Rab32对禽偏肺病毒C型复制的影响

doi:10.11843/j.issn.0366-6964.2024.09.028

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (9): 4041-4050.doi: 10.11843/j.issn.0366-6964.2024.09.028

Rab32对禽偏肺病毒C型复制的影响

冯旭飞¹^,²^,³^,⁴(), 亓宇翔³, 于瀚哲³, 张正洲³, 王如嘉³, 孟闯⁴, 董魁¹^,²^,*()

1. 山西医科大学公共卫生学院, 太原 030001
2. 山西医科大学煤炭环境致病与防治教育部重点实验室, 太原 030001
3. 扬州大学兽医学院(比较医学研究院), 扬州 225009
4. 扬州大学江苏省人兽共患病重点实验室, 扬州 225009

收稿日期:2023-11-10 出版日期:2024-09-23 发布日期:2024-09-27
通讯作者: 董魁 E-mail:xufei_feng1988@163.com;sxdongkui@163.com
作者简介:冯旭飞(1988-), 男, 山西吕梁人, 博士, 主要从事病原与宿主相互作用研究, E-mail: xufei_feng1988@163.com
基金资助:
江苏省高等学校基础科研(自然科学)面上项目(21KJB230009);江苏省人兽共患病学重点实验室开放课题(R2106);高等学校学科创新引智计划资助(D18007);江苏高校优势学科建设工程资助项目(PAPD);山西省高等教育“百亿工程”科技引导专项(BYBLD003-07);山西省高等教育“百亿工程”科技引导专项(BYBLK007-03)

Effect of Rab32 on the Replication of Avian Metapneumovirus Type C

Xufei FENG¹^,²^,³^,⁴(), Yuxiang QI³, Hanzhe YU³, Zhengzhou ZHANG³, Rujia WANG³, Chuang MENG⁴, Kui DONG¹^,²^,*()

1. School of Public Health, Shanxi Medical University, Taiyuan 030001, China
2. Key Laboratory of Coal Environmental Pathogenicity and Prevention of Ministry of Education, Shanxi Medical University, Taiyuan, 030001, China
3. College of Veterinary Medicine (Institute of Comparative Medicine), Yangzhou University, Yangzhou 225009, China
4. Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou 225009, China

Received:2023-11-10 Online:2024-09-23 Published:2024-09-27
Contact: Kui DONG E-mail:xufei_feng1988@163.com;sxdongkui@163.com

摘要/Abstract

摘要：

为探究Rab32对禽偏肺病毒C型(aMPV/C)复制的影响, 将A549细胞接种aMPV/C后进行激光共聚焦显微镜观察, 并用Image J软件对所采集图像的共定位系数进行分析; 将pCMV-Flag-Rab32、pCMV-Flag、siRab32以及siNC分别转染A549细胞后接种aMPV/C, 分别采用荧光定量PCR(qPCR)、Western blot和TCID₅₀检测收集样品中aMPV/C N基因转录水平、aMPV/C N和Rab32蛋白的表达水平以及病毒效价; 将pCMV-mcherry-Rab32分别与可融合表达GFP的aMPV/C结构蛋白的重组载体共转染A549细胞, 24 h后采用激光共聚焦显微镜观察; 将pCMV-Flag-Rab32分别与可融合表达GFP的aMPV/C结构蛋白的重组载体共转染HEK293T细胞, 36 h后收集细胞样进行免疫共沉淀试验。结果显示, Rab32与aMPV/C在细胞质中存在明显的共定位; Rab32与aMPV/C的共定位系数极显著高于未接毒组(P<0.01);过表达Rab32后aMPV/C N基因转录水平, Rab32和N蛋白表达水平以及病毒效价均极显著升高(P<0.01);而干扰Rab32表达后, N基因的转录水平, Rab32和N蛋白的表达水平以及病毒效价均极显著降低(P<0.01);Rab32可与多个aMPV/C蛋白(N、F、M、G、SH、P、M2-1、M2-2、L1、L2、L4)在细胞质中存在共定位, 而与L3蛋白无明显共定位; IP样品中表达N和M2-2蛋白的样品出现特异性条带, 其他蛋白无特异性条带。综上表明Rab32可能通过与aMPV/C N和M2-2蛋白之间的互作从而调控病毒的复制。相关研究结果可为深入阐明Rab32调控aMPV/C复制的分子机理提供研究基础。

关键词: Rab32, 禽偏肺病毒C型, 复制, 表达水平, 互作

Abstract:

To investigate the effect of Rab32 on the replication of avian metapneumovirus type C (aMPV/C), confocal images were obtained and co-localization coefficients were analyzed by Image J software after infected with A549 cells by aMPV/C. Furthermore, the plasmids of pCMV-Flag-Rab32 or pCMV-Flag as well as siRab32 or siNC RNA sequences were transfected into A549 cells before aMPV/C infection, respectively. The transcription level of aMPV/C N gene, the expression level of aMPV/C N and Rab32 proteins, and the viral titers in collected samples were detected by quantitative real-time PCR (qPCR), Western blot, and the half tissue culture infective dose (TCID₅₀) method, respectively. Then, the plasmids of pCMV-mcherry-Rab32 and GFP-tagged aMPV/C proteins were co-transfected into A549 cells before conducting confocal imaging, respectively. Subsequently, the plasmids of pCMV-Flag-Rab32 and GFP-tagged aMPV/C proteins were co-transfected into HEK293T cells before collecting cell samples at 36 h post-transfection and conducting the co-immunoprecipitation assay. The results showed that obvious co-localized fluorescent signals of Rab32 and aMPV/C were observed in the cytoplasm. The co-localization coefficients of Rab32 and N proteins were significantly higher than that of mock-infected group. The transcription level of the N gene, the expression level of Rab32 and N proteins, and the viral titers were significantly increased (P<0.01) after overexpression of Rab32, respectively. On the contrary, the transcription level of the N gene, the expression level of Rab32 and N proteins, and the viral titers were significantly decreased (P<0.01) after knockdown expression of Rab32, respectively. Obvious co-localized fluorescent signals of Rab32 and aMPV/C proteins (N, F, M, G, SH, P, M2-1, M2-2, L1, L2, and L4) were observed in the cytoplasm, while scarcely co-localized signals of Rab32 and L3 protein were found. Specific bands of N and M2-2 proteins were found in immunoprecipitation samples while no bands of other GFP-tagged aMPV/C proteins were found. These results indicating that Rab32 interacts with aMPV/C N and M2-2 proteins. In summary, Rab32 might affect aMPV/C replication by interacting with multiple viral proteins (N and M2-2). The relevant results can provide the basis for elucidating the mechanism of Rab32 regulating aMPV/C replication via protein interaction.

Key words: Rab32, avian metapneumovirus type c, replication, expression level, interaction

中图分类号:

S852.659.5

冯旭飞, 亓宇翔, 于瀚哲, 张正洲, 王如嘉, 孟闯, 董魁. Rab32对禽偏肺病毒C型复制的影响[J]. 畜牧兽医学报, 2024, 55(9): 4041-4050.

Xufei FENG, Yuxiang QI, Hanzhe YU, Zhengzhou ZHANG, Rujia WANG, Chuang MENG, Kui DONG. Effect of Rab32 on the Replication of Avian Metapneumovirus Type C[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4041-4050.

图/表 6

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引物名称 Primer name	序列(5′→3′) Sequences
aMPV/C-N F	GAGGTTCCCGAGGATTGACA
aMPV/C-N R	GATCACTCCCCACCTTAGCA
GAPDH F	GACAGTCAGCCGCATCTTCT
GAPDH R	GCGCCCAATACGACCAAATC

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Rab32对禽偏肺病毒C型复制的影响

Effect of Rab32 on the Replication of Avian Metapneumovirus Type C

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