Inhibiting Cellular Autophagy to Thwart Dengue Virus Packaging and Replication

描述(申请人提供)：登革热病毒是一种A类病原体，每年感染4000-1亿人。登革热感染的严重程度从自我限制的登革热到更致命的登革热、登革出血热和登革热休克综合征。尽管登革热感染长期以来仅限于热带和亚热带地区，但由于面对城市化和全球变暖，蚊子种群生态发生变化，该病毒已开始在以前的地理限制之外传播。此外，四种个体血清型的界限已经开始重叠，使更多的人面临登革出血热的风险，当二次感染与初次感染的血清型不同时，登革出血热的发病率会增加。目前还没有治疗方法或疫苗。传染性登革热病毒的形成严重依赖于自噬的细胞过程。在代谢应激条件下，自噬通过吞噬双膜小泡中的细胞质成分，并介导其溶酶体依赖的降解，在促进细胞存活方面发挥着重要作用。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也就不足为奇了。然而，某些微生物病原体，如登革热病毒，已被证明依赖于这一细胞过程或该过程的成分进行自身繁殖。因此，自噬的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物Spautin-1(特异性和强效的自噬抑制物-1)是由哈佛大学军营实验室发现的，它干扰了Beclin-1的稳定，而Beclin-1是形成自噬诱导复合体所必需的。柯克加德实验室已经证明，Spautin-1是一种有效的登革热病毒粒子组装抑制因子。在这项提案的R21部分，将通过对Spautin-1存在时形成的缺陷病毒颗粒的调查、Spautin耐药病毒的遗传分析以及对自噬扰动小鼠模型中的初次和继发感染的分析来剖析这种抑制的机制。在R33部分，袁和Kirkegaard实验室将合作测试新开发的Spautin-1衍生物，这些衍生物在小鼠的登革热发病和生长方面具有改进的药理学特性。虽然自噬在所有哺乳动物细胞中是一个正常的构成过程，但它被新发现的Spautin-1等分子暂时抑制可能允许清除急性感染的病原体，如破坏细胞自噬途径的登革病毒。 公共卫生相关性：登革热病毒每年感染4000万至1亿人，后果从亚临床发热到出血性致命性疾病。目前没有治疗方法，也没有疫苗。此外，很难找到针对登革病毒等RNA病毒的药物治疗方法，因为它们变异得如此之快，以至于抗药性发展得很快。然而，在感染登革热的人类细胞中抑制自噬(自噬)过程的新化合物对病毒具有深远的抑制作用，因为病毒颗粒在人类细胞内的组装严重依赖于这种“宿主”功能。描述了如何准确地确定病毒如何利用细胞自噬，以及如果这种宿主过程被药物靶向，耐药病毒是否会频繁出现。

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. PUBLIC HEALTH RELEVANCE: Dengue virus infects 40 to 100 million people each year, with outcomes ranging from subclinical fever to hemorrhagic, lethal disease. There is currently no treatment and no vaccine. Furthermore, it is difficult to find drug treatments for RNA viruses such as Dengue virus because they mutate so quickly that drug resistance develops rapidly. However, new compounds that inhibit the process of autophagy (self-eating) in the human cells infected by Dengue inhibit the virus profoundly, because the assembly of viral particles within human cells critically depends on this 'host' function. Work is described to determine exactly how cellular autophagy is used by the virus, and whether drug-resistant viruses will arise frequently if this host process is targeted pharmaceutically.