伪狂犬病病毒DNA聚合酶入核转运的分子机制

发布时间：2018-02-07 12:21

  本文关键词： 伪狂犬病病毒 DNA聚合酶 UL42 UL30 入核转运　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

【摘要】：伪狂犬病病毒(Pseudorabies virus,PRV)又被称为猪疱疹病毒1型或奥耶斯基病病毒,属于疱疹病毒科α-疱疹病毒亚科水痘病毒属。PRV可引起家畜和多种野生动物发生伪狂犬病,其中猪是该病的主要宿主和传染源。PRV在多个国家均有流行,对养猪业危害极大,造成严重的经济损失。PRV DNA复制是一个复杂的过程,需要病毒编码的多种酶蛋白协同参与,其中病毒DNA聚合酶发挥关键作用。PRV DNA聚合酶包含2个亚基:催化亚基UL30和辅助亚基UL42。UL30具有天然的酶活性,能催化病毒DNA的合成,而UL42能将聚合酶全酶固定在DNA模版上,增强全酶的持续合成能力。PRV DNA聚合酶在细胞质合成后必须转运到细胞核中才能发挥功能,这是启动病毒DNA复制的先决条件。然而,PRV DNA聚合酶入核转运的分子机制尚未阐明。作为PRV DNA聚合酶的辅助亚基,UL42是否是病毒复制所必需的也还不清楚。本研究旨在阐明PRV DNA聚合酶UL42与UL30亚基入核转运的分子机制,并明确辅助亚基UL42在PRV DNA复制过程中的作用。为阐明PRV DNA聚合酶UL42与UL30亚基的入核通路,本研究经免疫荧光试验证实,UL42能独立定位于细胞核中,UL30主要定位于细胞质中,然而当UL42与UL30共表达时,UL30的细胞质定位完全转移为细胞核定位,表明UL30的入核转运依赖于UL42。对UL42和UL30进行缺失和定向突变分析发现,UL42在354~370位氨基酸包含一个功能性和可转移性的双分型核定位信号(Nuclear localization signal,NLS),并证实K354、R355和K367是UL42双分型NLS保持完整结构和功能所必需的,然而UL30并无功能性NLS。免疫共沉淀试验结果表明,UL42与Importinα3(Impα3)和Impα4存在相互作用,且这一相互作用是由UL42双分型NLS介导的。经体外入核转运试验证实,UL42的核定位是一个温度、能量和受体依赖的过程,需要Impα和Impβ同时参与,表明UL42是经Impα/β通路入核的。当缺失NLS的UL42突变体(UL42ΔNLS)与UL30共表达时,UL42ΔNLS/UL30异源二聚体被完全限制在细胞质中,表明UL30利用UL42 NLS的功能入核。上述结果表明,PRV DNA聚合酶全酶复合体在细胞质中装配完成后,借助于UL42双分型NLS的功能,经Impα/β通路入核。研究表明,PRV UL42能在体外刺激UL30的催化活性,且它是UL30的细胞核协同转运蛋白,能在体外引导UL30入核,因此推测UL42是PRV复制的必需基因。本研究对PRV感染后UL42的表达动力学进行了分析,结果发现UL42是PRV的一个早期基因,在病毒感染后5 h就能检测到。根据UL42序列特征设计了3条特异性si RNAs,经Western blot试验证实它们都能在体外有效地抑制UL42的表达。在哺乳动物细胞中转染si RNAs后感染PRV,结果发现这3条si RNAs能剂量依赖性地抑制病毒感染后UL42的表达,且在下调UL42的表达后,PRV的复制显著降低,表明靶向UL42的RNAi能有效地降低UL42的表达,从而抑制PRV的复制。上述结果表明,UL42是PRV复制的必需基因。综上所述,本研究阐明了PRV DNA聚合酶入核转运的分子机制,证实了UL42是PRV复制的必需基因,对深入理解PRV的复制机制具有重要科学意义。
[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.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65
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本文编号：1494365