SARS-CoV-2非结构蛋白Nsp5转录组学分析验证  

Eukaryotic expression and transcriptomic analysis of the SARS-CoV-2 non-structural protein Nsp5

作　　者：郭佳峥 宗珊 向凌怡 乔嘉璐 彭倩 李卫玲[1] 孙宾莲 GUO Jiazheng;ZONG Shan;XIANG Lingyi;QIAO Jialu;PENG Qian;LI Weiling;SUN Binlian(Hubei Key Laboratory of Cognitive and Affective Disorders,Institute of Biomedical Sciences,School of Medicine,Jianghan University,Wuhan 430056;Department of Immunology,School of Medicine,Jianghan University,Wuhan 430056,China)

机构地区：[1]江汉大学医学部武汉生物医学研究院,认知与情感障碍湖北省重点实验室,湖北武汉430056 [2]江汉大学医学部免疫学教研室,湖北武汉430056

出　　处：《生物技术》2025年第1期30-36,24,共8页Biotechnology

基　　金：武汉市高校产学研项目(CXY202204);江汉大学科研专项资金项目(08190008);国家自然科学基金面上项目(31670167)。

摘　　要：[目的]探究严重急性呼吸综合征冠状病毒2(Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)非结构蛋白5(Nsp5)对宿主细胞基因mRNA的影响及其生物学意义。[方法]通过NCBI和Uniprot网站对当前流行的部分SARS-CoV-2突变株的Nsp5序列进行同源性分析,并对有和无过表达Nsp5的人非小细胞肺癌细胞A549细胞进行转录组测序及差异分析,对差异基因进行GO和KEGG功能分析,并对部分差异表达基因进行实时荧光定量反转录PCR(RT-qPCR)验证。[结果]序列比对分析发现Nsp5在SARS-CoV-2突变株中高度保守,保守性比例为99.67%;GO分析提示差异基因主要富集在第二信使介导的信号通路,氯离子通道复合物和细胞因子活性等方面;KEGG富集分析提示,差异表达基因主要参与信号通路如:PI3K-AKT、MAPK和细胞因子-细胞因子受体的相互作用;RT-qPCR实验验证了Nsp5可显著上调部分促炎相关基因CCL5、HILPDA(1.7倍)、SAT1(2倍)的表达;显著下调部分抑炎基因NSD1(1.9倍)、DHCR24(5.5倍)、DANJB1(2倍)、HNRNPM(3倍)、HYOU1-2(5.9倍)的表达。[结论]Nsp5蛋白可能通过上调炎症因子、下调抑炎因子的表达参与SARS-CoV-2感染诱导的炎症反应,为探索Nsp5在病毒与宿主相互作用中的角色提供了新线索。[Objective]To investigate the effects of Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)non-structural protein 5(Nsp5)on host cell gene mRNA and its biological significance.[Method]We conducted a homology analysis of the Nsp5 sequences from current circulating SARS-CoV-2 variants using the NCBI and Uniprot websites.Transcriptome sequencing and differential analysis were performed on human non-small cell lung cancer cells A549 with and without overexpression of Nsp5 with GO and KEGG analysis.The differentially expressed genes were verified by real-time fluorescent quantitative reverse transcription PCR(RT-qPCR).[Result]Sequence comparison analysis found that Nsp5 is highly conserved in SARS-CoV-2 variants,with a conservation ratio of 99.67%.GO analysis indicated that the differentially expressed genes are mainly enriched in second messenger-mediated signaling,chloride channel complex,and cytokine activity.KEGG enrichment analysis revealed that differentially expressed genes are primarily involved in signaling pathways such as PI3K-AKT,MAPK,and cytokine-cytokine receptor interaction.RT-qPCR experiments confirmed that Nsp5 significantly upregulates the expression of pro-inflammatory genes CCL5,HILPDA(1.7-fold),and SAT1(2-fold),while significantly downregulating the expression of anti-inflammatory genes NSD1(1.9-fold),DHCR24(5.5-fold),DANJB1(2-fold),HNRNPM(3-fold),and HYOU1-2(5.9-fold).[Conclusion]The Nsp5 protein may participate in the inflammation response under SARS-CoV-2 infection by upregulating inflammatory factors and downregulating anti-inflammatory factors.This finding provides new insights into the role of Nsp5 in the interaction between the virus and the host.

关 键 词：新型冠状病毒 Nsp5 保守性分析 高通量测序 转录组分析 蛋白表达验证 炎症反应 

分 类 号：R373[医药卫生—病原生物学]