pUL49调控HCMV宿主细胞周期和增殖的分子机制研究

发布时间：2018-03-05 08:36

  本文选题：人巨细胞病毒　切入点：pUL49蛋白　出处：《暨南大学》2016年博士论文　论文类型：学位论文

【摘要】：人巨细胞病毒(Human cytomegalovirus,HCMV)作为疱疹病毒的β-亚族成员,在人群中感染非常普遍。HCMV感染对健康人一般不致病,但对免疫低下人群如新生儿、多次输血、器官移植、艾滋病患者等会致病,甚至导致死亡。pUL49是HCMV病毒编码的蛋白,为病毒复制所必需,缺失pUL49开放阅读框(ORF)的BAC-Towne-ΔUL49病毒转染宿主细胞不能包装出子代病毒粒子,pUL49 mRNA的下调也会严重影响病毒的复制。尽管pUL49非常重要,但其在宿主细胞中的功能及作用机理目前还不明确。本课题为探索pUL49在宿主细胞中的功能,开展了如下研究:构建pUL-49基因过表达的慢病毒载体,包装、扩增、纯化病毒,感染U373细胞,筛选得到pUL49过表达的U373稳定细胞系。我们首先发现在pUL49稳定过表达的U373细胞中细胞增殖能力明显受到抑制,流式细胞检测发现pUL49过表达不诱导细胞凋亡、不影响凋亡相关蛋白的表达,但是使G1/S期细胞数量增加,说明pUL49过表达抑制U373细胞增殖不是通过诱导细胞凋亡,而是通过阻滞细胞周期于G1/S期实现的。为研究pUL49蛋白对U373细胞增殖抑制的分子机制,我们通过基于免疫共沉淀的蛋白质组学筛选得到33种与pUL49相互作用的宿主蛋白,其中一个侯选蛋白是FRK。FRK属于非受体蛋白酪氨酸激酶的一种,结构上与Src家族成员具有高度同源性;FRK具有磷酸化底物,抑制肿瘤细胞的增殖和迁移,阻滞细胞周期于G1期等作用。为了进一步确定二者之间的相互作用,通过免疫共沉淀技术确定pUL49和FRK在U373细胞中能够相互作用、间接免疫荧光技术确定pUL49和FRK共定位于细胞核。pUL49和FRK相互作用,两者又都抑制细胞增殖,提示pUL49可能通过FRK调控U373宿主细胞的生长增殖。我们设计了针对pUL49和FRK的siRNA,通过过表达和敲低实验组合发现pUL49过表达抑制细胞增殖;敲低FRK促进细胞增殖;在pUL49过表达的细胞中同时敲低pUL49可以部分抵消pUL49过表达引起的增殖抑制;pUL49过表达的同时敲低FRK也能部分抵消pUL49过表达引起的增殖抑制,两者趋势一致;在pUL49过表达的细胞中同时敲低FRK和pUL49,pUL49对增殖的抑制几乎全部被抵消,证实pUL49可通过FRK调控U373宿主细胞的增殖。同样以过表达和敲低实验组合发现pUL49通过FRK阻滞细胞周期于G1/S期。FRK阻滞细胞周期的其中一个机制是抑制细胞周期蛋白cyclin D1进入细胞核。我们在过表达pUL49的同时敲低FRK观察cyclinD1蛋白在细胞核内的分布情况,结果发现,pUL49过表达不影响cyclin D1蛋白的表达,但会使细胞核内的cyclin D1蛋白减少,细胞质中的cyclin D1蛋白增加,但同时敲低FRK使细胞核内的cyclin D1蛋白又会增加,相应地细胞质中的cyclin D1蛋白减少,说明pUL49调控U373宿主细胞的细胞周期是通过FRK调控cyclin D1的入核实现的。以高通量液相芯片技术检测若干调控细胞生长增殖的信号通路,发现pUL49过表达增加IRS-1(S636/S639)和P70S6K(T421/S424)蛋白的磷酸化,Western Blot进一步确定pUL49使IRS-1信号通路的IRS-1(S636/S639)、AKT(S473/T308)、PDK1(S241)、mTOR(S2448)、P70S6K(T421/S424)磷酸化。mTOR(S2448)、P70S6K(T421/S424)抑制剂均使细胞增殖进一步受到抑制,说明pUL49还需要通过IRS-1/AKT/mTOR/P70S6K信号通路来维持宿主细胞的生存。实验室前期研究表明缺失N端143个氨基酸后,pUL49的核定位可被影响,因此我们构建了pUL49及其缺失体pUL49(ΔN143)、pUL49(ΔN100)、pUL49(143)、pUL49(100)的真核表达载体。结果表明去除N端143个氨基酸后,pUL49(ΔN143)、pUL49(ΔN100)仍可使IRS-1(S636/S639)和P70S6K(T421/S424)磷酸化,证明pUL49蛋白的核定位对于IRS-1(S636/S639)和P70S6K(T421/S424)蛋白的磷酸化的调节不是必须的。总之,通过本课题研究,我们揭示了pUL49作为HCMV病毒的必须基因,通过FRK促使宿主细胞U373的细胞周期阻滞于G1/S期,从而抑制细胞的生长增殖,pUL49同时通过IRS-1/AKT/mTOR/P70S6K信号通路来维持宿主细胞的生存的分子机制。
[Abstract]:Human cytomegalovirus (Human cytomegalovirus HCMV) as the beta herpesvirus subfamily members,.HCMV infection is very common infection in healthy people generally do not cause the disease in the population, but in immunocompromised persons such as newborns, blood transfusion, organ transplantation, disease caused by AIDS patients may even lead to death,.PUL49 is HCMV encoding protein is essential for viral replication, deletion of pUL49 open reading frame (ORF) of BAC-Towne- UL49 virus transfection of host cells cannot package progeny virus particles, the down-regulation of pUL49 mRNA will seriously affect the replication of the virus. Although pUL49 is very important, but its function and role in the host cell in the mechanism is not clear at present this topic to explore pUL49 in the host cell function is carried out as follows: to construct a lentiviral vector, overexpression of pUL-49 gene amplification and purification, packaging, virus infected U373 cells, screened by P U373 stable cell lines overexpressing UL49. We first found stable cell proliferation in U373 cells in the expression of pUL49 was significantly inhibited, detected overexpression of pUL49 did not induce cell apoptosis, did not affect the expression of apoptosis related proteins, but the number of cells in G1/S phase increased, indicated that overexpression of pUL49 inhibited U373 cells not proliferation by inducing apoptosis, but through cell cycle arrest in G1/S phase. For the study of the molecular mechanism of pUL49 protein inhibited the proliferation of U373 cells, we through proteomic immunoprecipitation study screened 33 pUL49 proteins based on one candidate protein is a kind of FRK.FRK belongs to the non receptor protein tyrosine kinase, structure and member of the Src family is highly homologous with FRK; phosphorylation of substrate, inhibit the proliferation and migration of tumor cells, block The cell cycle in G1 phase and so on. In order to further determine the interaction between the two, pUL49 and FRK can determine interaction in U373 cells by CO immunoprecipitation, indirect immunofluorescence technique to determine pUL49 and FRK were located in the nucleus.PUL49 and FRK interaction, the two are both inhibition of cell proliferation, suggesting that pUL49 may be the growth and proliferation of FRK regulatory U373 cells of the host. We designed the siRNA for pUL49 and FRK, the overexpression and knockdown experiments showed that overexpression of pUL49 inhibits cell proliferation; knockdown of FRK promotes cell proliferation; in cells overexpressing pUL49 and knockdown of pUL49 can be partially offset by the overexpression of pUL49 induced proliferation inhibition at the same time; overexpression of pUL49 knockdown of FRK can be partially offset by the overexpression of pUL49 induced proliferation inhibition, both in the same trend; the over expression of pUL49 cells and the knockdown of FRK and pUL49, pUL49 The inhibitory effect on proliferation were almost totally offset, confirmed that pUL49 can be regulated by FRK U373 host cell proliferation. Similarly to overexpression and knockdown of pUL49 by FRK found that the experiment combined with cell cycle arrest in G1/S phase.FRK cell cycle arrest one mechanism is the inhibition of cyclin cyclin D1 into the nucleus. We in the over expression of pUL49 at the same time the knockdown of FRK to observe the distribution of cyclinD1 protein in the nucleus, the results showed that overexpression of pUL49 did not affect the expression of cyclin D1 protein, but the nucleus of cyclin D1 protein in the cytoplasm of cyclin decreased, D1 protein increased, but at the same time, the knockdown of FRK nuclear cyclin D1 protein will increase. The cytoplasmic cyclin protein D1 decreased, indicating the cell cycle regulation of pUL49 U373 host cells through nuclear FRK regulation of cyclin D1. With high throughput LIQUICHIP Technology Detection of signaling pathways regulating cell proliferation of pUL49, found that overexpression of IRS-1 (S636/S639) and P70S6K (T421/S424) protein phosphorylation, Western Blot pUL49 IRS-1 to further determine the signal pathway of IRS-1 (S636/S639), AKT (S473/T308), PDK1 (S241), mTOR (S2448), P70S6K (T421/S424) phosphate.MTOR (S2448), P70S6K (T421/S424) inhibitors were further inhibited cell proliferation, indicating that pUL49 is needed by IRS-1/AKT/mTOR/P70S6K signaling pathways maintaining host cell survival. Previous research showed that deletion of 143 amino acids in the N terminal after the nuclear localization of pUL49 can be influenced, so we constructed a pUL49 deletion and pUL49 (N143), pUL49 (N100), pUL49 (143), pUL49 (100) eukaryotic expression vector. The results showed that the removal of 143 amino acids in the N terminal, pUL49 (N143), pUL49 (N100) can make the IRS-1 (S636/S639) and P70S6K (T421/S424) Phosphorylation of pUL49 protein that nuclear localization for IRS-1 (S636/S639) and P70S6K (T421/S424) protein phosphorylation in the regulation is not necessary. In short, through this study, we revealed the pUL49 as the HCMV virus gene must, through cell cycle arrest of FRK to host cells U373 in G1/S, thus inhibiting the growth of the proliferation of cells, pUL49 through the IRS-1/AKT/mTOR/P70S6K pathway to maintain the molecular mechanisms of host cell survival.

【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R373

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