Zika Virus Pathogenesis and Selective Autophagy Induction to Inhibit Virus Production

在过去的一年里，寨卡病毒由于感染迅速蔓延而受到全世界的关注，其 与吉兰-巴利综合征的关联，其通过性行为和在子宫内传播给胎儿的能力， 患有小头畸形和其他严重脑缺陷的婴儿数量增加。的基因组 目前流行的病毒相对于最初从病毒中分离出来的病毒有 75 个氨基酸替换 1947 年乌干达的恒河猴 (MR766)，似乎只引起了零星和轻微的 疾病。这些替换中的任何一个是否对病毒复制适应性、发病机制或 人类之间的传播尚不清楚。因此，使用非洲分离株进行研究非常重要 以及最近的流行毒株。该提案的目标是阐明根本原因 控制流行病毒严重神经发病机制的机制并检验我们的假设 诱导自噬会抑制病毒感染。该提案汇集了以下知识 两名在相关领域久负盛名且经验丰富的研究人员。通过结合各领域的专业知识 Spector 病毒学实验室，特别是分子/细胞生物学和人类发病机制 巨细胞病毒（目前是神经出生缺陷的主要病毒原因）和干细胞分化，以及 在 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 [iPSC] 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 测序）和尖端计算技术，我们拥有 加深对这一重大医学问题的了解的独特机会。我们将确定 当细胞在不同分化阶段被感染时，病毒感染和细胞死亡的动力学 iPSC 转化为皮质神经元。我们还将利用我们在全基因组技术方面的优势来识别 宿主基因（包括编码和非编码RNA以及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之间的差异 菌株 MR766 和最近流行的分离株。也新颖且具有潜在的巨大治疗作用 意义在于我们提出的诱导自噬抑制 ZIKV 感染并促进细胞自噬的策略 生存和分化。具体来说，我们将测试是否用无毒的细胞处理 二糖海藻糖通过一种新的不依赖于 mTOR 的途径诱导自噬，会抑制 感染，正如我们在人类巨细胞病毒中发现的那样。我们还将测试另外 2 种自噬诱导剂， 不依赖mTOR的小分子SMER28和tat-beclin 1.本次目标的实现 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.