本文關(guān)鍵詞： 偽狂犬病病毒 DNA聚合酶 UL42 UL30 入核轉(zhuǎn)運　出處：《中國農(nóng)業(yè)科學院》2016年博士論文　論文類型：學位論文

【摘要】：偽狂犬病病毒(Pseudorabies virus,PRV)又被稱為豬皰疹病毒1型或奧耶斯基病病毒,屬于皰疹病毒科α-皰疹病毒亞科水痘病毒屬。PRV可引起家畜和多種野生動物發(fā)生偽狂犬病,其中豬是該病的主要宿主和傳染源。PRV在多個國家均有流行,對養(yǎng)豬業(yè)危害極大,造成嚴重的經(jīng)濟損失。PRV DNA復制是一個復雜的過程,需要病毒編碼的多種酶蛋白協(xié)同參與,其中病毒DNA聚合酶發(fā)揮關(guān)鍵作用。PRV DNA聚合酶包含2個亞基:催化亞基UL30和輔助亞基UL42。UL30具有天然的酶活性,能催化病毒DNA的合成,而UL42能將聚合酶全酶固定在DNA模版上,增強全酶的持續(xù)合成能力。PRV DNA聚合酶在細胞質(zhì)合成后必須轉(zhuǎn)運到細胞核中才能發(fā)揮功能,這是啟動病毒DNA復制的先決條件。然而,PRV DNA聚合酶入核轉(zhuǎn)運的分子機制尚未闡明。作為PRV DNA聚合酶的輔助亞基,UL42是否是病毒復制所必需的也還不清楚。本研究旨在闡明PRV DNA聚合酶UL42與UL30亞基入核轉(zhuǎn)運的分子機制,并明確輔助亞基UL42在PRV DNA復制過程中的作用。為闡明PRV DNA聚合酶UL42與UL30亞基的入核通路,本研究經(jīng)免疫熒光試驗證實,UL42能獨立定位于細胞核中,UL30主要定位于細胞質(zhì)中,然而當UL42與UL30共表達時,UL30的細胞質(zhì)定位完全轉(zhuǎn)移為細胞核定位,表明UL30的入核轉(zhuǎn)運依賴于UL42。對UL42和UL30進行缺失和定向突變分析發(fā)現(xiàn),UL42在354~370位氨基酸包含一個功能性和可轉(zhuǎn)移性的雙分型核定位信號(Nuclear localization signal,NLS),并證實K354、R355和K367是UL42雙分型NLS保持完整結(jié)構(gòu)和功能所必需的,然而UL30并無功能性NLS。免疫共沉淀試驗結(jié)果表明,UL42與Importinα3(Impα3)和Impα4存在相互作用,且這一相互作用是由UL42雙分型NLS介導的。經(jīng)體外入核轉(zhuǎn)運試驗證實,UL42的核定位是一個溫度、能量和受體依賴的過程,需要Impα和Impβ同時參與,表明UL42是經(jīng)Impα/β通路入核的。當缺失NLS的UL42突變體(UL42ΔNLS)與UL30共表達時,UL42ΔNLS/UL30異源二聚體被完全限制在細胞質(zhì)中,表明UL30利用UL42 NLS的功能入核。上述結(jié)果表明,PRV DNA聚合酶全酶復合體在細胞質(zhì)中裝配完成后,借助于UL42雙分型NLS的功能,經(jīng)Impα/β通路入核。研究表明,PRV UL42能在體外刺激UL30的催化活性,且它是UL30的細胞核協(xié)同轉(zhuǎn)運蛋白,能在體外引導UL30入核,因此推測UL42是PRV復制的必需基因。本研究對PRV感染后UL42的表達動力學進行了分析,結(jié)果發(fā)現(xiàn)UL42是PRV的一個早期基因,在病毒感染后5 h就能檢測到。根據(jù)UL42序列特征設(shè)計了3條特異性si RNAs,經(jīng)Western blot試驗證實它們都能在體外有效地抑制UL42的表達。在哺乳動物細胞中轉(zhuǎn)染si RNAs后感染PRV,結(jié)果發(fā)現(xiàn)這3條si RNAs能劑量依賴性地抑制病毒感染后UL42的表達,且在下調(diào)UL42的表達后,PRV的復制顯著降低,表明靶向UL42的RNAi能有效地降低UL42的表達,從而抑制PRV的復制。上述結(jié)果表明,UL42是PRV復制的必需基因。綜上所述,本研究闡明了PRV DNA聚合酶入核轉(zhuǎn)運的分子機制,證實了UL42是PRV復制的必需基因,對深入理解PRV的復制機制具有重要科學意義。
[Abstract]:Pseudorabies virus (Pseudorabies virus PRV) is also known as pig herpesvirus type 1 or Aujeszky's disease virus, herpes virus, alpha herpesvirus varicella virus.PRV can cause livestock and wild animal occurrence of pseudorabies virus, the pig is the main host of the disease and the source of infection in.PRV countries are popular, the pig industry of great harm, serious economic losses caused by.PRV DNA replication is a complex process, a variety of enzymes involved in virus encoding protein, which play a key role in viral DNA polymerase.PRV DNA polymerase contains 2 subunits: catalytic subunit UL30 and subunit UL42.UL30 assisted with enzyme activity natural and synthetic can catalyze the virus DNA, and UL42 can be fixed on the DNA template polymerase holoenzyme, enhanced holoenzyme processivity of.PRV DNA polymerase in the cytoplasm after synthesis must be translocated into the nucleus In order to function, which is the start of a prerequisite for viral DNA replication. However, the molecular mechanism of PRV DNA polymerase in nuclear transport has not been elucidated. As an auxiliary subunit of PRV DNA polymerase, UL42 is essential for virus replication is still unclear. The purpose of this study is to elucidate the PRV DNA polymerase UL42 and UL30 based on the molecular mechanism of nuclear transport, and clear auxiliary subunit UL42 in PRV DNA replication process. In order to elucidate the PRV DNA polymerase UL42 and UL30 subunit into the nucleus pathway, this study confirmed by immunofluorescence assay, UL42 independent nuclear localization, UL30 was mainly located in the cytoplasm, but when UL42 and UL30 co expression, cytoplasmic localization of UL30 transfer for nuclear localization, showed that UL30 transportation into the nucleus depends on the UL42. mutation analysis showed that deletion and orientation for UL42 and UL30, UL42 contains a functional and 354~370 amino acids Two types of nuclear localization signal can transfer the (Nuclear localization signal, NLS), and confirmed that K354, R355 and K367 are UL42 double type NLS to maintain the integrity of the structure and function of the necessary, but UL30 had no functional NLS. immunoprecipitation test results showed that UL42 and Importin alpha (Imp alpha 3 and 3) Imp alpha 4 interaction, and this interaction is composed of UL42 double type NLS mediated nuclear import. In vitro experiments confirmed that the nuclear localization of UL42 is a temperature dependent process, energy and receptors, Imp alpha and Imp beta to participate at the same time, indicating that UL42 is the Imp alpha / beta pathway to nuclei. When UL42 mutants lacking NLS (UL42 NLS) Co expressed with UL30, UL42 a NLS/UL30 heterologous two dimer is limited in the cytoplasm, showed that the UL30 using the UL42 function of NLS into the nucleus. The results showed that the PRV DNA polymerase holoenzyme complex in the cytoplasm is with, with the help of in UL42 Double type NLS function, the Imp alpha / beta pathway into the nucleus. The results show that the catalytic activity of PRV UL42 in vitro stimulation of UL30, and it is the nucleus of UL30 cotransporter UL30 in vitro can lead into the nucleus, suggesting that UL42 gene is required for the replication of PRV. The study of PRV infection the expression kinetics of UL42 were analyzed, the results showed that UL42 was an early gene PRV, 5 h could be detected after viral infection. According to the UL42 sequence characteristics of 3 specific Si RNAs design by Western blot test confirmed that they were capable of inhibiting UL42 in vitro effectively expressed in mammalian cells. Transfection of Si RNAs after PRV infection, the results show that the 3 Si RNAs to express UL42 dose dependently inhibited the virus after infection, and the down-regulation of UL42 expression after PRV replication significantly decreased, suggesting that targeting UL42 RNAi can effectively reduce the expression of UL42, thereby inhibiting PR V replication. The above results show that UL42 is the essential gene for PRV replication. In conclusion, this study elucidated the molecular mechanism of PRV DNA polymerase entry and transportation, confirmed that UL42 is the essential gene for PRV replication, and has important scientific significance for understanding PRV replication mechanism.

【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S852.65
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本文編號：1494365