猪繁殖与呼吸综合征病毒诱导巨噬细胞糖酵解促进病毒复制  

Porcine reproductive and respiratory syndrome virus infection induces glycolysis of macrophages to facilitate viral replication

作　　者：段滇宁[1] 李雅楠 梁燕娇 黄诗婷 刘建奎[1,2,3] 邱龙新 陈洪博[1,2,3] DUAN Dianning;LI Yanan;LIANG Yanjiao;HUANG Shiting;LIU Jiankui;QIU Longxin;CHEN Hongbo(College of Life Science,Longyan University,Longyan 364012,Fujian,China;Key Laboratory of Preventive Veterinary Medicine and Biotechnology(Longyan University),Longyan 364012,Fujian,China;Engineering Research Center for the Prevention and Control of Animal Original Zoonosis,Longyan 364012,Fujian,China)

机构地区：[1]龙岩学院生命科学学院,福建龙岩364012 [2]福建省预防兽医学与生物技术高校重点实验室(龙岩学院),福建龙岩364012 [3]动物源性人兽共患病防控福建省高校工程研究中心,福建龙岩364012

出　　处：《生物工程学报》2024年第12期4546-4556,共11页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(32102628);福建省自然科学基金(2021J01568,2022J011157)。

摘　　要：为探究糖酵解对猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)在猪肺泡巨噬细胞(porcine alveolar macrophages,PAMs)中复制的作用,以PRRSV感染PAMs为研究对象,采用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)、实时荧光定量PCR、病毒滴度测定和蛋白质免疫印迹试验(Western blotting)检测PAMs感染PRRSV后糖代谢的变化,PRRSV蛋白和病毒滴度及细胞相关基因和蛋白的表达水平;并应用靶向低氧诱导因子-1(hypoxia inducible factor-1α,HIF-1α)特异性siRNA分析HIF-1α在病毒复制中的作用。结果显示,PRRSV感染增强PAMs糖酵解,上清液葡萄糖摄取和乳酸含量显著升高(P<0.05或0.01),细胞内ATP显著降低(P<0.05),己糖激酶2(hexokinase 2,HK2)、6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3,PFKFB3)和丙酮酸激酶M2型(pyruvate kinase isozyme type M2,PKM2)含量显著升高(P<0.05或0.01);抑制糖酵解会显著降低PRRSV蛋白表达和病毒滴度(P<0.01);特异性siRNA敲低HIF-1α的表达后,糖酵解和PRRSV滴度显著降低(P<0.05);抑制糖酵解过程后逆转了PRRSV抑制干扰素信号通路。本研究为探索糖酵解在PRRSV复制过程中的功能提供了理论支持。This work aims to explore the effect of glycolysis on the replication of porcine reproductive and respiratory syndrome virus(PRRSV)in porcine alveolar macrophages(PAMs).The changes of glucose metabolism,PRRSV protein levels,PRRSV titers,and the relative expression levels of genes and proteins in PAMs were analyzed by ELISA,qPCR,virus titration,and Western blotting after PRRSV infection.The effect of hypoxia-inducible factor-1α(HIF-1α)on PRRSV replication was subsequently assessed by specific siRNAs targeting to HIF-1α.The results showed that PRRSV infection enhanced glycolysis,elevated the levels of glucose uptake and lactate in the supernatant(P<0.05 and 0.01,respectively),reduced ATP production(P<0.05),and up-regulated the expression of hexokinase 2(HK2),6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3),and pyruvate kinase isozyme type M2(PKM2)in PAMs(P<0.05 and 0.01,respectively).Glycolysis inhibitors down-regulated the expression of PRRSV proteins and reduced virus titers(P<0.01).The knockdown of HIF-1αby siRNAs inhibited glycolysis and lowered PRRSV titers(P<0.05).In addition,the interferon pathways inhibited by PRRSV infection were reversed by the inhibition of glycolysis.These findings may facilitate further investigation of the role of glycolysis in PRRSV replication.

关 键 词：猪繁殖与呼吸综合征病毒 糖酵解 缺氧诱导因子1Α 巨噬细胞 

分 类 号：S852.651[农业科学—基础兽医学]