本文選題：人巨細(xì)胞病毒　切入點(diǎn)：pUL49蛋白　出處：《暨南大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：人巨細(xì)胞病毒(Human cytomegalovirus,HCMV)作為皰疹病毒的β-亞族成員,在人群中感染非常普遍。HCMV感染對(duì)健康人一般不致病,但對(duì)免疫低下人群如新生兒、多次輸血、器官移植、艾滋病患者等會(huì)致病,甚至導(dǎo)致死亡。pUL49是HCMV病毒編碼的蛋白,為病毒復(fù)制所必需,缺失pUL49開(kāi)放閱讀框(ORF)的BAC-Towne-ΔUL49病毒轉(zhuǎn)染宿主細(xì)胞不能包裝出子代病毒粒子,pUL49 mRNA的下調(diào)也會(huì)嚴(yán)重影響病毒的復(fù)制。盡管pUL49非常重要,但其在宿主細(xì)胞中的功能及作用機(jī)理目前還不明確。本課題為探索pUL49在宿主細(xì)胞中的功能,開(kāi)展了如下研究:構(gòu)建pUL-49基因過(guò)表達(dá)的慢病毒載體,包裝、擴(kuò)增、純化病毒,感染U373細(xì)胞,篩選得到pUL49過(guò)表達(dá)的U373穩(wěn)定細(xì)胞系。我們首先發(fā)現(xiàn)在pUL49穩(wěn)定過(guò)表達(dá)的U373細(xì)胞中細(xì)胞增殖能力明顯受到抑制,流式細(xì)胞檢測(cè)發(fā)現(xiàn)pUL49過(guò)表達(dá)不誘導(dǎo)細(xì)胞凋亡、不影響凋亡相關(guān)蛋白的表達(dá),但是使G1/S期細(xì)胞數(shù)量增加,說(shuō)明pUL49過(guò)表達(dá)抑制U373細(xì)胞增殖不是通過(guò)誘導(dǎo)細(xì)胞凋亡,而是通過(guò)阻滯細(xì)胞周期于G1/S期實(shí)現(xiàn)的。為研究pUL49蛋白對(duì)U373細(xì)胞增殖抑制的分子機(jī)制,我們通過(guò)基于免疫共沉淀的蛋白質(zhì)組學(xué)篩選得到33種與pUL49相互作用的宿主蛋白,其中一個(gè)侯選蛋白是FRK。FRK屬于非受體蛋白酪氨酸激酶的一種,結(jié)構(gòu)上與Src家族成員具有高度同源性;FRK具有磷酸化底物,抑制腫瘤細(xì)胞的增殖和遷移,阻滯細(xì)胞周期于G1期等作用。為了進(jìn)一步確定二者之間的相互作用,通過(guò)免疫共沉淀技術(shù)確定pUL49和FRK在U373細(xì)胞中能夠相互作用、間接免疫熒光技術(shù)確定pUL49和FRK共定位于細(xì)胞核。pUL49和FRK相互作用,兩者又都抑制細(xì)胞增殖,提示pUL49可能通過(guò)FRK調(diào)控U373宿主細(xì)胞的生長(zhǎng)增殖。我們?cè)O(shè)計(jì)了針對(duì)pUL49和FRK的siRNA,通過(guò)過(guò)表達(dá)和敲低實(shí)驗(yàn)組合發(fā)現(xiàn)pUL49過(guò)表達(dá)抑制細(xì)胞增殖;敲低FRK促進(jìn)細(xì)胞增殖;在pUL49過(guò)表達(dá)的細(xì)胞中同時(shí)敲低pUL49可以部分抵消pUL49過(guò)表達(dá)引起的增殖抑制;pUL49過(guò)表達(dá)的同時(shí)敲低FRK也能部分抵消pUL49過(guò)表達(dá)引起的增殖抑制,兩者趨勢(shì)一致;在pUL49過(guò)表達(dá)的細(xì)胞中同時(shí)敲低FRK和pUL49,pUL49對(duì)增殖的抑制幾乎全部被抵消,證實(shí)pUL49可通過(guò)FRK調(diào)控U373宿主細(xì)胞的增殖。同樣以過(guò)表達(dá)和敲低實(shí)驗(yàn)組合發(fā)現(xiàn)pUL49通過(guò)FRK阻滯細(xì)胞周期于G1/S期。FRK阻滯細(xì)胞周期的其中一個(gè)機(jī)制是抑制細(xì)胞周期蛋白cyclin D1進(jìn)入細(xì)胞核。我們?cè)谶^(guò)表達(dá)pUL49的同時(shí)敲低FRK觀察cyclinD1蛋白在細(xì)胞核內(nèi)的分布情況,結(jié)果發(fā)現(xiàn),pUL49過(guò)表達(dá)不影響cyclin D1蛋白的表達(dá),但會(huì)使細(xì)胞核內(nèi)的cyclin D1蛋白減少,細(xì)胞質(zhì)中的cyclin D1蛋白增加,但同時(shí)敲低FRK使細(xì)胞核內(nèi)的cyclin D1蛋白又會(huì)增加,相應(yīng)地細(xì)胞質(zhì)中的cyclin D1蛋白減少,說(shuō)明pUL49調(diào)控U373宿主細(xì)胞的細(xì)胞周期是通過(guò)FRK調(diào)控cyclin D1的入核實(shí)現(xiàn)的。以高通量液相芯片技術(shù)檢測(cè)若干調(diào)控細(xì)胞生長(zhǎng)增殖的信號(hào)通路,發(fā)現(xiàn)pUL49過(guò)表達(dá)增加IRS-1(S636/S639)和P70S6K(T421/S424)蛋白的磷酸化,Western Blot進(jìn)一步確定pUL49使IRS-1信號(hào)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)抑制劑均使細(xì)胞增殖進(jìn)一步受到抑制,說(shuō)明pUL49還需要通過(guò)IRS-1/AKT/mTOR/P70S6K信號(hào)通路來(lái)維持宿主細(xì)胞的生存。實(shí)驗(yàn)室前期研究表明缺失N端143個(gè)氨基酸后,pUL49的核定位可被影響,因此我們構(gòu)建了pUL49及其缺失體pUL49(ΔN143)、pUL49(ΔN100)、pUL49(143)、pUL49(100)的真核表達(dá)載體。結(jié)果表明去除N端143個(gè)氨基酸后,pUL49(ΔN143)、pUL49(ΔN100)仍可使IRS-1(S636/S639)和P70S6K(T421/S424)磷酸化,證明pUL49蛋白的核定位對(duì)于IRS-1(S636/S639)和P70S6K(T421/S424)蛋白的磷酸化的調(diào)節(jié)不是必須的�？傊�,通過(guò)本課題研究,我們揭示了pUL49作為HCMV病毒的必須基因,通過(guò)FRK促使宿主細(xì)胞U373的細(xì)胞周期阻滯于G1/S期,從而抑制細(xì)胞的生長(zhǎng)增殖,pUL49同時(shí)通過(guò)IRS-1/AKT/mTOR/P70S6K信號(hào)通路來(lái)維持宿主細(xì)胞的生存的分子機(jī)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.

【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R373

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