【作者】 伍兵；

【导师】 王宾；

【作者基本信息】 中国农业大学 ， 微生物学， 2014， 博士

【摘要】 在漫长的生物进化历史中,病毒也一直和宿主共同进化。在这过程中,宿主发展了多种免疫应答机制以清除病毒,而病毒则共进化出花样繁多的逃逸策略来躲避宿主的免疫应答,保护自身的生存利益。天然免疫包括天然免疫细胞如树突状细胞(Dendritic cells, DCs)、巨噬细胞以及抗病毒因子是宿主抵抗病毒入侵的第一道防线,起到了直接抵抗病毒的作用。对宿主和病毒相互作用的探索将有助于更好地理解病毒感染和发病的机制,也为抗病毒疫苗和药物研发提供新的思路。Ⅰ型单纯疱疹病毒(Herpes simplex virus-1, HSV-1),是一种DNA病毒。在长久的进化中,HSV-1也进化出多种策略逃逸宿主的天然免疫,从而造成裂解感染并导致口腔疱疹、脑炎和肺炎等疾病,以及在急性感染粘膜组织后于附近的神经元细胞建立潜伏感染。HSV-1感染进入细胞的机制过去有许多研究,但对于其如何利用细胞内吞途径逃避或抑制免疫反应的机理仍然有许多不清楚的地方。研究发现HSV-1感染会导致DCs的增多和一氧化氮(Nitric Oxide, NO)分泌的增加。而一些病毒,如SV40可以利用细胞膜窖小窝(caveolae)结构作为进入细胞的入口。Caveolin-1(小窝蛋白-1,Cav-1),是caveolae的基本结构蛋白。先前的研究发现Cav-1可能在病毒感染时参与病毒与宿主的相互作用,抑制先天炎症细胞因子,并促进病毒的复制。但是这种相互作用如何影响宿主对病毒的防卫,Cav-1、DCs在HSV-1感染中具有什么作用目前还不清楚。本研究发现Caveolin-1缺失的(Caveolin-1knockout, Cav-1KO)小鼠能够更好地抵抗病毒感染。致死剂量病毒感染时,相比于野生型(Wild Type, WT)小鼠,Cav-1KO能更好地保护小鼠,表现为肺部中更低的病毒滴度和较轻的肺部病理损伤。通过气道内过继转移Cav-1knockdown和Cav-1overexpression的DC细胞实验发现,过继转移Cav-1knockdown的DC细胞的WT小鼠具有更高的存活率。除此之外,相比于Cav-1单敲除小鼠,DCs和Cav-1双敲除(CD11c-DTR/Cav-1-/-)小鼠的存活率显著降低。表明DCs在Cav-1KO抵抗HSV-1感染中具有重要作用,暗示HSV-1利用DC细胞上表达的Cav-1促进病毒感染。进一步通过q-PCR筛选实验发现WT小鼠肺细胞和DC细胞中iNOS(诱导性一氧化氮合酶)的表达受到显著抑制。同时发现Cav-1明显抑制了小鼠血清、支气管灌洗液和DCs培养上清中HSV-1诱导的NO水平。而且用SMT (iNOS抑制剂)预处理Cav-1KO小鼠抑制iNOS和NO的表达明显降低了Cav-1单敲除体现的抗病毒能力。表明Cav-1KO对小鼠的保护依赖于DC细胞中NO的分泌。通过激光共聚焦和Co-IP实验进一步揭示在病毒感染的DC细胞中,Cav-1与iNOS、HSV-1在caveolae中存在共定位。这些结果说明病毒可以利用DC细胞上的Cav-1将iNOS束缚在caveolae中从而降低iNOS和NO表达水平,进而帮助病毒逃逸。通过以上结果,我们首次阐述了HSV-1利用Cav-1抑制DC细胞中的iNOS的表达并阻碍NO的分泌,从而达到逃逸宿主抗病毒反应的机制。Cav-1的缺失导致caveolae结构的破坏和iNOS表达升高,进而NO的大量分泌保护Cav-1KO小鼠免于HSV-1的致死感染。因此,Cav-1可能是HSV-1用来逃避宿主免疫的一条路径。本研究的发现可能提供一种新的有价值的抗病毒治疗策略。更多还原

【Abstract】 Viruses coevolve with their host and exploit diverse pathways to evade host immune responses. Host also develops various immune responses to counteract viral infections. In addition, innate immunity contain DCs, macrophages and antiviral cytokines plays a key role in the initiation and modulation of antiviral innate responses. Understanding the interactions of host-virus will provide us new insight for the mechnism of viral infections, viral pathogenesis and vaccines design.Herpes simplex virus type1(HSV-1) is a dsDNA virus belongs to the a-herpesvirus family, which causes oral herpes, encephalitis, keratitis, neonatal herpes and pneumonia disease and establishes latency in the neurons after acute infection of mucosal tissues. Previous reports found that HSV-1infection led to DC accumulations and NO productions which play a role in antiviral effects. In addition, Caveolin-1(Cav-1), the principal element of caveolae structure, is implicated in the viral-host interaction. Several viruses, including SV40, exploit caveolae as a gateway to enter cell and suppress host responses to facilitate their infections.The roles of Cav-1, DCs in HSV-1infection have not been fully elucidated.Here, we identify that Cav-1KO mice are more resistant to viral infection. Compared to wild type (WT) of animals, Cav-1KO could increase the protection upon HSV-1infection, accompanied with alleviative lung injury and lower viral load. The experiment of Cav-1knockdown and Cav-1overexpression DCs intra-tracheally showed Cav-1kd DC received WT mice increased the survival, indicated that the DCs played a role in Cav-1KO mediated resistant to HSV-1infection. And HSV-1may exploit Cav-1to avoid clearance. In addition, increased survival of Cav-1-/-mice was correlated with augmented iNOS and NO production in lungs and DCs. In turns, DCs and Cav-1deficiencies (CD11c-DTR/Cav-1-1-) or Cav-1-/-mice that were treated with an iNOS inhibitor exhibited a significantly reduced protection as compared to Cav-1single deletion only. Finally, we also observed that Cav-1co-localized with iNOS and HSV-1in caveolae in the viral infected DCs by confocal microscopy and Co-IP analysis.Our findings for the first time describe a novel mechanism of interaction of HSV-1with the Cav-1protein to suppress iNOS/NO in DCs and ultimately avoiding anti-viral responses of host. In addition, the disruption of caveolae lead to the increase of iNOS and NO in DCs, resulting in the protection of Cav-1KO against HSV-1infection. Thus, Cav-1could act as a gateway for HSV-1evasion. The data also provide a valuable target for drug design and therapeutic approach against herpes virus infections.更多还原