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RhoA分子在登革2型病毒感染过程中的作用研究

Role of RhoA in Dengue Virus Serotype 2 Infection

【作者】 宋富强

【导师】 胡晓梅;

【作者基本信息】 第三军医大学 , 微生物学, 2011, 硕士

【摘要】 登革病毒(dengue virus,DV)属于黄病毒属、是一种包膜的单股正链RNA病毒。根据包膜蛋白的抗原性不同,可将登革病毒分为四个血清型,即DV1~4。DV以蚊虫为主要传播媒介广泛流行于热带和亚热带地区。每年,登革病毒会导致数百万人感染,引起登革热(dengue fever,DF)和登革出血热/登革休克综合症(dengue hemorrhagic fever/ dengueshock syndrome, DHF/DSS)。DF是自限性发热性疾病。而DHF/DSS则是威胁患者生命的重症,其主要特征是血管通透性显著增加,导致血浆渗漏。每年大约有50万DHF/DSS患者,如未及时治疗,病死率可上升至50%。因而对其致病机理和预防手段的研究已成为亟待解决的前沿课题。研究表明,宿主细胞骨架在病毒的感染过程中发挥重要作用,而构成细胞骨架的主要成分微丝、微管、中间纤维在不同病毒的感染过程中分别扮演着不同的角色。我们小组前期实验证实微丝和波形蛋白纤维在DV的感染及复制过程中发挥着重要作用。DV的感染可以引起微丝骨架的改变,用微丝药物破坏其聚合和解离的平衡,可以导致DV感染受到抑制说明微丝聚合和解离的平衡对DV的感染具有重要意义。而调节微丝骨架的关键因子是Rho GTP酶,作为分子开关Rho GTP酶在无活性的GDP和有活性的GTP两种形式间循环,调节细胞的形态、生长、运动以及细胞周期等。Rho GTP酶家族中研究的比较多的有3个成员:RhoA、Cdc42和Rac1。我们对波形蛋白纤维的研究发现,ECV304细胞在感染DV2后,细胞中的波形蛋白会发生重排,重排的波形蛋白从细胞边缘回缩、环绕于细胞核周围,并与病毒抗原共存,用丙烯酰胺长时间作用细胞后,破坏波形蛋白会影响DV2的复制增殖。目前研究发现波形蛋白纤维的重排是由激酶磷酸化所致,而RhoA及其下游激酶ROCK (Rho associated coiled-coil forming protein kinase)在波形蛋白纤维磷酸化中扮演着重要角色。为此,本课题以RhoA/ ROCK通路为研究对象,采用构建RhoA突变体及RhoA和ROCK特异性抑制剂干扰RhoA/ ROCK通路的方法,观察对DV感染与复制的不同环节的影响,从而为解析DV感染的分子机制提供理论依据,为预防和控制DV感染提供新思路。本研究的主要实验内容和结果如下:1.利用突变体观察RhoA功能的异常对DV病毒感染的影响本实验用DV2感染ECV304细胞及稳定表达RhoA突变体的ECV304细胞株:ECV304N、ECVWtRhoA、ECVV14RhoA和ECVN19RhoA(MOI=1),通过病毒噬斑计数法分别检测感染后1 h细胞内和24h细胞内外的病毒滴度。结果显示在病毒感染1小时后ECVN19RhoA、ECVWtRhoA、ECVV14RhoA细胞内的病毒滴度均低于ECV304和ECV304N,其中ECVV14RhoA、ECVWtRhoA下降比较明显,而ECVN19RhoA下降较少,相较于ECV304N的病毒滴度分别下降了49.71%、51.76%、20.59%。在病毒感染24小时后,细胞上清中的病毒滴度ECVV14RhoA、ECVWtRhoA和ECVN19RhoA均低于ECV304N和ECV304,其中ECVV14RhoA、ECVWtRhoA下降比较明显,而ECVN19RhoA下降不明显,相较于ECV304N的病毒滴度分别下降了76.6%、77.2%、23%;细胞内病毒滴度的变化与上清趋势相符,即ECVV14RhoA、ECVWtRhoA下降比较明显,而ECVN19RhoA下降不明显。相较于ECV304N的病毒滴度分别下降了58.75%、62.32%、20.29%。结果提示:RhoA功能的异常对DV2的感染有明显的抑制作用。2.利用C3转移酶抑制RhoA活性观察对DV感染的影响利用C3转移酶抑制ECV304细胞内RhoA活化后实施感染实验,收集病毒穿入细胞1 h时的细胞样本和感染后24 h培养上清及细胞样本进行病毒滴度检测,结果显示:药物组病毒穿入1h后细胞内的病毒滴度相较于对照组的病毒滴度下降了88.46%;药物组病毒感染24h培养上清和细胞内的病毒滴度相较于对照组分别下降了12.52%、82.35%。与免疫荧光及共聚焦显微镜观察到的结果相符,即药物处理组病毒抗原阳性的细胞数目明显低于对照组。结果说明抑制RhoA活化可明显抑制DV穿入宿主细胞及DV的复制增殖。3.采用G-lisa检测DV病毒感染过程中RhoA分子活性的变化用灭活DV2(56℃,30min)和DV2分别感染ECV304细胞。在感染30min,1h,8h及24时收集细胞样品,采用G-LISATM RhoA Activation Assay Biochem KitTM(Cytoskeleton)试剂盒检测胞内RhoA分子的活性。结果显示:在感染30min及1h时RhoA活性较对照组有显著升高,其中感染30min时最为明显,感染后1h开始下降。而在感染8h和24h后RhoA分子活性较对照组没有明显变化。说明RhoA活性主要在病毒穿入细胞的过程中被激活,而在病毒的复制增殖过程中没有变化。4.利用Y-27632抑制ROCK活性观察对DV感染的影响使用ROCK活性抑制剂Y-27632抑制ROCK活性后实施感染实验,收集感染后8 h和24 h培养上清和细胞样本进行病毒滴度检测,结果显示:与对照组相比药物处理组(6个浓度处理)细胞上清和细胞样本中病毒滴度均没有明显变化。与免疫荧光及共聚焦显微镜观察到结果一致,即药物处理组与对照组之间病毒抗原阳性的细胞数目没有明显差异。说明抑制ROCK活性对DV的复制增殖无明显影响。综上所述,RhoA在DV感染过程中具有重要作用,DV在进入ECV304细胞的过程中激活RhoA。上述实验结果为深入理解DV与宿主细胞的相互作用、阐明DV的致病机制提供了重要的实验依据。

【Abstract】 Dengue virus (DV) belongs to the family Flavivirus and there are four serotypes (DV1-4). They cause a mild-to-debilitating febrile illness (classical dengue fever, DF) or life-threatening syndrome (dengue haemorrhagic fever/dengue shock syndrome, DHF/DSS). Annually there are millions of infections and at tens of thousands of deaths. The infecting DV is able to produce an acute febrile syndrome characterized by clinically significant vascular permeability(DHF/DSS). An estimated 500 000 cases of DHF require hospitalisation each year, and up to 50% may die if not properly diagnosed and treated. However, no currently licensed vaccines or antiviral drugs are available for dengue because its pathogenic mechanisms are still not fully understood.The previous study showed that the cytoskeleton play an important role during viral infection. The cytoskeleton network is composed of actin, microtubules(MT), intermediate filaments(Ifs) and their motor protein and other elements. Our study have found that actin and vimentin play an important role in DV2 infection. DV2 infection could induce rearrangement of actin and vimention, but the mechanism needs to be further confirmed. The present study show that the rearrangement of actin is regulated by signal transduction pathways that depend on small GTPases Rho. Of the 22 identified mammalian Rho GTPases, the best characterized are RhoA (stress fibers), Rac1 (lamellipodia) and Cdc42 (filopodia). At the same time, the rearrangement of vimentin mainly due to phosphorylation and RhoA and its downstream kinase ROCK is a key molecule in regulation of this process. Therefore, in this study, the possibility of RhoA / ROCK involved in DV2 infection was investigated. We found that RhoA was activated during DV2 entry target cells by using RhoA activation assay kit. Perturbing the activation of RhoA with pharmacological agents and RhoA mutants can interfere with DV2 infction. Moreover, inactivation ROCK may not interfere DV2 infection. These results indicated that RhoA may play an essential role in DV2 infection. RESULTES1. RhoA is involved in DV2 infectionIn our experiment, RhoA mutants including ECVWtRhoA, ECVV14RhoA, ECVN19RhoA and ECV304N cells were infected with the DV2. We detected the supernatant and intracellular virus titers after1 h, 24h p.i. by the virus plaque counts. The results showed that , after 1 hour of viral infection , virus titers of cell fraction in ECVV14RhoA, ECVWtRhoA, ECVN19RhoA cells were lower than ECV304 and ECV304N group, compared with ECV304N control ,the viral titers were reduced 49.71%, 51.76%, 20.59% respectively. At the same time, after 24 hours of viral infection, supernatant virus titer in ECVN19RhoA, ECVWtRhoA, ECVV14RhoA cell were also lower than ECV304N and ECV304, compared with ECV304N control,the titers decreased by 76.6% and 77.2%, 23% respectively. Meanwhile, the same change trend was observed in virus titer of cell fraction. The titers of cell fraction were also reduced by 58.75%, 62.32%, 20.29% in ECVV14RhoA, ECVWtRhoA, ECVN19RhoA groups Compared with that in ECV304N cells. The results suggest that RhoA is involved in DV2 infection in ECV304 cells.2. Effects of exoenzyme C3 transferase on DV2 infections cycleUsing exoenzyme C3 transferase inhibit the activation of RhoA in ECV304 cells ,then cells were infected with DV2. we detected the supernatant and intracellular virus titers after1 h, 24h p.i. by the virus plaque counts. The results showed that at1 h p.i. , Compared with the control group, titers of cell fraction of the drug-treated group was significantly decreased 88.46%; Meanwhile, at 24h p.i. , compared with the control group , titers of supernatant and cell fraction of drug-treated group were also reduced 12.52%, 82.35% respectively. This results were consistent with our obsearvation using confocal microscopy. The results suggest that inhibition of RhoA activation could inhibit the DV into the host cell and the DV replication proliferation.3. G-lisa detect the activity of RhoA molecular during DV infectionECV cells were infected with Inactivated DV2 (56℃, 30min) and DV2. Collected the cell samples at the time 30min, 1h, 8h and 24h afer infection. Using G-LISATM RhoA Activation Assay Biochem KitTM (Cytoskeleton) kit to detect the intracellular activity of RhoA molecule. The results showed that compared with control group , RhoA activity increased significantly at 30min and 1h after the infection. However, at 8h and 24h after infection the molecular activity of RhoA nearly did not change compared with control group. This result suggest that RhoA activity is activated mainly in the course of the virus into the cell, and has not changed in the proliferation process of viral replication.4. Inhibiting Rock activity by Y-27632 to observe the impact on DV infectionUsing the ROCK inhibitor Y-27632 inhibited ROCK activity ,then taking the infection experiments, we collected supernatant and cell samples after 8 h and 24 h infection for virus titer test. Results showed that titers of supernatant and cell fraction have no significant difference between the groups drugs-treated and the control group. The results were consistent with our obsearvation using confocal microscopy. These results suggest that inhibition of ROCK activity have no effect on the replication of DV2.In conclusion, RhoA molecular plays an important role in DV2 infection. RhoA was activated during DV2 entry cells.

【关键词】 登革病毒ECV304细胞RhoA/ROCKC3转移酶Y-27632中间纤维波形蛋白
【Key words】 DVECV304RhoA/ROCKY-27632vimentinIFsC3 transferase
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