非洲猪瘟病毒MGF110-5-6L基因功能分析

Functional analysis of MGF110-5-6L gene of African swine fever virus

非洲猪瘟(African swine fever, ASF)是由非洲猪瘟病毒(African swine fever virus, ASFV)感染猪只引起的一种高度接触性传染病,最急性型致死率可高达100%。ASFV结构非常复杂,编码超过160多种蛋白质,其中多基因家族(multigene families, MGFs)蛋白包括MGF100、MGF110、MGF300、MGF360和MGF505/530等,且MGF360和MGF505可抑制宿主干扰素(interferon, IFN)的产生以及影响ASFV的致病性。但关于MGF110家族相关基因的功能尚无报道,因此本研究选取了MGF110家族中MGF110-5-6L基因进行了相关功能的探究。应用同源重组技术构建了MGF110-5-6L基因缺失的突变体病毒(ASFV-ΔMGF110-5-6L),通过检测该突变体病毒在原代猪肺泡巨噬细胞(porcine alveolar macrophages, PAMs)中的复制水平来探究其在ASFV复制周期中的功能,结果表明,其复制动力学曲线与亲本病毒ASFV HLJ/2018(ASFV-WT)一致,说明缺失MGF110-5-6L基因不影响ASFV在PAMs中的复制。为了探究MGF110-5-6L蛋白在ASFV复制周期中的生物学功能,将ASFV-WT和ASFV-ΔMGF110-5-6L分别感染PAMs,在感染后4,12,20 h收集样品,进行转录组测序。结果显示,与ASFV-WT感染组相比,ASFV-ΔMGF110-5-6L感染组中差异表达基因主要为和IL-17信号通路、肿瘤坏死因子(TNF)信号通路以及细胞因子与细胞因子受体之间的相互作用相关。本研究构建了ASFV MGF110-5-6L基因缺失毒株,并通过转录组测序发现,MGF110-5-6L基因作为ASFV复制非必需基因具有拮抗细胞因子表达的功能,不仅为探究MGF110家族成员的生物学功能提供科学数据,还为深入揭示ASFV拮抗宿主细胞天然免疫反应的分子机制提供参考。

African swine fever(ASF),caused by African swine fever virus(ASFV),is a highly contagious infectious disease of domestic pigs and wild boar with a mortality approaching to 100%.ASFV is very complex in structure and encodes more than 160 proteins,and multi-gene family proteins encoded by ASFV including MGF100,MGF110,MGF300,MGF360 and MGF505/530,and MGF360 and MGF505 proteins can inhibit the production of host interferon(IFN)and affect the pathogenicity of ASFV.However,little is known about the function of genes of MGF110,focusing on thisscientific issue,we explored the functions of MGF110-5-6L,a member of MGF110.In this study,the MGF110-5-6L gene-deleted ASFV mutant(ASFV-ΔMGF110-5-6L)was constructed by homologous recombination technique.The biological characteristics of ASFV-ΔMGF110-5-6L was investigated by evaluating the replication level in primary porcine alveolar macrophages(PAMs).The results showed that the replication kinetics was consistent with the wild-type ASFV HLJ/2018(ASFV-WT),indicating that deletion of the MGF110-5-6 Lgene did not affect the replication of ASFV in PAMs.To investigate the effects of the MGF110-5-6 Lprotein on the host signaling pathways,PAMs were infected with ASFV-WT or ASFV-ΔMGF110-5-6L,and were collected at 4,12 and 20 h post-infection for transcriptome sequencing.Transcriptome analysis showed that the differentially expressed genes in the ASFV-WT-or ASFV-ΔMGF110-5-6L-infected PAMs were mainly enriched on the signaling pathways involved in interleukin 17(IL-17)and tumor necrosis factor(TNF)and cytokine-cytokine receptor interaction compared with the ASFV-WT.In this study,the MGF110-5-6L-deleted ASFV was constructed,and MGF110-5-6L was confirmed as a non-essential gene for ASFV replication and has the function of inhibiting the expression of cytokine,which not only provides scientific data for exploring the biological functions of MGF110,but also provides a reference for further revealing the molecular mechanism of ASFV antagonizing the innate immune response of host cells.