【作者】 陈全刚；

【导师】 方六荣；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2013， 博士

【摘要】 细胞自噬是一种溶酶体依赖性的降解途径,在维持细胞内环境稳定的过程中发挥着重要作用。越来越多的研究显示,细胞自噬在不同病毒感染的过程中扮演着不同的角色,同样病毒对细胞自噬的调控机制也不尽相同。此外,细胞自噬还跟天然免疫存在着密切的关系。内质网是蛋白质合成的重要场所,在蛋白的翻译和修饰过程中发挥着重要作用,病毒复制过程中往往导致大量未折叠或错误折叠蛋白的堆积而引起内质网应激,进而诱导未折叠蛋白反应(UPR), UPR同样跟病毒的增殖存在重要的关系。此外,UPR可以调控细胞自噬的水平。猪繁殖与呼吸综合征(PRRS)以引起妊娠母猪繁殖障碍和仔猪呼吸道症状为主要特征,具有较高的死亡率。尽管随着研究的深入取得了一系列的研究成果,但针对猪繁殖与呼吸综合征病毒(PRRSV)感染引起的免疫抑制及其对细胞的具体调控机制仍不甚明了。鉴于此,本研究深入探讨了PRRSV感染后自噬及内质网应激的诱导情况。主要研究内容包括：1. PRRSV感染诱导自噬体的形成为确定PRRSV感染是否诱导自噬体的形成,首先通过Western Blot的方法检测PRRSV感染后II型微管结合蛋白轻链3(LC3-Ⅱ)的表达变化,结果显示PRRSV感染明显诱导了LC3-I向LC3-II的转化,初步揭示了PRRSV感染可以诱导细胞自噬；进一步我们通过激光共聚焦显微镜观察PRRSV感染后EGFP-LC3的定位,结果发现,PRRSV感染MARC-145细胞后融合表达蛋白EGFP-LC3发生明显的点状聚集,进一步证明PRRSV感染可以诱导细胞自噬；最后,通过透射电镜对细胞的超微结构进行观察,同时在阳性对照组及PRRSV感染组中观察到自噬体的存在,直接证明了PRRSV感染后可以诱导自噬体的形成。2. PRRSV感染促进自噬体的降解两种途径可引起细胞内自噬体的聚集：细胞自噬被诱导、自噬体的降解被抑制。为验证PRRSV感染诱导白噬体聚集的具体机制,首先通过Lyso-Tracker对溶酶体进行染色,观察EGFP-LC3与溶酶体的共定位情况,证实PRRSV感染诱导的自噬体可以与溶酶体发生共定位,初步推断PRRSV感染能促进自噬体的降解。进一步,通过Western Blot分别对p62的降解情况和LC3-Ⅱ的周转进行了分析,结果证实PRRSV感染后促进了p62的降解同时也促进了LC3-Ⅱ的周转。充分表明了PRRSV感染可以促进自噬体的降解。3.细胞自噬促进PRRSV的增殖研究中分别通过自噬抑制剂3-MA和自噬激活剂雷帕霉素(Rapa)处理细胞,检测抑制或激活细胞自噬对PRRSV增殖的影响。结果发现诱导细胞自噬促进PRRSV的增殖,而3-MA抑制细胞自噬后,PRRSV的增殖受到抑制。进一步,通过设计干扰分子分别干扰ATG7和Beclin-1的表达,进一步证明细胞自噬在PRRSV增殖过程中的促进作用。另外,通过抑制剂和干扰分子分别抑制自噬溶酶体的形成,同样显著抑制了PRRSV的增殖。以上结果表明细胞自噬能够促进PRRSV的增殖。4.参与诱导细胞自噬的PRRSV编码蛋白为筛选参与诱导细胞自噬的PRRSV编码蛋白,首先用紫外光(UV)灭活的PRRSV感染MARC-145细胞,结果发现UV灭活的PRRSV不具有诱导细胞自噬的能力,初步推断诱导细胞自噬的为PRRSV的非结构蛋白。进一步通过转染PRRSV编码蛋白的真核表达质粒,并通过Western Blot检测LC3-Ⅱ的表达变化情况,结果显示多种PRRSV编码的非结构蛋白均可诱导细胞自噬。5. PRRSV诱导的细胞自噬参与免疫抑制PRRS是一种强烈的免疫抑制性疾病,现已证实,PRRSV感染可以抑制由poly(I:C)诱导的干扰素的表达,而在对细胞自噬研究的过程中现已证实细胞自噬可以引起干扰素的下调。为此,我们通过荧光素酶报告质粒检测PRRSV诱导的细胞自噬是否参与其引起的免疫抑制,结果表明细胞自噬被抑制后PRRSV引起的免疫抑制得到恢复,这表明PRRSV诱导的细胞自噬参与其引起的免疫抑制。6. PRRSV感染MARC-145细胞诱导内质网应激PRRSV感染细胞后,通过电镜观察其内质网的形态变化情况。发现PRRSV感染后可以引起内质网的扩张,推断PRRSV感染可以引起内质网应激；进一步通过Western Blot检测了糖调节蛋白78和94(GRP78和GRP94)的表达变化情况,证实PRRSV感染后可显著诱导GRP78和GRP94的表达。表明PRRSV感染诱导了内质网应激并诱发了内质网应激反应。7. PRRSV感染MARC-145细胞激活PERK途径活化的PERK可以磷酸化转录起始因子2α (eIF2α),为此本研究通过Western Blot检测了PRRSV感染后eIF2α的磷酸化水平,证实了PRRSV感染MARC-145细胞后eIF2α磷酸化水平显著升高,而总的eIF2α未发生变化。进一步我们检测了ATF4的mRNA的表达变化情况,证实了磷酸化的eIF2α可以诱导ATF4的表达,最后研究证实了PRRSV感染后GADD34的表达水平也明显升高,这充分证明了PRRSV感染MARC-145细胞后,PERK的通路被激活。8. PRRSV感染MARC-145细胞激活IRE1途径首先通过半定量的方法检测了IRE1所诱导的Xbp1mRNA的切割情况,并最早在PRRSV感染24h后检测到Xbp1s的存在,证实了PRRSV感染可以诱导Xbp1的切割,上述结论进一步通过Xbp1的荧光素报告质粒得到了佐证。之后通过荧光素酶报告质粒对受Xpb1调控的两个反应元件(ERSE和UPRE)的活化进行验证,结果表明ERSE和UPRE在PRRSV感染后都被激活,最后通过荧光定量验证了受Xpb1调控的EMEM在PRRSV感染后同样被激活,充分证明了PRRSV感染可以激活IRE1途径。9. PRRSV感染MARC-145细胞不激活ATF6途径ATF6的激活要通过切割实现,研究中通过Western Blot检测了PRRSV感染后ATF6的切割情况。发现PRRSV感染并不诱导ATF6的切割,初步推测PRRSV感染不激活ATF6途径；进一步通过荧光定量PCR检测了ATF6下游分子的表达变化情况,结果发现PRRSV感染不能诱导其下游分子calnexin, calreticulin和PDI的表达,进一步证实PRRSV感染不能激活ATF6信号通路。10.内质网应激反应促进PRRSV增殖内质网应激反应通常会影响病毒的增殖,为检测PRRSV诱导的内质网应激对其自身增殖的影响,本研究分别通过内质网应激的抑制剂和干扰分子阻断内质网应激反应,并进一步通过空斑验证其对PRRSV增殖的影响。结果显示,无论是抑制内质网应激反应,还是分别干扰其单条通路均能抑制PRRSV的增殖。11. PRRSV感染诱导内质网应激介导的凋亡PERK诱导的CHOP的表达通常被认为是内质网应激诱导凋亡的重要标志。为此,我们首先通过CHOP的荧光素报告质粒证实了PRRSV感染后可以显著激活CHOP的表达,进一步通过荧光定量PCR对CHOP的mRNA的表达水平进行了验证,同样证实了PRRSV感染可以诱导CHOP的mRNA的表达。此外,本研究进步通过ELISA试剂盒证实了PRRSV感染可以诱导caspase3的活化,直接证明了PRRSV感染可以诱导细胞凋亡。而当CHOP的表达被干扰后,caspase3的活化受到抑制,充分证明PRRSV感染可以诱导由内质网应激引起的细胞凋亡。12. PRRSV诱导的内质网应激调控细胞自噬研究发现,内质网应激跟细胞自噬存在着密切的调控关系,内质网应激可以通过多种途径诱导细胞自噬。本研究初步探究了PRRSV诱导的内质网应激与细胞自噬的关系,通过抑制剂及干扰分子研究证实,PRRSV诱导的内质网应激调控细胞自噬的水平,而PERK和IRE1途径参与其中。更多还原

【Abstract】 Autophagy is a lysosomal-dependent degradation pathway which plays an important role in the maintenance of intracellular homeostasis. Cellular autophagy function to limit viral replication and pathogenesis. However, some viruses can counteract and/or subvert the host autophagy machinery in order to enhance their own growth or survival. In addition, autophagy plays specific roles in immune.The endoplasmic reticulum is an important place for protein synthesis, assembly and maturation. Virus infection often leads to a large number of unfolded or misfolded protein accumulation and leading ER stress response (unfolded protein response UPR). The UPR also regulates the replication of the virus. Moreover, the UPR can further activate the cellular autophagy.Porcine reproductive respiratory syndrome (PRRS) characterized by severe reproductive failure in sows, respiratory distress high mortality in piglets and growing pigs often leads a higher mortality rate. There have been many researches about PRRSV, but little was known about themechanism of immunosuppression during PRRSV infection.Hence, in the study we investigated whether the cellular autophagy and ER stress response were activated during PRRSV infection. The main contents include:1. PRRSV infection induced the formation of autophagyTo determine whether PRRSV infection triggers cellular autophagy, we first examined the expression of LC3-II. The results showed that PRRSV infection induce the conversion of LC3-I to LC3-II, suggested that PRRSV infection may induce cellular autophagy. Next, laser confocal microscopy was performed to investigated the punctate accumulation of EGFP-LC3, the results showed that cells with both Nsp2-positive and EGFP-positive showed punctate accumulation of LC3, this further indicated that PRRSV infection induce the cellular autophagy. Lastly, the cells ultrastructure was observed by TEM during PRRSV infection, directly demonstrate autophayg was induced during PRRSV infection.2. PRRSV infection facilitates the degradation of the autophagosomeTwo pathways can lead to the accumulation of autophagosome, induced the formation or inhibited the degradation of autophagosome. To investigated the mechanism of autophagosome accumulation during PRRSV infection. degradation pathway. First, lysosome was stained by Lyso-Tracker and the distribution of lysosome and EGFP-LC3were observed. We founded the clearly co-localization of lysosome and EGFP-LC3in PRRSV-infected cells. Furthermore, the turnover of LC3and the expression of p62were detected by Western Blot. Taken together, the results suggested that PRRSV infection facilitate the degradation of the autophagosome.3. Induction of autophagy enhances PRRSV replicationCells treated by3-Methyladenine (3-MA) or rapamycin (Rapa) to inhibit/induce the cellular autophagy, then, plaque assay was performed to analysis the effects of PRRSV replication. The results suggested that induction of autophagy enhance the PRRSV replication. Another, we performed RNA interference experiments to knockdown the expression of two critical genes of the autophagy pathways:ATG7and Beclin-1. We founded the siRNA can inhibit the autophagy induced by PRRSV and leading to the repression of PRRSV replication.4. PRRSV encodes protein which involved in the induction of autophagyTo screens the protein which participate in the PRRSV-induced autophagy, we detected whether the UV-inactivated virus can activate the autophagy. We founded that the PRRSV inactivated by UV cannot induce the autophagy. We concluded that PRRSV encoded non-structural proteins play a major role in autophagy induction. Furthermore, overexpression Nsps of PRRSV demonstrated that several PRRSV-encoded proteins were involved in the autophagy induction.5. Knockdown of autophagy enhances the innate immune response in PRRSV-infected MARC-145cellsPRRS is a strong immunosuppressive diseases. It has been demonstrated that PRRSV infection inhibits the expression of IFN which induced by poly(I:C). Several researches reported that activate the autophagy result of decreased IFN production. Hence, the luc reporter was used to investigate whether PRRSV-induced cellular autophagy was involved in the immunosuppressive. The result indicated that PRRSV-induced autophagy impairs innate immune response, inhibit of autophagy enhances the innate immune response in PRRSV-infected cells.6. PRRSV infection elicits ER dilation and over expression of the ER chaperone, GRP79and GRP94To observed cellular changes that may accompany response to PRRSV infection, ultrastructure analysis using electron microscopy was performed. As shown in the result, compared to the mock infection, the most prominent change observed is the expansion of the ER in PRRSV infection cells. The result suggested that the ER stress would be induced during PRRSV infection. Then, glucose-regulated protein78and94(GRP78and GRP94) expression were both enhanced at various time-points after PPRSV infection Taken together, the results indicate that PRRSV infection induce ER stress.7. PRRSV infection activates the PERK/eIF2a/ATF4signaling pathwaywe tested the expression of eIF2a which can be phosphorylated by active PERK, and found that the expression of phosphorylated form of eIF2a was enhanced at12h post-infection and remained elevated throughout the infection time course.In addition, qRT-PCR were performed to measure the levels ofActivating transcription factor4(ATF4) and GADD34mRNA which can be induced by eIF2a. The results suggested that, there were a great increase in the level of ATF4and GADD34mRNA after PRRSV infection or treated with TG. Taken together, the results implied that PRRSV infection can activate the PERK signal pathway and may resulting in a translational attenuation.8. PRRSV infection activates the IRE1/Xbpl branch of UPRWe tested the splicing of the Xbpl mRNA by RT-PCR using primers which overlapping the26-bp intron. The results showed that, the448-bp fragment amplified from Xbp1s mRNA was detected starting at24h after PRRSV infection, and the level of this mRNA continued to increase through48hpi. Dual-luciferase reporter assay was performed to evaluate the level of Xbpl splicing and the similar results were found.This further demonstrated that PRRSV infection induce the splicing of Xbpl mRNA. In addition, we found both ERSE and UPRE response elements were activated significantly during PRRSV infection, and the ER degradation-enhancing a-mannosidase-like protein (EDEM), which transcription is solely dependent on Xbp was induced. These results indicated that PRRSV infection activate the IRE1pathway leading to the splicing of Xbpl RNA and resulting in activation of downstream target genes.9. PRRSV infection cannot activate the ATF6pathwayIn response to ER stress, the90-kDa precursor of ATF6was cleaved into a50-kDa protein which functions as a transcription factor. To examine the effects of PRRSV on the ATF6pathway, Western Blot was performed to assess the status of ATF6,both90-or50-kDa ATF6protein were detected in MARC-145cells which treated with TG for4h. However, corresponding to the mock-infection cells, the50-kDa cleavage products was undetectable at any time during PRRSV infection. In addition, the expression of calnexin, calreticulin, ERp57and PDI were no obvious change during PRRSV infection. These results demonstrated that PRRSV infection induce the ER stress but cannot activate the ATF6pathway.10. ER stress modulates the PRRSV replicationPlaque assay were performed to determine the effect of4-PBA on PRRSV replication, the dates showed that4-PBA reduced PRRSV growth significantly at micromolar concentrations. To extend these studies, we performed RNA interference (RNAi) experiments to knockdown the PERK and IRE1which can be activated during PRRSV infection. The result showed that knockdown of PERK or IRE1significantly decrease PRRSV yield. Based on these results, we concluded that the UPR induced by PRRSV plays a positive role to its replication.11. PRRSV infection induces the CHOP expression leading ER stress-mediated apoptosisTranscription factor CHOP/GADD153can be activated in cells suffering from ER stress, and CHOP activation appears to contribute to subsequent cell growth arrest and apoptosis. To investigate the activation of CHOP during PRRSV infection, MARC-145cells were co-transfeced with CHOP-luc and pRL-TK. The results suggest that, he CHOP promoter was activated during PRRSV infection. Besides, to demonstrate induction of CHOP at mRNA level, we performed qRT-PCR. The results showed that treated by TG induce the transcription of CHOP, and CHOP mRNA induced by PRRSV was observed at24h and sustained to48h. The results indicated that PRRSV infection activate the expression of CHOP. Caspase3is the effector caspase activated by proteolytic cleavage and ER stress-mediated apoptotic signals ultimately converge on it. Here, we demonstrate that, caspase3was activated during PRRSV infection. In addition, this activation can be blocked by siRNA of CHOP,Taken together, our results indicated that the persistent activation of UPR pathways by PRRSV leading to the induction of apoptosis.12. Induction of complete autophagic response by PRRSV via the unfolded protein responseUPR, the major ER stress pathway, is a potent stimulus of autophagy. We have demonstrated that PRRSV could induce ER stress to activate PERK and IRE1arms of the UPR. To test whether PRRSV indeed induced the accumulation of autophagy via the induction of ER stress, we treated cells with siRNA and4-PBA, we then examined whether the suppression of UPR would affect the lipidation of LC3by PRRSV. All the results indicated that ER stress was important for the lipidation of LC3induced by PRRSV.更多还原