【摘要】：RNA病毒種類繁多,比較常見的RNA病毒有埃博拉病毒、甲型流感病毒、中東呼吸綜合癥冠狀病毒以及登革病毒等。近幾年來(lái)這些RNA病毒的爆發(fā)和流行對(duì)人類健康已經(jīng)構(gòu)成嚴(yán)重的威脅,尤其是最近西非爆發(fā)的埃博拉病毒致死率已經(jīng)達(dá)到40%。在RNA病毒感染過程中病毒和宿主都會(huì)通過編碼一些小分子來(lái)調(diào)節(jié)對(duì)方,其中包括microRNA分子。MicroRNA作為一類小片段的RNA分子具有重要的調(diào)節(jié)作用,通常一個(gè)microRNA可以同時(shí)調(diào)節(jié)上百個(gè)基因的表達(dá),這種高效的調(diào)節(jié)方式常常被病毒加以利用并輔助其感染。雖然已知很多microRNA都能夠參與病毒感染的過程,但是對(duì)其發(fā)揮作用的具體機(jī)制仍然不是十分清楚。為了研究RNA病毒感染過程中microRNA發(fā)揮的作用,分別對(duì)埃博拉病毒和甲型流感病毒進(jìn)行分析研究。在埃博拉病毒中,利用生物信息學(xué)的方法對(duì)埃博拉病毒基因組序列進(jìn)行分析,發(fā)現(xiàn)在病毒基因組中含有一段潛在的microRNA編碼序列。進(jìn)一步利用埃博拉病毒感染者血清的RNA-Seq數(shù)據(jù)對(duì)預(yù)測(cè)的序列進(jìn)行驗(yàn)證并結(jié)合體外實(shí)驗(yàn)驗(yàn)證結(jié)果,發(fā)現(xiàn)該預(yù)測(cè)的序列能夠編碼出兩條成熟的microRNA分子并分別命名為Zebov-miR-1-5p和Zebov-miR-1-3p。其中Zebov-miR-1-5p的序列與人內(nèi)源性has-miR-155-5p的序列具有相似性,而且能夠有效抑制宿主細(xì)胞中KPNA1基因的表達(dá)。KPNA1作為入核輔助因子能夠調(diào)節(jié)STAT1的信號(hào)傳遞功能,埃博拉病毒通過此途徑抑制干擾素信號(hào)釋放并最終逃逸宿主細(xì)胞對(duì)病毒感染的免疫監(jiān)視。與埃博拉病毒不同,在甲型流感病毒感染中,病毒并不能編碼任何microRNA分子,但是被感染的宿主細(xì)胞中,部分microRNA的表達(dá)會(huì)隨著病毒復(fù)制的增加而逐漸降低。通過熒光素酶報(bào)告實(shí)驗(yàn)表明,表達(dá)降低的microRNA中,miR-23a能夠靶向于流感病毒基因組的PA、PB1以及PB2中的部分序列。流感病毒聚合酶相關(guān)基因的表達(dá)下降能夠抑制病毒在宿主體內(nèi)的復(fù)制速度。利用miR-23a的這個(gè)特性在流感病毒感染的小鼠中注射miR-23a可以用于改善流感病毒對(duì)小鼠的感染程度。結(jié)合以上兩種RNA病毒與microRNA之間的調(diào)控關(guān)系,分析認(rèn)為不同的RNA病毒在其感染過程中具有不同的調(diào)節(jié)方式。其中既有通過主動(dòng)編碼microRNA逃逸宿主的免疫監(jiān)視,也有利用宿主表達(dá)的microRNA調(diào)節(jié)自身復(fù)制速度以延長(zhǎng)感染時(shí)間。因此RNA病毒在利用microRNA的方式上存在不同的策略,通過不同的策略在宿主與病毒感染過程中達(dá)到一個(gè)平衡并有利于病毒的擴(kuò)增和傳播。當(dāng)對(duì)RNA病毒的這些調(diào)節(jié)方式深入研究后,我們就可以利用病毒感染中的這些特點(diǎn)有針對(duì)性地設(shè)計(jì)出藥物以干擾病毒的感染過程。
[Abstract]:RNA viruses include Ebola virus, influenza A virus, Middle East Respiratory Syndrome coronavirus and dengue virus. The outbreaks and epidemics of these RNA viruses have posed a serious threat to human health in recent years, especially since the recent outbreak of Ebola in West Africa has led to a mortality rate of 40 per cent. In the process of RNA infection, both the virus and the host regulate each other by encoding small molecules, including microRNA molecules. MicroRNAs play an important role as a class of small fragments of RNA molecules. Usually, a single microRNA regulates the expression of hundreds of genes at the same time. Although many microRNA are known to be involved in the process of viral infection, the mechanism of its role is still unclear. In order to study the role of microRNA in the process of RNA infection, Ebola virus and influenza A virus were analyzed. In Ebola virus, the genome sequence of Ebola virus was analyzed by bioinformatics, and a potential microRNA coding sequence was found in the genome of Ebola virus. The predicted sequence was further verified by RNA-Seq data from the sera of Ebola virus infected patients and combined with the results of in vitro experiments. It was found that the predicted sequence could encode two mature microRNA molecules named Zebov-miR-1-5p and Zebov-miR-1-3p., respectively. The sequence of Zebov-miR-1-5p is similar to that of human endogenous has-miR-155-5p, and it can effectively inhibit the expression of KPNA1 gene in host cells. KPNA1 can regulate the signal transduction function of STAT1 as an auxiliary factor. In this way, Ebola virus inhibits the release of interferon signals and eventually escapes host cells from immune surveillance of virus infection. Unlike the Ebola virus, the virus does not encode any microRNA molecules in influenza A infection, but the expression of microRNA in infected host cells decreases with the increase of viral replication. The results of luciferase report showed that the down-expressed microRNA could target the PA,PB1 of influenza virus genome and some sequences of PB2. Decreased expression of influenza virus polymerase-related genes can inhibit the replication of the virus in the host. The use of this characteristic of miR-23a can be used to improve the degree of influenza virus infection in mice by injecting miR-23a into mice infected with influenza virus. According to the regulatory relationship between the two RNA viruses and microRNA, it is concluded that different RNA viruses have different regulation modes in the process of infection. Both immune surveillance of host by active coding of microRNA and microRNA expressed by host adjusts the rate of self-replication to prolong infection time. Therefore, RNA virus has different strategies in the way of using microRNA, through different strategies in the host and virus infection process to achieve a balance and conducive to the expansion and transmission of the virus. When the regulation of RNA virus is deeply studied, we can use these characteristics of virus infection to design drugs to interfere with the infection process of the virus.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R373
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本文編號(hào)：2250230