非洲猪瘟病毒D1133L蛋白增加宿主波形蛋白磷酸化而促进病毒在猪巨噬细胞中的复制

D1133L protein of African swine fever virus promotes virus replication in pig macrophages by increasing vimentin phosphorylation in the host

摘要:

非洲猪瘟(African swine fever, ASF)是由非洲猪瘟病毒(African swine fever virus, ASFV)感染引起家猪和野猪的一种高死亡率的传染性疾病。ASFV具有庞大的基因组,其中非结构蛋白pD1133L被预测为其编码的6个解旋酶之一。本实验室应用免疫沉淀-质谱联用(immunoprecipitation-mass spectrometry, IP-MASS)技术筛选与pD1133L互作的宿主细胞蛋白,发现细胞波形蛋白(vimentin, VIM)为pD1133L互作的宿主蛋白之一,但尚不清楚宿主蛋白VIM对ASFV复制的影响。【目的】探究ASFV与VIM的相互调控作用,揭示VIM促进ASFV复制的机制。【方法】通过免疫共沉淀(co-immunoprecipitation, Co-IP)试验验证pD1133L与VIM存在互作关系;外源过表达VIM蛋白以及设计并合成VIM的siRNA探究VIM对ASFV复制的影响;利用Western blotting以及荧光定量PCR (quantitative real-time PCR, qPCR)方法检测ASFV对VIM蛋白水平以及转录水平的影响;通过Western blotting、间接免疫荧光试验(immunofluorescence assay, IFA)探究巨噬细胞感染ASFV后VIM磷酸化水平变化以及亚细胞定位变化情况;CCK-8试剂盒检测VIM磷酸化抑制剂KN-93处理的最佳浓度,并利用Western blotting以及IFA检测KN-93对VIM磷酸化、亚细胞定位以及对ASFV复制影响。【结果】VIM过表达促进ASFV复制,敲低VIM的表达则抑制ASFV复制;ASFV感染抑制VIM蛋白水平以及转录水平表达,且呈时间依赖性;ASFV感染后VIM发生磷酸化修饰且发生亚细胞定位改变,从而促进ASFV复制。【结论】证实了ASFV与宿主蛋白VIM之间的相互调控作用;初步确定ASFV感染后VIM受到ASFV pD1133L调控,亚细胞定位发生重排向核周聚集从而促进ASFV复制的机制。

关键词:非洲猪瘟病毒;D1133L蛋白;波形蛋白;蛋白互作;磷酸化

Abstract:

African swine fever (ASF) caused by African swine fever virus (ASFV) is a severe infectious disease affecting both domestic pigs and wild boar. ASFV has a large genome, and the non-structural protein pD1133L is predicted to be one of the six helicases the genome encodes. We used the IP-MASS technology to screen the host proteins interacting with pD1133L and found that vimentin (VIM) is one of the host proteins that interacted with pD1133L. However, it remains unclear how the VIM affects ASFV replication. 【Objective】 To investigate the mutual regulation between ASFV and VIM and disclose the mechanism by which VIM enhances ASFV replication. 【Methods】 We employed the Co-IP assay to examine the interaction between pD1133L and VIM. Furthermore, we examined the effects of VIM on ASFV replication by designing and synthetizing VIM siRNAs and overexpressing VIM. Western blotting and quantitative real-time PCR (qPCR) were employed to determine the impact of ASFV on the protein and mRNA levels of VIM. Western blotting and indirect immunofluorescence assay (IFA) were used to explore the changes in the phosphorylation level and subcellular localization of VIM in macrophages infected with ASFV. The CCK-8 kit was used to determine the optimal concentration of KN-93, a VIM phosphorylation inhibitor, for treatment. The effects of KN-93 on the phosphorylation and subcellular localization of VIM and the replication of ASFV were examined by Western blotting and IFA. 【Results】 The overexpression of VIM promoted the replication of ASFV, while the knockdown of VIM inhibited ASFV replication. In addition, ASFV infection down-regulated both the protein and mRNA levels of VIM in a time-dependent manner. After ASFV infection, VIM was modified by phosphorylation and changed in subcellular localization, thereby promoting ASFV replication. 【Conclusion】 This study confirms the interaction between ASFV and the host protein VIM. After ASFV infection, pD1133L leads to the rearrangement of the subcellular localization of VIM towards paranuclear aggregation, which promotes ASFV replication.

Key words:African swine fever virus (ASFV);D1133L protein;vimentin;protein-protein interaction;phosphorylation