Inhibiting Cellular Autophagy to Thwart Dengue Virus Packaging and Replication

描述（由申请人提供）：登革热病毒是A类病原体，每年感染4000万至1亿人。登革热感染的严重程度从自限性登革热到更致命的登革热出血热和登革热休克综合征不等。虽然登革热感染长期以来仅限于热带和亚热带地区，但由于城市化和全球变暖，蚊子种群的生态变化，该病毒已开始在其以前的地理限制之外传播。此外，四种血清型的界限开始重叠，使更多的人面临登革出血热的风险，其发病率在与初次感染不同的血清型的二次感染后增加。目前没有治疗方法或疫苗。感染性登革病毒的形成主要依赖于自噬的细胞过程。自噬通过将细胞质成分吞噬在双膜囊泡中并介导其溶酶体依赖性降解，在促进细胞在代谢应激条件下存活中起重要作用。在这种能力下，细胞自噬可以作为先天免疫反应的一个组成部分，摧毁许多细胞内病原体，这并不奇怪。然而，某些微生物病原体，如登革病毒，已被证明依赖于这种细胞过程，或过程的成分，为自己的繁殖。因此，自噬抑制剂应该有助于控制这些感染。最近发表的自噬小分子抑制剂Spaclin-1（特异性和有效的自噬抑制剂-1）是由袁俊英（哈佛大学）的实验室发现的，可以干扰自噬诱导复合物形成所需的beclin-1的稳定。Kirkegaard实验室已经表明Spaeulase-1是感染性登革病毒体组装的有效抑制剂。在本提案的R21部分中，将通过研究在Spaetin-1存在下形成的缺陷病毒颗粒、Spautin抗性病毒的遗传分析以及自噬扰动小鼠模型中的原发性和继发性感染来剖析这种抑制的机制。在R33部分，Yuan和Kirkegaard实验室将合作测试新开发的Spaetin-1衍生物，这些衍生物对小鼠的登革热发病机制和生长具有改进的药理学特性。虽然自噬是所有哺乳动物细胞中的正常组成过程，但它被新发现的Spaeplasty-1等分子暂时抑制可能允许清除破坏细胞自噬途径的急性感染病原体，如登革热病毒。

DESCRIPTION (provided by applicant): Dengue virus, a category A pathogen, infects 40-100 million people each year. Dengue infection severity ranges from the self-limiting Dengue fever to the more lethal forms of the disease, Dengue hemorrhagic fever and Dengue shock syndrome. Although Dengue infections have long been limited to tropical and subtropical areas, the virus has begun to spread outside its former geographic limitations due to the changing ecology of mosquito populations in the face of urbanization and global warming. Furthermore, the boundaries for the four individual serotypes have begun to overlap, putting more people at risk for Dengue hemorrhagic fever, whose incidence is increased upon secondary infection with a different serotype from the primary infection. No treatment or vaccine currently exists. The formation of infectious Dengue virus is critically dependent upon the cellular process of autophagy. Autophagy plays an important role in promoting cell survival under metabolic stress conditions by engulfing cytoplasmic constituents in double-membraned vesicles and mediating their lysosome-dependent degradation. In this capacity, it is not surprising that cellular autophagy can act as a component of the innate immune response, destroying many intracellular pathogens. However, certain microbial pathogens, such as Dengue virus, have been shown to depend on this cellular process, or constituents of the process, for their own propagation. Therefore, inhibitors of autophagy should help to control these infections. The recently published small-molecule inhibitor of autophagy Spautin-1 (specific and potent autophagy inhibitor-1) was found by the laboratory of Junying Yuan (Harvard University) to interfere with the stabilization of beclin-1, required for the formation of autophagy-induction complexes. The Kirkegaard laboratory has shown that Spautin-1 is a potent inhibitor of the assembly of infectious Dengue virions. In the R21 portion of this proposal, the mechanism of this inhibition will be dissected through investigation of the defective viral particles formed in he presence of Spautin-1, genetic analysis of Spautin-resistant viruses, and analysis of primary and secondary infections in mouse models of autophagy perturbation. In the R33 portion, the Yuan and Kirkegaard laboratories will collaborate to test newly developed Spautin-1 derivatives with improved pharmacological properties on Dengue pathogenesis and growth in mice. Although autophagy is a normal constitutive process in all mammalian cells, its temporary inhibition by molecules such as the newly discovered Spautin-1 is likely to allow the clearance of acutely infecting pathogens such as Dengue virus that subvert the cellular autophagy pathway.