【作者】 刘畅；

【导师】 耿运琪；

【作者基本信息】 南开大学 ， 微生物学， 2009， 博士

【摘要】 潜伏感染是慢病毒逃脱宿主免疫监视和免疫清除的重要机制。宿主通过先天免疫以及获得性免疫抵抗外界病毒的感染。干扰素调节是先天免疫的重要组成部分,干扰素可以诱导一系列干扰素刺激基因的大量表达。干扰素刺激基因所编码干扰素刺激蛋白的高水平表达可以抑制病毒的复制,这种抑制是导致慢病毒建立潜伏感染的原因之一。诸多外界因素可以影响慢病毒的感染状态,使慢病毒由潜伏感染转向裂解感染。其中其他病毒与慢病毒的超感染便是影响因素之一,其他病毒的超感染往往可以促进慢病毒的复制。本研究以牛免疫缺陷病毒(BIV)为慢病毒模型,对一种干扰素刺激蛋白ISG15在BIV潜伏感染中的作用进行了初步的探讨。ISG15是一种可以被Ⅰ型干扰素诱导表达的小分子蛋白,属于类泛素修饰蛋白家族。与泛素类似,ISG15可以共价地连接到一些蛋白上,进行蛋白的翻译后修饰,该修饰称之为蛋白质的ISGylation修饰。ISG15以及ISGylation修饰参与多种重要的生物学过程,其中包括:先天免疫、肿瘤发生以及怀孕建立等。各种属间ISG15的氨基酸序列同源性并不高。目前,对于牛ISG15(bovineISG5,bISG15)的研究主要集中于其在母牛怀孕的建立方面,而bISG15在先天免疫以及抗病毒方面的研究基本上是空白。我们首先建立了bISG15的相关检测体系,包括bISG15的体外启动子Luciferase报告系统、bISG15 mRNA的RT-PCR检测系统以及bISG15表达的Western-blot检测系统等。在此基础上,对bISG15在胎牛肺细胞(FBLs,一种BIV的饲养细胞)中的表达、BIV与bISG15的相互影响(包括BIV感染对bISG15表达水平的影响、bISG15对BIV复制的抑制、BIV调控蛋白bTat对bISG15抑制的抵抗等)进行了初步的研究。最后,我们研究了RNA病毒牛病毒性腹泻病毒(BVDV)和DNA病毒牛疱疹病毒Ⅰ型(BHV-1)对bISG15表达的影响,并对其分子机制进行了初步探讨。本文获得了以下重要结果及结论:1.通过RT-PCR以及Western-blot检测系统证实,FBLs中bISG15本底表达水平不高,但通过poly I:C或LPS刺激可以引起bISG15表达水平的上调;2.通过免疫荧光以及染色体免疫沉淀实验证实,FBLs中bISG15表达水平的上调与干扰素调节因子3(IRF-3)密切相关,并通过体外转染实验证明IRF-3过表达可以激活bISG15的启动子;3.BIV感染FBLs,可以引起bISG15的少量上调,而随感染进行bISG15的增加表达水平逐渐下降;4.bISG15参与细胞的抗病毒过程,可以抑制BIV的复制,说明bISG15在BIV建立潜伏感染的过程中发挥一定作用;另一方面,BIV又可抑制bISG15的表达,该抑制作用可能与BIV的调控蛋白Tat有关;5.BVDV和BHV-1感染FBLs可以抑制bISG15表达的本底水平,而BHV-1的即早期蛋白bICP0可以抑制IRF-3对bISG15启动子的激活,据此推测,BVDV和BHV-1超感染可以通过抑制干扰素通路促进BIV的复制。更多还原

【Abstract】 Lantent infection is the strategy how lentiviruses escape from immune sruveillance and immune clearance of the host. Innate immunity and adaptive immunity play important roles in defending the virus infection, while the interferon regulation is an important part of innate immunity. Interferon can stimulate the expression of hundrands of interferon stimulated genes (isgs). The over expression of interferon stimulated protein can inhibit the replication of viruses. The inhibition can lend lentivirus to establish latent infection. Latent infection of lentiviruses can be broken by many factors, including virus super-infection. Some virus super-infection with lentivirus can stimulate the replication of the lentivirus.In this work, we used BIV as a lentivirus model, and did a preliminary study on the function of bovine ISG15 (bISG15) in latent infection of BIV.ISG15 is a small molecular protein whose expression can be up-regulated by type I interferon. It also belongs to the family of ubiquitin-like modifiers. Like ubiquitin, ISG15 can be conjugated to other proteins to modify them. The process of ISG15 modification is called ISGylation. ISG15 and ISGylation play important roles in many processes including innate immunity, cancer and pregnancy. The amino acid sequences analysis shows that the cross-species conservation of ISG15 is not high. Recently, most studies on bovine ISG15 (bISG15) were focused on its role in pregnancy. However, studies about the functions that bISG15 is involeved in innate immune response or antiviral effect was rarely done.In order to relieve the relation between bISG15 and viruses which infected cattle, we established the RT-PCR assay system to detect the mRNA level of bISG15, the Western-blot assay to detect the expression level of bISG15 and reporting system of bISG15 gene promoter in vitro. These assay systems were used to detect the expression of bISG15 in FBLs which were used in the culture of BIV. Then, we did some research work in the relationship between bISG15 and BIV, and the infuence of BVDV or BHV-1 infection on bISG15 expression. The main findings and significance of this study are: 1. RT-PCR and Westen-blot assayes suggested that the basal expression of bISG15 in FBLs was too low to be detected, and the expression of bISG15 was induced by poly I:C or LPS. 2. Immunofluorescence assay and ChIP assay demonstrated that IRF-3 played a role in inducing the expression of bISG15 in FBLs. The Luciferase assay showed that the over-expression of bovine IRF-3 could active the promoter of bISG15 gene. 3. Infection of BIV can up-regulated the expression of bISG15 weakly in FBLs. 4. The replication of BIV in FBLs can partly repressed by bISG15. bISG15 plays a role in the establishment of latent infection of BIV. Meanwhile, bTat, which is the regulatory protein of BIV, seems to be involed in the repression. 5. BVDV or BHV-1 infection can repress the expression of bISG15. bICP0, which is the protein of BHV-1, can repress the activation of bISG15 gene promoter by IRF-3. BVDV or BHV-1 can help BIV replicating though repressing the interferon signal pathway.更多还原