与新城疫病毒NP蛋白互作宿主细胞蛋白的筛选和鉴定

doi:10.11843/j.issn.0366-6964.2025.08.032

Abstract

Abstract:

Newcastle disease is an acute contact infectious disease caused by Newcastle disease virus (NDV), which is characterized by respiratory and digestive symptoms. The widespread epidemic of NDV has caused huge economic losses to the poultry industry. NP is a nucleocapsid protein of NDV, which plays an important role in viral replication, immune response and cellular autophagy, while the host proteins interacting with NP remains unclear. The study was aim to screen the host proteins interacting with NDV NP and explore the effects of the host proteins on the replication of NDV, as well as to provide a theoretical basis for disease-resistant drug development. The eukaryotic expression plasmid of NDV NP was successfully constructed with the eukaryotic expression vector pXJ40. The host proteins interacting with NDV NP were screened by co-immunoprecipitation, mass spectrometry, GST-pull down, and laser confocal microscopy. The effects of the host proteins on NDV replication were explored by overexpression and knockdown in DF1cells. A total of 211 potential host proteins interacting with NP were screened by mass spectrometry. Enrichment analysis of the differential proteins revealed their possible biological functions and involvement in biological processes. Further detailed analysis of the interaction between HSP70 and NP confirmed the direct interaction of the two proteins and mapped the domain responsible for this binding to the SBD region in HSP70. NDV infection down-regulates HSP70 expression in host cells. Overexpression of HSP70 significantly inhibited viral replication at both the protein and transcriptional levels. However, both knockdown of endogenous HSP70 and use of HSP70 inhibitors significantly promoted NDV replication. This study indicated that the host protein HSP70 interacts with NDV NP and the overexpression of HSP70 could significantly inhibit NDV replication and act as a negative regulator of NDV infection, which provided a new idea for the design of antiviral drug targeting HSP70.

Key words: Newcastle disease virus, NP, HSP70 protein, co-immunoprecipitation

CLC Number:

S852.659.5

HAN Yin, XIE Ziwei, YAN Shuke, XU Fei, CHEN Ruiai. Screening and Identification of Host Proteins Interacting with NP of Newcastle Disease Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3942-3957.

Figures/Tables 13

Table 1

Table 2

Table 3

Fig. 1

Fig. 2

Fig. 3

Table 4

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

References 27

1	DIMITROV K M , RAMEY A M , QIU X , et al. Temporal, geographic, and host distribution of avian paramyxovirus 1 (Newcastle disease virus)[J]. Infect Gene Evolut, 2016, 39, 22- 34. doi: 10.1016/j.meegid.2016.01.008
2	BUTT S L , MOURA V , SUSTA L , et al. Tropism of Newcastle disease virus strains for chicken neurons, astrocytes, oligodendrocytes, and microglia[J]. BMC Vet Res, 2019, 15 (1): 317. doi: 10.1186/s12917-019-2053-z
3	AMARASINGHE G K , AYLLON M A , BAO Y , et al. Taxonomy of the order Mononegavirales: update 2019[J]. Arch Virol, 2019, 164 (7): 1967- 1980. doi: 10.1007/s00705-019-04247-4
4	PANTUA H D , MCGINNES L W , PEEPLES M E , et al. Requirements for the assembly and release of Newcastle disease virus-like particles[J]. J Virol, 2006, 80 (22): 11062- 11073. doi: 10.1128/JVI.00726-06
5	STEWARD M , VIPOND I B , MILLAR N S , et al. RNA editing in Newcastle disease virus[J]. J Gen Virol, 1993, 74 (Pt 12): 2539- 2547.
6	KARSUNKE J , HEIDEN S , MURR M , et al. W protein expression by Newcastle disease virus[J]. Virus Res, 2019, 263, 207- 216. doi: 10.1016/j.virusres.2019.02.003
7	GOFF P H , GAO Q , PALESE P . A majority of infectious Newcastle disease virus particles contain a single genome, while a minority contain multiple genomes[J]. J Virol, 2012, 86 (19): 10852- 10856. doi: 10.1128/JVI.01298-12
8	LONEY C , MOTTET-OSMAN G , ROUX L , et al. Paramyxovirus ultrastructure and genome packaging: cryo-electron tomography of sendai virus[J]. J Virol, 2009, 83 (16): 8191- 8197. doi: 10.1128/JVI.00693-09
9	LIAO T , CHEN Y , GUO L , et al. The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency[J]. PLoS Pathog, 2024, 20 (2): e1012027. doi: 10.1371/journal.ppat.1012027
10	CHENG J H , SUN Y J , ZHANG F Q , et al. Newcastle disease virus NP and P proteins induce autophagy via the endoplasmic reticulum stress-related unfolded protein response[J]. Sci Rep, 2016, 6, 24721. doi: 10.1038/srep24721
11	ROSENZWEIG R , NILLEGODA N B , MAYER M P , et al. The Hsp70 chaperone network[J]. Nat Rev Mol Cell Biol, 2019, 20 (11): 665- 680. doi: 10.1038/s41580-019-0133-3
12	SU Y S , HSIEH P Y , LI J S , et al. The heat shock protein 70 family of chaperones regulates all phases of the enterovirus A71 life cycle[J]. Front Microbiol, 2020, 11, 1656. doi: 10.3389/fmicb.2020.01656
13	MACEJAK D G , SARNOW P . Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells[J]. J Virol, 1992, 66 (3): 1520- 1527. doi: 10.1128/jvi.66.3.1520-1527.1992
14	XU T , LIN Z , WANG C , et al. Heat shock protein 70 as a supplementary receptor facilitates enterovirus 71 infections in vitro[J]. Microb Pathog, 2019, 128, 106- 111.
15	HAN C , XIE Z , LV Y , et al. Direct interaction of the molecular chaperone GRP78/BiP with the Newcastle disease virus hemagglutinin-neuraminidase protein plays a vital role in viral attachment to and infection of culture cells[J]. Front Immunol, 2023, 14, 1259237. doi: 10.3389/fimmu.2023.1259237
16	SHOKEEN K , SRIVATHSAN A , KUMAR S . Lithium chloride functions as Newcastle disease virus-induced ER-stress modulator and confers anti-viral effect[J]. Virus Res, 2021, 292, 198223. doi: 10.1016/j.virusres.2020.198223
17	YE T , JIANG K , WEI L , et al. Oncolytic Newcastle disease virus induces autophagy-dependent immunogenic cell death in lung cancer cells[J]. Am J Cancer Res, 2018, 8 (8): 1514- 1527.
18	ZHANG D , DING Z , XU X . Pathologic mechanisms of the Newcastle disease virus[J]. Viruses, 2023, 15 (4): 864. doi: 10.3390/v15040864
19	ZHAN Y , YU S , YANG S , et al. Newcastle disease virus infection activates PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways to benefit viral mRNA translation via interaction of the viral NP protein and host eIF4E[J]. PLoS Pathog, 2020, 16 (6): e1008610. doi: 10.1371/journal.ppat.1008610
20	KAUR R , BATRA J , STUCHLIK O , et al. Heterogeneous ribonucleoprotein A1 (hnRNPA1) interacts with the nucleoprotein of the influenza A virus and impedes virus replication[J]. Viruses, 2022, 14 (2): 199. doi: 10.3390/v14020199
21	OGLESBEE M J , LIU Z , KENNEY H , et al. The highly inducible member of the 70 ku family of heat shock proteins increases canine distemper virus polymerase activity[J]. J Gen Virol, 1996, 77 (Pt 9): 2125- 2135.
22	KUMSTA C , JAKOB U . Redox-regulated chaperones[J]. Biochemistry, 2009, 48 (22): 4666- 4676. doi: 10.1021/bi9003556
23	AGOSTINI I , POPOV S , LI J , et al. Heat-shock protein 70 can replace viral protein R of HIV-1 during nuclear import of the viral preintegration complex[J]. Exp Cell Res, 2000, 259 (2): 398- 403. doi: 10.1006/excr.2000.4992
24	TAKASHIMA K , OSHIUMI H , MATSUMOTO M , et al. DNAJB1/HSP40 suppresses melanoma differentiation-associated gene 5-mitochondrial antiviral signaling protein function in conjunction with HSP70[J]. J Innate Immun, 2018, 10 (1): 44- 55. doi: 10.1159/000480740
25	SUN M , YU Z , MA J , et al. Down-regulating heat shock protein 27 is involved in porcine epidemic diarrhea virus escaping from host antiviral mechanism[J]. Vet Microbiol, 2017, 205, 6- 13. doi: 10.1016/j.vetmic.2017.04.031
26	LE Y , JIA P , JIN Y , et al. The antiviral role of heat shock protein 27 against red spotted grouper nervous necrosis virus infection in sea perch[J]. Fish Shellfish Immunol, 2017, 70, 185- 194. doi: 10.1016/j.fsi.2017.08.032
27	HAUSER H , SHEN L , GU Q L , et al. Secretory heat-shock protein as a dendritic cell-targeting molecule: a new strategy to enhance the potency of genetic vaccines[J]. Gene Ther, 2004, 11 (11): 924- 932. doi: 10.1038/sj.gt.3302160

引物名称 Primer name	序列 Primer sequences
pXJ40-Flag-NP-F	5′-gataagtccggatccaagcttATGTCGTCTGTTTTTGACGAATACG-3′
pXJ40-Flag-NP-R	5′-agttctgctttaataagatctTCAGTACCCCCAGTCAGTGTCG-3′
pXJ40-Myc-HSP70-F	5′-gaagaggatctgtccggatccATGTCTGGCAAAGGGCCG-3′
pXJ40-Myc-HSP70-R	5′-tctggtaccgagctcctgcagTTAATCTACTTCTTCAATGGTTGGGC-3′
pXJ40-Myc-HSP70-NBD-F	5′-gaagaggatctgtccggatccGGCAAAGGGCCGGCCATC-3′
pXJ40-Myc-HSP70-NBD-R	5′-tctggtaccgagctcctgcagTTCAGACTTGTCTCCCATGAGGA-3′
pXJ40-Myc-HSP70-SBD-F	5′-gaagaggatctgtccggatccTTGGATGTCACCCCCCTGT-3′
pXJ40-Myc-HSP70-SBD-R	5′-tctggtaccgagctcctgcagGCGACCCTTGTCATTGGTGA-3′
pXJ40-Flag-F	5′-ACATCCTGGTCATCATCCTG-3′
pXJ40-Flag-R	5′-ACCACAACTAGAATGCAGTG-3′
pXJ40-Myc-F	5′-ATCCTGCCTTTCTCTTTATGGT-3′
pXJ40-Myc-R	5′-TAACCGTATTACCGCCTTTGAGTG-3′

引物名称 Primer name	引物序列 Primer sequences
q-DHN3-F	5′-CTCATAGCAGGCTCCTGTCG-3′
q-DHN3-R	5′-CAGTTGTGCGATCATGCGAG-3′
q-NP-F	5′-TGAGCACATCATTCTGGCGA-3′
q-NP-R	5′-CAGATACTCGTTGGGCAGCA-3′
q-HSP70-F	5′-ATTCTTGCGTGGGTGTCTTC-3′
q-HSP70-R	5′-GATGGTGTTGGTGGGGTTC-3′
q-β-Actin-F	5′-CTGTGCCCATCTATGAAGGCTA-3′
q-β-Actin-R	5′-ATTTCTCTCTCGGCTGTGGTG-3′

名称Name	靶序列Target sequence
si-HSP70 1#	CCCGCTTACTTCAACGACT
si-HSP70 2#	CAAGAAGCAGAGAAATACA
si-HSP70 3#	CAAGAAGCAGAGAAATACA

登录号 Accession No.	基因名称 Gene Name	大小/ku MW	覆盖率/% Coverage	肽段 Peptides
A0A1C9KD18	ANXA2	38.6	71	27
Q7SX63	HSP70	69.9	49	31
H9KZT7	ILF2	54.3	3	1
F1NJS6	KPNA2	55.9	28	10
F1NVA4	NPM1	43.7	28	10
A0A1D5NYB2	PABPC1	70.8	36	23
A0A1D5PJW4	PSMA7	27.9	44	10
Q6DV79	STAT3	88.1	14	7

[1]	LIU Sha, YANG Caichun, ZHANG Xiaoyu, CHEN Qiong, LIU Xiong, CHEN Hongbo, ZHOU Huanhuan, SHI Liangyu. Population Genetic Structure and Genome-wide Runs of Homozygosity Analysis in Meihuaxing Pigs Based on 80K SNP Chip [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3749-3760.
[2]	ZHANG Jialiang, HUANG Chang, YANG Yonglin, YANG Hua, BAI Wenlin, MA Yuehui, ZHAO Qianjun. Genetic Structure and Wool Trait Selection Signatures Analysis of Chinese Sheep Populations Based on 50K Liquid SNP Chip [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3164-3176.
[3]	MIAO Junjie, ZHANG Riquan, WU Houyi, YOU Xinming, HUANG Yiwen, HUANG Xiaoying, GUO Zhenyang, LIU Jianlin, XIAO Weihua, GUO Tianhua, CHEN Hao, KANG Dongliu. Genome-Wide SNP Analysis Revealed the Characteristics of Germplasm Resources and Genetic Diversity of Jinggang Black-Palm Geese [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3199-3209.
[4]	WU Jianliang, SU Yang, MAO Ruihan, ZHOU Lei, YAN Tiantian, LI Zhi, LIU Jianfeng. Design and Effect Evaluation of A Whole-Genome Low-Density SNP Chip in Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2733-2740.
[5]	YAO Boyuan, YANG Zhiwen, SUN Yapeng, YANG Yanan, ZHANG Yaru, WANG Xinrong. Analysis of Novel Transcripts, Alternative Splicing, and SNP in Porcine Heart Tissue Based on RNA-Seq Technology [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1664-1675.
[6]	HUANG Cheng, YANG Zhiyuan, LIN Jian, CHENG Huimin, WANG Mi, MAO Huilin, WANG Guoliang, LIU Guiming, ZHAO Jicheng, LIU Yuehuan. Construction and Efficacy Evaluation of mRNA Vaccines against H9 Subtype Avian Influenza Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1843-1853.
[7]	ZHOU Taizeng, YANG Yiting, ZHU Yuehua, QIAN Hongxi, LIU Yihui, GAN Mailin, ZHU Li, SHEN Linyuan. Genome-wide Association Study of Stillbirths and Mummies in Sows [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1231-1241.
[8]	GAO Zhiqiang, LAI Ping'an, SONG Yueqian, CHONG Yan, GUO Youran, BAI Zilong, GUO Huimin, WANG Lin, PU Jing, SHI Xiju, REN Tong, ZHAO Xiangpeng. Studies and Application of Multi-target Nucleic Acid Mass Spectrometry Detection Method for Avian Influenza/Newcastle Disease Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1386-1395.
[9]	YANG Xiaowen, NING Wenqing, ZHOU Shizhong, YUAN Yaqin, HOU Xuexin, DING Jiabo. Establishment of a Quantitative Real-time PCR Detection Method for Trimethoprim-Sulfamethoxazole-resistance Strains of Brucella melitensis [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1465-1472.
[10]	YIN Qiong, GAO Mingchao, YAO Xiumei, LIU Kunyu, LIU Wei, JIE Hongwei, LI Hua, YE Fei. Correlation Analysis between Melanin Content in Breast Muscle and PMEL17 Gene of Muchuan Black-bone Chickens [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 168-177.
[11]	Guangxuan FAN, Tianjiao WANG, Yimeng DONG, Hongliang WANG, Ning DING, Xinhao WANG, Xiumei XING. Molecular Genealogy Construction and Population Genetic Structure Analysis of Jilin Sika Deer Based on SNP Loci [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3925-3935.
[12]	Ruiqi ZHANG, Yanqin PANG, Zaishan LI, Xiuguo SHANG, Ganqiu LAN, Jinbiao GUO, Yunxiang ZHAO. Research on Feeding Capacity Selection of Lactating Sows Based on Intelligent Precision Feeding [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 2890-2900.
[13]	DUAN Yixin, ZHANG Linyun, ZHAO Yongju. The Evaluated Methods and Influencing Factors of SNP Heritability and Its Application in Farmer Animal Breeding [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1854-1865.
[14]	CUI Shengdi, WANG Kai, ZHAO Zhenjian, CHEN Dong, SHEN Qi, YU Yang, WANG Junge, CHEN Ziyang, YU Shixin, CHEN Jiamiao, WANG Xiangfeng, TANG Guoqing. Identification of Candidate Genes for Pork Texture Traits Using GWAS Combined with Co-localisation of DNA Methylation [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1945-1957.
[15]	SUN Wenli, WANG Haoqi, ZE Licuo, GAO Yufan, ZHANG Feifan, ZHANG Jian, DUAN Mengqi, SHANG Peng, QIANG Bayangzong. Polymorphism of Pro-Inflammatory Factors (IL-1β, IL-6, TNF-α) in Tibetan Pigs and Its Association Analysis with Immune Traits [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1958-1969.

Screening and Identification of Host Proteins Interacting with NP of Newcastle Disease Virus

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 13

References 27

Related Articles 15

Recommended Articles

Metrics

Comments