细胞外基质蛋白ABI3BP抗水疱性口炎病毒初步作用研究

doi:10.24920/004319

摘要/Abstract

摘要：

目的探讨细胞外基质蛋白ABI3BP对水疱性口炎病毒（vesicular stomatitis virus，VSV）基因组复制和先天免疫信号通路的影响。

方法在人皮肤成纤维细胞BJ-5ta中转染小干扰RNA（siRNA）敲低ABI3BP基因，建立ABI3BP基因缺失的VSV-GFP病毒感染细胞模型。通过鬼笔环肽细胞免疫荧光实验，检测敲低ABI3BP后细胞内肌动蛋白（F-actin）形态结构的变化。在ABI3BP基因缺失的VSV-GFP病毒感染细胞模型上，利用RT-qPCR方法检测细胞中病毒mRNA水平的变化。利用免疫印迹法（Western blotting）检测IRF3和TBK1磷酸化水平的变化。

结果成功建立敲减ABI3BP基因的VSV-GFP感染的细胞模型。鬼笔环肽细胞免疫荧光染色表明，敲减ABI3BP基因后，细胞内F-actin的表达发生结构重排。采用不同剂量的病毒感染细胞，与对照组相比，ABI3BP敲低细胞中基因拷贝数分别增加2.5~3.5倍（P<0.01）和2.2~4倍（P<0.01），VSV-GFP病毒蛋白表达水平显著上调；在ABI3BP基因敲低的细胞模型上，VSV-GFP病毒感染导致Ⅰ型干扰素免疫通路关键免疫分子p-IRF3 和p-TBK1蛋白磷酸化水平明显下调。

结论本研究表明细胞外基质蛋白ABI3BP对维持细胞内F-actin纤维网状结构具有重要作用。ABI3BP缺失促进RNA病毒的复制，ABI3BP是I型干扰素通路激活的重要调控分子。

关键词: 细胞外基质蛋白, 肌动蛋白, 水疱性口炎病毒, 病毒复制

Abstract:

Objective To explore the influence of extracellular matrix protein ABI-interactor 3-binding protein (ABI3BP) on vesicular stomatitis virus (VSV) genome replication and innate immune signaling pathway.

Methods The small interfering RNA (siRNA) was transfected to knock down ABI3BP gene in human skin fibroblast BJ-5ta cells. VSV-green fluorescent protein (VSV-GFP)-infected cell model was established. The morphological changes and F-actin stress fiber formation were detected on ABI3BP knockdown cells by phalloidin immunofluorescence staining. The mRNA level of virus replication was detected by RT-qPCR in BJ-5ta cells after VSV-GFP infection; western blotting was performed to detect the changes in interferon regulatory factor 3 (IRF3) and TANK-binding kinase 1 (TBK1) phosphorylation levels.

Results The VSV-GFP-infected BJ-5ta cell model was successfully established. Efficient knockdown of ABI3BP in BJ-5ta cells was achieved. Phalloidin immunofluorescence staining revealed structural rearrangement of intracellular F-actin after ABI3BP gene knockdown. Compared with the control group, the gene copy number of VSV-GFP in ABI3BP knockdown cells increased by 2.2 - 3.5 times (P<0.01) and 2.2 - 4.0 times (P<0.01) respectively when infected with VSV of multiplicity of infection 0.1 and 1. The expression of viral protein significantly increased in ABI3BP knockdown cells after virus infection. The activation of type-I interferon pathway, as determined by phosphorylated IRF3 and phosphorylated TBK1, was significantly decreased in ABI3BP knockdown cells after VSV-GFP infection.

Conclusions Extracellular matrix protein ABI3BP plays an important role in maintaining the formation and rearrangement of actin structure. ABI3BP gene deletion promotes RNA virus replication, and ABI3BP is an important molecule that maintains the integrity of type I interferon pathway.

Key words: ABI-interactor 3-binding protein, extracellular matrix protein, actin, vesicular stomatitis virus, viral replication

Xiang-Bo Meng, Mei-Hua Chen, Nuo Xu, Tian-Qi Li, Shuai-Chen Li, Sun-Xin Zhou, Huan Chen, Tong Zhang. Antiviral Effect of Extracellular Matrix Protein ABI3BP on Vesicular Stomatitis Virus and Its Mechanism: A Preliminary Study In Vitro[J].Chinese Medical Sciences Journal, 2024, 39(1): 1-8.

图/表 7

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siRNA	Nucleic acid sequence
si-ABI3BP #1	5’GGTGGAATTTGGAGTAAGA3’
si-ABI3BP #2	5’GCCATCAGGTGCTGATAGA3’
si-ABI3BP #3	5’GAAGGTGGAATTTGGAGTA3’

Gene	Primer sequence
hABI3BP	Forward: 5’TTGCCAAGTCACTGTCCCAAT3’
	Reverse: 5’GTGGCTGGACAGATTTGAAAAAT3’
GAPDH	Forward: 5’GAGTCAACGGATTTGGTCGT3’
	Reverse: 5’TTGATTTTGGAGGGATCTCG3’
VSV-G	Forward: 5’ATGGCGTACTTCCAGATGG3’
	Reverse: 5’CTCGGTTCAAGATCCAGGT3’

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