【作者】 杨莘；

【导师】 童光志；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2014， 博士

【摘要】 猪繁殖与呼吸综合征（Porcine reproductive and respiratory syndrome，PRRS）是由猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus，PRRSV）感染引起一种急性、高度传染的病毒性传染病，对世界养猪业造成巨大的经济损失。2006年，我国出现高致病性猪繁殖与呼吸综合征病毒（HP-PRRSV），加大了对养猪业的危害。目前，病毒仍在持续的进化，在世界范围内受到广泛关注。PRRSV难以控制的原因之一在于病毒的致病和免疫机理并不十分清楚，因此，难以研发安全、有效的疫苗来对该病进行防控。病毒侵入机体后，机体对侵入的病毒产生一定的限制和清除作用。病毒如何调控细胞内相关信号通路或利用自身编码蛋白来逃避机体免疫系统的识别和清除，从而有利于自身的感染和复制，成为近年来针对PRRSV研究的热点。PI3K/Akt信号通路是细胞内重要的信号转导通路，多种病毒感染过程中均能够活化该通路来有利于自身的复制和增殖。SAMHD1作为最新发现的天然免疫限制性因子，具有三磷酸水解酶的活性，能够降解细胞内dNTP，阻止I型人免疫缺陷病毒（HIV-1）的逆转录过程，抑制病毒在髓系细胞中的感染与复制。作为猪肺泡巨噬细胞（PAM）嗜性的PRRSV，感染过程中是否需要PI3K/Akt信号通路参与以及与SAMHD1的蛋白相互作用机制如何，这些都是针对PRRSV病毒感染亟待研究的问题。本研究发现，HP-PRRSV HuN4株感染过程能够激活PI3K/Akt信号通路促进自身的感染与增殖。病毒在感染过程中活化PI3K/Akt信号通路具有细胞特异性，活化的PI3K/Akt与病毒的感染增殖无关，而与病毒诱导的细胞病变的形成密切相关。HP-PRRSV感染通过Akt下游FoxO1和Bad通路来促进细胞的存活，下游的GSK-3信号分子与病毒感染增殖密切相关。HuN4-F112病毒在GSK-3抑制剂持续存在的条件下进行传代，结果发现在连续传代14代后，病毒滴度可恢复到与未添加抑制剂的对照组一致的水平。对抑制剂耐受的PRRSV突变株进行全基因测序和序列对比发现，抑制剂耐受病毒基因组中共有18个核苷酸发生突变，其中Nsp2、Nsp12、GP3和GP4蛋白中6个核苷酸的突变可导致病毒氨基酸的突变，暗示这6个氨基酸可能在病毒复制过程中，单独或相互协调对GSK-3信号通路进行调节。为了探索SAMHD1对HP-PRRSV增殖的影响，本实验通过RACE方法克隆获得猪SAMHD1全基因及其编码序列。原核表达纯化重组猪SAMHD1蛋白，并以此为免疫原制备了具有特异性反应的猪SAMHD1单克隆抗体。对SAMHD1的遗传进化、亚细胞定位和组织分布分析发现，SAMHD1具有严格的分支界限且是一种核内定位蛋白，猪SAMHD1几乎在所有组织中均有表达，但在扁桃体、淋巴结、肺脏、肝脏中出现高表达。在MARC-145细胞中过表达SAMHD1，westernblot分析和间接免疫荧光（IFA）实验发现，HuN4的感染和增殖受到显著抑制，细胞培养上清中病毒滴度明显降低。蛋白磷酸化分析发现，过表达SAMHD1主要以非磷酸化的形式存在。实时荧光定量RT-PCR检测发现，过表达SAMHD1能够明显抑制病毒基因组cRNA链的合成，并且能够显著上调ISG15和ISG56的转录水平。因此，我们推测SAMHD1能够发挥其三磷酸水解酶的活性以及调控干扰素相关因子的表达拮抗HuN4病毒的感染增殖。SAMHD1表达调控机制研究发现，TLR3和RIG-I/MDA5通路参与SAMHD1表达。干扰素调节因子3（IRF-3）的磷酸化入核在调控SAMHD1的表达方面具有重要作用。实验发现，IFN-α能够诱导PAM和MARC-145细胞中SAMHD1上调表达。过表达TBK1上调SAMHD1的表达和启动子的荧光素酶活性，将其磷酸化功能性位点Ser172突变后，其诱导SAMHD1表达和启动子荧光素酶活性的能力降低。过表达IRF-3上调SAMHD1启动子的荧光素酶活性，但只有活化形式的IRF-3能够上调SAMHD1的表达。干扰IRF-3表达后，IFN-α诱导SAMHD1上调表达受到显著抑制；恢复IRF-3的表达，SAMHD1的诱导表达又恢复到与对照组一致的水平。抑制IRF-3磷酸化入核后，IFN-α和NDV感染均无法诱导SAMHD1的上调表达。HuN4感染调控SAMHD1的机制研究发现，HuN4感染MARC-145细胞中IRF-3的磷酸化受到显著抑制，同时SAMHD1的表达并未出现明显变化，在感染后期出现了一定程度的下降。而HuN4感染PAM细胞时，能够显著上调IRF-3的磷酸化活性和SAMHD1的表达。通过对IRF-3上游相关通路的活化分析发现，HuN4感染主要通过RIG-I/MDA5/TBK1通路激活IRF-3并上调SAMHD1的表达。PAM细胞中抑制IRF-3的磷酸化入核，HuN4感染诱导的SAMHD1表达受到显著抑制，但HuN4的增殖在感染早期出现了显著增加。可见，SAMHD1在拮抗HuN4病毒在PAM细胞中的增殖具有一定的作用。对SAMHD1磷酸化分析发现，HuN4感染MARC-145细胞过程中，SAMHD1一直处于磷酸化的状态，而PAM细胞中SAMHD1并没有出现磷酸化。免疫沉淀实验发现，MARC-145细胞中，CyclinA2/CDK1复合体能够与SAMHD1互作，促进SAMHD1的磷酸化。HuN4感染能够上调CDK1的活性；抑制CyclinA2/CDK1复合体的活性，HuN4病毒的增殖受到了显著抑制。MARC-145细胞中下调SAMHD1的表达，HuN4病毒的增殖并没有增加。以上结果表明，HuN4感染过程中，能够通过抑制IRF-3的磷酸化和促进CyclinA2/CDK1的活性，来抑制SAMHD1的表达和功能，促进自身的增殖。总之，本研究证实激活PI3K/Akt信号通路与HP-PRRSV感染密切相关，天然免疫限制性因子SAMHD1能够拮抗HP-PRRSV的感染并且病毒感染过程能够调控SAMHD1活性来促进自身的感染，这些研究结果对于进一步揭示病毒的感染机理以及进一步进行抗PRRSV研究具有积极的意义。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS), casued by porcine reproductive andrespiratory syndrome virus (PRRSV), is an acute and highly contagious viral disease and posessignificant economic losses to the pork industry worldwide. In2006, an outbreak of HP-PRRSVdescribed as ‘‘pig high fever disease’’ occurred in China and caused disastrous loses to the farmers. Atpresent, the virus is sustaining evolution and is currently a major concern for the swine industryworldwide. Due to the pathogenesis and immunological mechanism of PRRSV is in a complex manner,PRRSV is particularly difficult to control and the development a safe and effective vaccine for PRRSVhas difficulties. When viruses invade cells, various cellular mechanisms are activated to inhibit viralreplication. Most recently, the studies of PRRSV are focus on the mechanisms that how the virus toevade the recognition and elimination of host innate immune reponse, through the cell signalingpathways or encoded proteins. Phosphatidylinositol-3-kinase (PI3K)/Akt is an important cellularpathway, and has been shown to participate in various replication steps of multiple viruses. The sterilealpha motif and HD domain1(SAMHD1) protein has been identified as a novel innate immunityrestriction factor, function as a triphosphohydrolase that depletes cells of deoxynucleotide triphosphates(dNTPs), inhibits lentiviral complementary DNA (cDNA) synthesis and blocks HIV-1infection inmyeloid-lineage cells. Porcine alveolar macrophages (PAMs) are the target cells of PRRSV, whether thePI3K/Akt pathway is regulated by PRRSV and the relationship of PRRSV and SAMHD1require muchmore attentions and researches.In the study, our data provide new evidences of a novel role for the PI3K/Akt pathway inHP-PRRSV strain HuN4infection. The PRRSV regulates the PI3K/Akt pathway by differentmechanisms in PAMs and MARC-145cells. The PI3K/Akt pathway has a negligible effect on theprolifeation of PRRSV in MARC-145cells, but regulates the formation of virus-induced CPE. Thedownstream targets of Akt, FoxO1and Bad were regulated by PRRSV through the control of thePI3K/Akt pathway to control host-cell survival and the activity of GSK-3was essential for the virusinfection. To select the mutant virus which was conferred the drug resistance, GSK-3inhibitor X wasused to suppress the HuN4-F112virus replication in MARC-145for14passages. After fourteenpassages, the viral titer increased and restored to level of the control without pharmaceutical treatment.Then complete genome of the selected mutant virus which tolerated to the GSK-3inhibitor X wassequenced. Alignment of the complete genome sequence of the parental virus to the mutant virus withGSK-3inhibitor X resistance indicated that there are eighteen nucleotides changes. Six of them led tonon-synonymous mutation, one was found in both Nsp12and GP3, and two in Nsp2and GP4,respectively. The complete genome sequence analysis revealed that some amino acids in Nsp2, Nsp12,GP3and GP4were essential for the interaction with GSK-3individually or coordinately, which wasrequired for the virus replication.To explore the antiviral effect of SAMHD1on PRRSV infection, the porcine SAMHD1complete gene sequence was first cloned from porcine PBMCs by RACE PCR. The whole recombinant porcineSAMHD1protein was acquired by prokaryotic expression. Specific MAbs against porcine SAMHD1were prepared using the purified recombinant porcine SAMHD1as immunogen. In our analysis of thephylogenesis, subcellular localization and tissue distribution, the SAMHD1sequences were clearlyseparated in evolutionary terms and SAMHD1was localized to the nucleus. SAMHD1mRNA wasdetected in all of the swine tissues examined, and higher levels of SAMHD1mRNA were detected inthe tonsil, lung, liver, and lymph node tissues. The western blotting and immunofluorescence analysesshowed that proliferation of HP-PRRSV strain HuN4, was efficiently blocked in MARC-145cellsoverexpressing the porcine SAMHD1and viral titers were significantly reduced, compared to theHuN4-infected control cells. Analysis of the phosphorylation of SAMHD1found that the overexpressedSAMHD1was mainly in nonphosphorylated status. Real-time quantitative RT-PCR analysis showedthat the synthesis of HuN4genome cRNA was inhibited and the transcripts of ISG15and ISG56wasobviously upregulated in MARC-145cells overexpressing SAMHD1. The results suggested thatoverexpresstion of SAMHD1may perform its dNTP triphosphohydrolase functions to inhibit thesynthesis of cRNA and upregulates the expression of the antiviral genes to block the proliferation ofPRRSV.Here, we show that TLR-3and RIG-I/MDA5pathways are involved in SAMHD1expression, thephosphorylation and nucleus translocation of interferon regulation factor3(IRF-3) plays major role inSAMHD1expression. SAMHD1is inducible expression in porcine alveolar macrophages (PAMs) andMARC-145cells by IFN-α. Overexpression of TBK1enhances the SAMHD1promoter luciferaseactivity and upregulates expression. The mutation in the phosphorylation site S172A of TBK1impairedthe inducible expression of SAMHD1. Overexpression of IRF-3enhances the SAMHD1promoterluciferase activity, but only active form of IRF-3can play a role in inducing SAMHD1expression.Degradation of IRF-3expression by RNA interference and inhibition its phosphorylation and nucleartranslocation by BX795reveal that the IFN-α and NDV infection inducible upregulation of SAMHD1expression was significantly impaired. Complementary IRF-3expression, the inducible expression ofSAMHD1was restored restored to level of the control.Our data found that the phosphorylation of IRF-3was significantly blocked in MARC-145cellsinfected with HuN4. Meanwhile, the expression of SAMHD1was also in basic level. In contrast, thephosphorylation of IRF-3and expression of SAMHD1was significantly upregulated in PAMs infectedwith HuN4. The RIG-I/MDA5/TBK1pathway was participated in the activation of IRF-3by HuN4infection. Inhibition of the phosphorylation and nuclear translocation of IRF-3, the inducible expressionof SAMHD1was significantly reduced in PAMs infected with HuN4, and the proliferation of HuN4virus was increased at the early stage of infection. These findings indicate that SAMHD1plays animportant role in blocking HuN4virus proliferation in PAMs. Analysis the phosphorylation ofSAMHD1found that SAMHD1was phosphorylated in MARC-145infected with HuN4virus, but notin PAMs. Immunoprecipitation asssy showed that SAMHD1was phosphorylated by CyclinA2/CDK1complex in MARC-145cells. The activity of CDK1was upregulated by HuN4virus infection. The proliferation of HuN4virus was obviously inhibited by CyclinA2/CDK1inhibitor. Degradation of theexpression of SAMHD1, the proliferation of HuN4in MARC-145cells was not affected. The resultsshowed that the expression and activity of SAMHD1was inhibited in MARC-145cells infected byHuN4virus, via inhibiting the phosphorylation of IRF-3and promoting the activity of CyclinA2/CDK1.In summary，our data showed that the activation of PI3K/Akt is regulated by HP-PRRSV. As aninnate immunity restriction factor, SAMHD1could inhibit the proliferation of HP-PRRSV, and theactivity of SAMHD1was regulated by PRRSV for effective infection. To investigate the regulatorymechanism of SAMHD1by PRRSV makes sense to explore the pathogenic mechanism of infection andfurther highlights the antiviral researches of PRRSV.更多还原