【作者】 王娟娟；

【导师】 戴建锋；

【作者基本信息】 苏州大学 ， 免疫学， 2015， 硕士

【摘要】 登革病毒(dengue virus,DENV)属于黄病毒科(Flavivirade)黄病毒属(Flavivirus),是目前世界上分布最广泛的由蚊类传播的虫媒病毒。截至目前,还没有安全有效的疫苗或者抗病毒药物来治疗登革病毒引起的疾病。鞘糖脂是由神经酰胺和糖类构成的糖脂分子,普遍分布于真核细胞的膜类结构上。最近,有研究发现鞘糖脂通过调节病毒感染宿主细胞的多个步骤来参与病毒感染。然而,鞘糖脂在登革病毒感染中的具体作用仍然不明确。在本研究中,我们采用小鼠黑色素瘤B16细胞和缺失鞘糖脂的突变体GM95细胞来研究鞘糖脂在登革病毒感染中的作用。结果发现与野生型B16细胞相比,缺失鞘糖脂的GM95细胞对登革病毒极不敏感:GM95细胞中病毒的复制水平,细胞上清中的活病毒量,都比野生型对照B16细胞中显著降低。用抑制鞘糖脂合成的抑制剂大豆皂苷或者PPMP来处理B16细胞,或者用RNAi的方法干扰B16细胞中三种主要鞘糖脂合成酶(mt3gal5,ma4Gal T or mb3Gn T),都能抑制登革病毒感染。相反地,在GM95细胞中加入外源鞘糖脂GM3,能部分的恢复登革病毒感染。同时,我们发现B16细胞感染登革病毒后,代表性的酸性鞘糖脂GM3的含量显著上升,鞘糖脂合成酶基因的表达也显著上调。这些结果提示了鞘糖脂在登革病毒感染上皮细胞B16过程中发挥作用。为了研究鞘糖脂参与登革病毒感染细胞的具体机制,我们用病毒基因定量PCR测定和罗丹明染料标记登革病毒等方法,来研究B16和GM95细胞对登革病毒的结合和内吞效率。结果表明,GM95细胞结合和内吞登革病毒的能力与B16细胞相比并无显著性差异。进一步实验发现,鞘糖脂对登革病毒的RNA复制起重要作用。向B16和GM95细胞中转染DNA型登革病毒复制子后,复制子在B16细胞中的复制水平显著地高于GM95细胞。此外,在登革病毒感染的B16细胞胞质中,病毒复制复合物的组成成分之一非结构蛋白NS4A与鞘糖脂GM3在内质网膜区域有部分共定位。这些实验数据表明,鞘糖脂不参与上皮细胞对登革病毒的结合和内吞作用,但影响病毒复制。靶向鞘糖脂的合成可成为潜在的抑制登革病毒感染的新思路。更多还原

【Abstract】 Dengue virus(DENV), a member of the flavivirus genus of the Flaviviridae family, causes the most prevalent arthropod-borne viral disease of humans worldwide. Currently, there is no approved vaccine, nor any antiviral drugs for the treatment of dengue. Glycosphingolipids(GSLs), amphipathic molecules consisting of a ceramide and sugar residues, are ubiquitous cellular components of eukaryotic membrane structures. Recently, GSLs were reported to involve in virus infection by regulating various steps of viral-host cell interaction. However, the distinct role of GSLs during DENV infection remains unclear. In this study, we used mouse melanoma B16 cells and their GSL-deficient mutant counterpart GM95 cells to study the influence of GSLs on DENV infection. We have found that GSL-deficient GM95 cells were highly resistant to DENV infection when compared with B16 wild type cells. Pretreatment of B16 cells with GSLs synthesis inhibitor or impairing the critical enzymes(mt3gal5, ma4 Gal T or mb3 Gn T) required for the synthesis of three major groups of GSLs by RNAi both significantly inhibited dengue virus infection. Instead, the decreased DENV infection was partially rescued by addition of exogenous GSLs into GM95 cells. DENV binding and entry, as determined by q PCR of viral genes or TRITC-labeled virus imaging, were not impaired in GM95 cells when compared with B16 wt cells. We further confirmed that GLSs are critical for DENV RNA replication by directly transfecting a DNA-based dengue virus replicon into B16 and GM95 cells. DENV replicon showed a 50 fold-increase in B16 cells when compared to GM95 cells. Furthermore, GSLs were co-localized with DENV nonstructural protein 4A(NS4A), a known component of viral replication complex(RC) inside the cytoplasm of B16 cells. Taken together, these data indicated that GSLs are not required for DENV virus attachment and endocytosis, however, participate in virus replication. Targeting GSLs could be a novel strategy to inhibit DENV infection.更多还原