Zika Virus Pathogenesis and Selective Autophagy Induction to Inhibit Virus Production

在过去的一年里，寨卡病毒由于迅速传播的感染而获得了全世界的关注，其 与格林-巴利综合征有关，它通过性传播和宫内传播给胎儿的能力， 患有小头畸形症和其他严重脑缺陷的婴儿数量增加。该病毒的基因组 目前流行的病毒有75个氨基酸替换相对于最初从一个 1947年乌干达的恒河猴(MR766)，似乎只造成了零星的和轻微的 疾病。这些替换中的任何一个是否对病毒复制适合性、发病机制或 人类之间的传播是未知的。因此，重要的是利用非洲的孤立进行研究 以及最近流行的病毒株。这项提议的目的是澄清潜在的 控制流行性病毒严重神经发病的机制和验证我们的假说 自噬的诱导将抑制病毒感染。这项建议汇集了以下知识 两名在相关领域经验丰富的资深调查人员。通过将专业知识与 SPECTOR实验室病毒学，特别是分子/细胞生物学和人类发病机制 巨细胞病毒(目前导致神经出生缺陷的主要病毒原因)和干细胞分化，以及 YEO实验室研究人类干细胞生物学(包括人类诱导多能干细胞的分化 细胞[IPSCs]到大脑皮层神经元)、基因表达和RNA的高通量分析 处理(包括单细胞rna序列)和尖端计算技术，我们拥有 这是一个对这一重大医学问题有更多了解的难得机会。我们将确定 不同分化阶段细胞感染病毒和细胞死亡的动力学 IPSCs到大脑皮层神经元。我们还将利用我们在全基因组技术方面的优势来识别 寄主基因(包括编码和非编码RNA和microRNA)表达的变化 感染。重要的是，我们将比较早期非洲寨卡病毒感染的差异 菌株MR766和最近流行的分离株。也很新颖，可能有很好的治疗效果 重要的是我们提出了诱导自噬的策略来抑制ZIKV感染并促进细胞 生存和分化。具体地说，我们将测试是否用无毒的 二糖海藻糖通过一种新的不依赖mTOR的途径诱导自噬，将抑制 感染，就像我们已经发现的人类巨细胞病毒一样。我们还将测试另外两个自噬诱导剂，一个 不依赖mTOR的小分子SMER28和TAT-BECLIN1.目标的实现 该提案将促进制定旨在预防和治疗寨卡病毒的新战略 感染。

In the past year, Zika virus has gained worldwide attention due to the rapidly spreading infection, its association with Guillain-Barré syndrome, its ability to be transmitted sexually and in utero to the fetus, and the increased number of infants with microcephaly and other severe brain defects. The genome of the current epidemic virus has 75 amino acid substitutions relative to the virus originally isolated from a rhesus monkey in Uganda in 1947 (MR766), which appears to have caused only sporadic and mild disease. Whether any of these substitutions has effects on viral replication fitness, pathogenesis, or spread between humans is unknown. Thus it is important that studies be done using the African isolate as well as more recent epidemic strains. The goal of this proposal to elucidate the underlying mechanisms governing the severe neural pathogenesis of the epidemic virus and to test our hypothesis that induction of autophagy will inhibit the virus infection. This proposal brings together the knowledge of two well-established and experienced investigators in related fields. By combining the expertise in the Spector lab in virology, specifically the molecular/cell biology and pathogenesis of human cytomegalovirus (currently the major viral cause of neural birth defects), and stem cell differentiation, and in the Yeo lab on human stem cell biology (including differentiation of human induced pluripotent stem cells [iPSCs] to cerebral cortex neurons), high-throughput analysis of gene expression and RNA processing (including single cell RNA seq), and cutting edge computational technologies, we have a unique opportunity to gain greater understanding of this major medical problem. We will determine the kinetics of viral infection and cell death when cells are infected at different stages of differentiation of iPSCs to cortical neurons. We will also leverage our strengths in genome-wide technologies to identify changes in expression of host genes (including coding and noncoding RNAs and microRNAs) during infection. Importantly, we will compare the differences between infection with the early African Zika virus strain MR766 and a more recent epidemic isolate. Also novel and potentially of great therapeutic significance is our proposed strategy of inducing autophagy to inhibit ZIKV infection and promote cell survival and differentiation. Specifically, we will test whether treatment of cells with the nontoxic disaccharide trehalose, which induces autophagy via a novel mTOR-independent pathway, will inhibit the infection, as we have found for human cytomegalovirus. We will also test 2 other autophagy inducers, a mTOR-independent small molecule SMER28 and tat-beclin 1. Accomplishment of the goals of this proposal will facilitate the development of new strategies designed to prevent and treat Zika virus infection.