ROCK激酶在Ⅱ型登革病毒感染诱导波形蛋白纤维重排中的作用研究

发布时间：2018-05-16 07:50

本文选题：登革病毒 + ECV304细胞　；参考：《第三军医大学》2012年硕士论文

【摘要】：登革病毒(dengue virus,DENV)是黄病毒科黄病毒属的单股正链RNA病毒,根据其E蛋白的抗原性不同,可分为四种血清型,即DENV1-4。登革病毒主要通过蚊媒传播,广泛流行于热带和亚热带地区,引起人类登革热(dengue fever,DF)和登革出血热/休克综合症(dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS)。全世界每年约有5000万至1亿感染者,其中约50万为DHF/DSS患者,如未及时治疗致死率可高达50%,登革病毒感染已成为亟待解决的公共卫生问题。而目前对于DENV感染仍无有效的疫苗和特异性药物,预防主要依赖于对蚊媒的控制。因此深入研究DENV与宿主细胞相互作用的机制,将有助于阐明DENV的致病机制和寻找防治的突破点。病毒是一类具有高度寄生性的微生物,它完全依赖宿主细胞的能量和代谢系统,获取生命活动所需的物质与能量。病毒的复制和装配通常在细胞内的特定区域进行,多种病毒在核周区域或者细胞质中形成由细胞骨架、细胞器、特定的细胞内膜构成的特殊结构,称为“病毒工厂”(virus factories),其功能可能是为病毒的复制和装配提供了相对独立的空间,以排除宿主细胞蛋白酶和细胞器对复制过程的干扰。黄病毒科病毒的病毒工厂构成与内质网、高尔基体和线粒体密切相关,而DENV在宿主细胞中形成的病毒工厂的构成尚不清楚。有研究表明,多种病毒的病毒工厂构建过程与波形蛋白纤维密切相关。我们的前期工作显示:DENV2感染可诱导ECV304细胞波形蛋白纤维发生重排,波形蛋白丝从细胞边缘回缩并环绕细胞核,与病毒蛋白呈共同分布,利用丙烯酰胺破坏波形蛋白纤维,可明显抑制DENV2的复制和产生,但其机制尚不清楚。波形蛋白纤维的重排通常是由激酶磷酸化其头部区结构域所致,研究表明激活ROCK激酶(Rho-associatedcoiled coil-containing kinase,ROCK)使波形蛋白头部磷酸化位点Ser71磷酸化而发生重排,但其是否在DENV2感染诱导波形蛋白纤维重排中发挥作用需要进一步研究。基于ROCK激酶在波形蛋白纤维重排中的重要作用,以及DENV2感染引起波形蛋白纤维重排这一现象,结合前期实验结果,本研究拟以ROCK激酶为主要研究对象,观察波形蛋白纤维、内质网、高尔基体和线粒体与DENV2病毒工厂构成的关系,检测DENV2感染后宿主细胞波形蛋白磷酸化水平及ROCK激酶活性的变化的改变,并利用特异性抑制剂抑制ROCK激酶活性,验证DENV2感染可能激活ROCK激酶活性、诱导波形蛋白磷酸化和重排、参与形成病毒工厂的推论,阐明ROCK激酶在DENV2感染诱导波形蛋白纤维重排中的作用,为预防和控制DENV2感染提供新思路。本研究的主要实验内容和结论如下: 1.形态学观察波形蛋白纤维及相关细胞器与DENV2病毒工厂构成的关系本实验使用标记物质特异性标记细胞波形蛋白、内质网、高尔基体和线粒体,以细胞免疫荧光染色观察波形蛋白纤维及相关细胞器与DENV2病毒工厂构成的关系。结果显示:对照组波形蛋白纤维从细胞核至细胞边缘呈网状分布,而DENV2感染组细胞波形蛋白纤维网状结构塌陷发生重排,主要表现为(1)波形蛋白纤维丝由细胞边缘回缩至细胞核一侧聚集,形成包绕病毒NS1/E蛋白的笼状结构(2)波形蛋白纤维丝由细胞边缘回缩环绕细胞核,与病毒NS1/E蛋白呈明显共分布；对照组细胞内质网在胞质内呈均匀弥漫分布,而DENV2感染组细胞内质网回缩聚集于核周,与病毒NS1/NS3蛋白呈明显共分布；高尔基体形态及分布未发生明显改变,在感染后48h可观察到与病毒E蛋白呈共分布；线粒体在感染后48h形态由管网状变为点泡状,没有与病毒蛋白共分布。结果提示高尔基体可能与病毒结构蛋白的转运和成熟有关,线粒体可能参与了病毒导致的线粒体途径的凋亡过程,两者似乎并不参与DENV2病毒工厂的形成。而波形蛋白纤维和内质网可能共同参与了DENV2在ECV304细胞中病毒工厂的形成。 2.DENV2感染诱导的波形蛋白纤维重排与波形蛋白Ser71磷酸化密切相关 ROCK激酶可以特异性地使波形蛋白头部磷酸化位点Ser71磷酸化,从而导致波形蛋白纤维发生重排,因此本实验使用能识别Ser71磷酸化波形蛋白的单克隆抗体,以细胞免疫荧光染色观察DENV2感染后Ser71磷酸化波形蛋白与病毒蛋白的分布关系。结果显示:Ser71磷酸化的波形蛋白纤维分布与DENV2感染诱导波形蛋白重排形成的结构相似,且与病毒蛋白明显共存。这提示DENV2诱导的波形蛋白纤维重排与波形蛋白Ser71磷酸化密切相关,DENV2感染可能通过激活ROCK激酶使波形蛋白Ser71磷酸化导致波形蛋白纤维发生重排。 3.DENV2感染导致ECV304细胞内波形蛋白纤维发生重排,使用Y-27632抑制ROCK激酶活性能显著抑制波形蛋白纤维重排现象。免疫荧光染色结果显示:对照组细胞各时相点波形蛋白纤维形态没有明显变化,从细胞核至细胞边缘呈网状分布,而DENV2感染组细胞感染后30min时即可观察到波形蛋白纤维网状结构开始塌陷,纤维丝由细胞边缘回缩,感染后1h时波形蛋白纤维重排至核周区域,聚集于细胞核一侧,感染后8h时波形蛋白纤维环绕细胞核,感染后24h时波形蛋白纤维以上述两种重排后的形态共同存在,并呈浓缩分布使局部荧光强度增强。这一结果提示波形蛋白纤维发生重排与DENV2在ECV304细胞中的感染与复制周期密切相关。使用ROCK激酶特异性抑制剂Y-27632抑制ROCK激酶活性后进行感染实验,以免疫荧光染色观察波形蛋白纤维的形态变化。结果显示:只加入DENV2的对照组中波形蛋白纤维发生重排,而Y-27632药物处理组各时间点的波形蛋白纤维形态保持了原有的纤维网状结构,未发生明显的形态变化。这提示ROCK激酶在DENV2感染诱导的波形蛋白纤维重排中发挥了重要作用,使用药物抑制ROCK激酶活性能显著抑制DENV2感染诱导的波形蛋白纤维重排。 4. DENV2感染导致波形蛋白Ser71磷酸化水平显著升高,使用Y-27632抑制ROCK激酶活性能显著抑制波形蛋白Ser71磷酸化为进一步研究波形蛋白Ser71磷酸化在DENV2感染中的作用,本实验使用能识别Ser71磷酸化波形蛋白的单克隆抗体,以Western blot检测DENV2感染后ECV304细胞内波形蛋白表达及Ser71磷酸化水平的变化。结果显示: DENV2感染过程中各时相点的波形蛋白表达没有明显差异,而Ser71磷酸化水平在感染后30min时升高至对照组的176.3%,8h时增高至对照组的333.2%达到峰值,12h时呈明显下降的趋势,达到对照组的203.5%,其余时间点相比对照组没有显著差异。这一结果提示DENV2感染过程中波形蛋白表达量未受影响,但Ser71磷酸化水平变化与DENV2的感染和复制过程密切相关。使用ROCK激酶特异性抑制剂Y-27632抑制ROCK激酶活性后进行感染实验,以Western blot检测波形蛋白Ser71磷酸化水平的变化。结果显示:与只加入DENV2的对照组相比,Y-27632药物处理组各时间点的Ser71磷酸化波形蛋白水平显著降低,感染后30min时降低至对照组的51.42%,1h时降低至42.42%,8h时降低至54.01%,24h时降低至46.76%。结果提示ROCK激酶在DENV2感染复制过程中发挥了重要作用,使用药物持续抑制ROCK激酶活性能显著抑制DENV2感染诱导的波形蛋白Ser71磷酸化,从而抑制波形蛋白纤维发生重排。 5. DENV2感染导致ROCK激酶活性水平显著升高本实验使用ROCK激酶活性检测试剂盒,分别检测灭活病毒感染的对照组和一般感染组ECV304细胞中不同时相点ROCK激酶活性水平。结果显示:在感染后15min、30min和8h时ROCK激酶活性较对照组显著升高,15min时升高了243.75%,30min时下降至177.19%,8h时升高至216.18%,其余时间点ROCK激酶水平较对照组没有明显变化。这一结果提示ROCK激酶活性主要是在DENV2吸附和穿入ECV304细胞的过程中明显升高,之后回复初始水平,而在病毒从ECV304细胞中释放的关键时间点8h时再次明显上升,然后降低至初始水平。综上所述,本实验初步确定DENV2感染导致ECV304细胞波形蛋白纤维重排,并和内质网一起参与DENV2病毒工厂的形成；病毒感染过程中ROCK激酶活性明显升高,并引发其所对应波形蛋白Ser71磷酸化位点的磷酸化水平升高；使用药物抑制ROCK激酶活性能显著抑制DENV2感染诱导的波形蛋白Ser71磷酸化和波形蛋白纤维重排。上述实验结果初步验证了DENV2感染可能激活ROCK激酶活性、诱导波形蛋白磷酸化和重排、参与形成病毒工厂的推论,阐明ROCK激酶在DENV2感染诱导波形蛋白纤维重排中的作用。
[Abstract]:Dengue virus (DENV) is a single strand RNA virus of the yellow virus of the family yellows. According to the antigenicity of the E protein, it can be divided into four serotypes. That is, the DENV1-4. dengue virus is spread mainly through mosquito vectors and is widely prevalent in tropical and subtropical regions, causing human dengue fever (dengue fever, DF) and dengue hemorrhagic / shock heald. Dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). There are about 50 million to 100 million infected people in the world each year, about 500 thousand of which are DHF/DSS patients, if the mortality rate can be as high as 50%, and dengue virus infection has become an urgent problem for public health. At present, there is still no effective vaccine and specificity for DENV infection. Drug prevention is mainly dependent on the control of mosquito vectors. Therefore, a thorough study of the mechanism of the interaction between DENV and host cells will help to elucidate the pathogenesis of DENV and to find a breakthrough in the prevention and treatment.
Viruses are highly parasitic microorganisms that depend entirely on the energy and metabolic systems of the host cells to obtain the material and energy needed for life activities. The replication and assembly of the virus is usually carried out in specific regions of the cell, and many viruses form the cytoskeleton, organelles, and specific fines in the perinuclear region or cytoplasm. The special structure of the endometrium, known as the "virus factories", has the function of providing a relatively independent space for the replication and assembly of the virus to exclude the interference of the host cell protease and organelles to the replication process. The virus factory of the flavivirid virus is composed of the endoplasmic reticulum, the Golgi body and the mitochondria. The composition of the virus factory formed by DENV in the host cells is not clear. Studies have shown that the process of plant construction of a variety of viruses is closely related to the vimentin fiber. Our previous work showed that DENV2 infection could induce rearrangement of vimentin fibers in ECV304 cells, and vimentin filament retracted from the edge of the cell and ring. There is a common distribution between the nucleus and the virus protein. Using acrylamide to destroy the vimentin fiber can obviously inhibit the replication and production of DENV2, but its mechanism is not clear. The rearrangement of vimentin fiber is usually caused by the kinase phosphorylation of its head region, and the study shows that the activation of ROCK kinase (Rho-associatedcoiled coil-containing) is shown. Kinase, ROCK) rearranged the phosphorylation site of vimentin on the phosphorylation site Ser71, but it needs further study whether it plays a role in the DENV2 infection induced vimentin fiber rearrangement.
Based on the important role of ROCK kinase in the rearrangement of vimentin fiber and the rearrangement of vimentin (vimentin) caused by DENV2 infection, combined with the previous experimental results, this study aims to study the relationship between vimentin fiber, endoplasmic reticulum, Golgi body and mitochondria and the DENV2 virus factory, and the detection of DENV 2 the changes in the level of vimentin phosphorylation and ROCK kinase activity after infection, and the inhibition of ROCK kinase activity by specific inhibitors, verify that DENV2 infection may activate ROCK kinase activity, induce vimentin phosphorylation and rearrangement, participate in the formation of virus factory, and clarify that ROCK kinase induces a waveform egg in DENV2 infection. The role of white fiber rearrangement is a new way to prevent and control DENV2 infection.
The main contents and conclusions of this study are as follows:
1. morphological observation of vimentin fibers and associated organelles and their relationship with DENV2 virus factories
The relationship between vimentin fiber and related organelles and the DENV2 virus factory was observed by labeling material specifically with vimentin, endoplasmic reticulum, Golgi apparatus and mitochondria. The results showed that the vimentin fiber in the control group was reticulate from the nucleus to the edge of the cell, and DENV2 infection was found in the control group. The rearrangement of the reticular formation of vimentin fibers in the group cells was manifested as (1) the vimentin filament was retracted from the edge of the cell to the nucleus of the nucleus and formed a cage like structure wrapped around the virus NS1/E protein (2) the vimentin filament was retracted from the cell edge to encircled the nucleus of the cell, and the virus NS1/E protein was convolently co distributed; the control group was in the control group. The endoplasmic reticulum was distributed uniformly in the cytoplasm, while the endoplasmic reticulum retracted in the DENV2 infection group was clustered at the perinuclear cycle, and was obviously co distributed with the virus NS1/NS3 protein. The morphology and distribution of the Golgi bodies did not change obviously. After infection, the 48h could be observed with the virus E protein, and the mitochondria in the form of 48h were changed from the tube reticulation after infection. The results suggest that the Golgi bodies may be related to the transport and maturation of the viral structural proteins, and the mitochondria may participate in the apoptosis process of the mitochondrial pathway caused by the virus, and they do not seem to be involved in the formation of the DENV2 virus factory. The wave protein fiber and endoplasmic reticulum may participate in the DENV 2 the formation of a virus factory in ECV304 cells.
The rearrangement of vimentin fibers induced by 2.DENV2 infection is closely related to vimentin Ser71 phosphorylation.
ROCK kinase can specifically phosphorylate the phosphorylation site Ser71 of vimentin head, leading to the rearrangement of vimentin fibers. Therefore, this experiment uses a monoclonal antibody that recognizes the Ser71 phosphorylation of vimentin. The distribution of Ser71 phosphorylated vimentin and viral protein after DENV2 infection is observed by cell immunofluorescence staining. The results showed that the distribution of Ser71 phosphorylated vimentin fiber was similar to the structure of vimentin rearrangement induced by DENV2 infection, and it coexisted with viral proteins. This suggests that the rearrangement of vimentin induced by DENV2 is closely related to the phosphorylation of vimentin Ser71. DENV2 infection may lead to vimentin Ser71 by activating ROCK kinase. Phosphorylation leads to the rearrangement of vimentin fibers.
3.DENV2 infection leads to rearrangement of vimentin fibers in ECV304 cells. Inhibition of ROCK kinase activity by Y-27632 can significantly inhibit the rearrangement of vimentin fibers.
The results of immunofluorescence staining showed that the morphology of vimentin fibers in each phase of the control group did not change significantly, and the cells from the nucleus to the cell edge were reticulate, while the reticular formation of vimentin fibers could be observed in the DENV2 infection group 30min after infection, and vimvil was retracted from the edge of the cell, and vimentin was found at 1h after infection. The fibers rearranged into the perinuclear region, gathered at the nucleus of the nucleus, and the vimentin fiber was surrounded by the nucleus after 8h infection. After 24h infection, the vimentin fiber existed together in the form of the two rearrangements, and the concentration distribution enhanced the local fluorescence intensity. The results suggested that the rearrangement of the fibrin fiber and DENV2 in the ECV304 cells. Infection in the medium is closely related to the cycle of replication.
ROCK kinase specific inhibitor Y-27632 was used to inhibit the activity of ROCK kinase, and the morphological changes of vimentin fiber were observed by immunofluorescence staining. The results showed that the vimentin fiber was rearranged in the control group only with DENV2, while the form of vimentin fiber at each time point in the Y-27632 drug treatment group remained the same. ROCK kinase plays an important role in the rearrangement of vimentin fiber induced by DENV2 infection, and the inhibition of ROCK kinase activity by drug can significantly inhibit the rearrangement of vimentin fiber induced by DENV2 infection.
4. DENV2 infection resulted in a significant increase in vimentin Ser71 phosphorylation level, and inhibition of ROCK kinase activity by Y-27632 significantly inhibited vimentin Ser71 phosphorylation.
In order to further study the role of vimentin Ser71 phosphorylation in DENV2 infection, this experiment uses a monoclonal antibody that recognizes Ser71 phosphorylation of vimentin, and uses Western blot to detect the changes in the expression of vimentin and the level of Ser71 phosphorylation in ECV304 cells after DENV2 infection. The results show that the waveform eggs of each phase point in the DENV2 infection process are shown. There was no significant difference in white expression, but the level of phosphorylation of Ser71 increased to 176.3% of the control group at 30min after infection, and the increase to 333.2% of the control group reached the peak value at 8h, and the trend of 12h decreased significantly, reaching 203.5% of the control group. The other time points were not significantly different from those in the control group. This result suggests that vimentin in the DENV2 infection process is a result of vimentin. The expression level was not affected, but the level of Ser71 phosphorylation was closely related to the infection and replication process of DENV2.
The infection experiment was carried out after the inhibition of ROCK kinase activity with the ROCK kinase specific inhibitor Y-27632. The changes in the phosphorylation level of vimentin Ser71 were detected with Western blot. The results showed that the level of Ser71 phosphorylated vimentin decreased significantly at each time point of Y-27632 drug treatment group compared with that of the control group only with DENV2, and 30min decreased after infection. Low to 51.42% of the control group, 1H decreased to 42.42%, 8h decreased to 54.01%, and the decrease to 46.76%. at 24h suggested that ROCK kinase played an important role in the replication of DENV2 infection. The use of drug continuous inhibition of ROCK kinase activity could significantly inhibit the Ser71 phosphorylation induced by DENV2 infection, thus inhibiting the onset of vimentin fibers. Rearrangement.
5. DENV2 infection leads to a significant increase in ROCK kinase activity.
In this experiment, the ROCK kinase activity detection kit was used to detect the activity level of ROCK kinase in the control group and the ECV304 cells of the general infection group, respectively. The results showed that the activity of ROCK kinase in 15min, 30min and 8h after infection was significantly higher than that of the control group, the 15min increased by 243.75%, and the 30min decreased to 177.19% when 30min, 8h. The level of ROCK kinase increased to 216.18% at the rest of the time and did not change significantly compared with the control group. This result suggested that the activity of ROCK kinase increased significantly in the process of DENV2 adsorption and penetration of ECV304 cells, and then returned to the initial level, while the critical time point 8h released from the virus was obviously increased again, and then decreased. Low to initial level.
To sum up, this experiment preliminarily determined that DENV2 infection leads to the rearrangement of vimentin fiber in ECV304 cells, and participates with the endoplasmic reticulum in the formation of the DENV2 virus factory. The activity of ROCK kinase in the process of virus infection is obviously increased, and the phosphorylation level of the corresponding vimentin Ser71 phosphorylation site is raised, and the use of drugs to inhibit ROCK excitation The enzyme activity could significantly inhibit the phosphorylation of vimentin Ser71 induced by DENV2 infection and the rearrangement of vimentin fiber. The results showed that DENV2 infection may activate ROCK kinase activity, induce vimentin phosphorylation and rearrangement, participate in the inference of the virus factory, and clarify the weight of vimentin fiber induced by ROCK kinase in DENV2 infection. The role of the platoon.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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