheng, LIU Xiaoli, GU Changqin, ZHANG Wanpo, CHENG Guofu, HU Xueying. Ultrastructural Changes in Renal Tissue of Piglets Infected with PRRSV [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(4): 1079-1085.