Rapid Phenotyping of the ZIKV Genome

1.项目摘要/摘要 最近寨卡病毒(ZIKV)的爆发令世界感到惊讶，因为它通过 美国及其与出生缺陷的关系，如新生儿小头畸形和其他神经系统疾病 成人的功能障碍，如格林-巴利综合征(GBS)。然而，我们目前面临的挑战是 我们对ZIKV的功能知之甚少，也不知道为什么ZIKV突然变成了 致病性太强了。快速评估ZIKV功能的尝试受到了广泛的主机的进一步挑战 ZIKV感染的细胞。在这里，我们提出了一种独特的组合研究方法来应对这些挑战。 具体地说，我们计划使用分裂酵母作为替代系统来快速分析ZIKV的功能 基因组，然后是人类神经细胞的即时翻译和寨卡病毒感染模型。 裂解酵母是一种简单的单细胞真核生物，已被广泛用作模式生物 研究人类细胞生物学和病毒学。这是一个研究高度保守的细胞活动的经过充分测试的模型。 如所描述ZIKV对细胞生长、细胞周期调节、染色体生物学 和细胞死亡。因此，研究裂殖酵母中的这些ZIKV效应不仅与ZIKV疾病有关。 但我们也可以加快ZIKV基因组的功能特征。 我们是唯一有资格进行这项拟议研究的机构，因为我们在 使用分裂酵母对病毒基因组进行分析，以及在有关病毒感染的哺乳动物研究中。在……里面 事实上，我们是第一个确定HIV-1基因组特征并在分裂酵母中开发模型系统进行研究的人 HIV-1病毒蛋白R(VPR)和蛋白酶(PR)。我们的研究得到了进一步加强，因为有一个 多学科团队，其专业知识涵盖拟议研究的整个范围。因此，我们应该 在拟议的研究中取得成功的可能性很高。 我们推测ZIKV疾病如小头症或GBS是由病毒致病性引起的。 所有ZIKV固有的因素。或者，新的ZIKV变种已经作为基因的结果而出现 导致适应高致病性的突变。为了验证这一假设，我们将重点关注两个 特定目标(SA)。SA1是用来对ZIKV基因组进行快速表型以鉴定致病性的 因子(S)，这将进一步与基因上独特的寨卡病毒变异株进行比较，以回答这个问题 观察到的ZIKV致病性是一种固有的病毒特性还是新获得的病毒功能。这个 SA2将开发抗ZIKV药物测试和筛选系统，以对抗病毒致病因子和 ZIKV NS2B/NS3酶，后者已被用作其他黄病毒的治疗靶点。 拟议实验的成功完成将1)提供全基因组范围的描述 ZIKV表型，2)识别与单个ZIKV蛋白相关的病毒致病因子，以及3)发展 基于裂解酵母细胞的抗寨卡病毒药物测试和筛选系统。

1. Project Summary/Abstract The recent Zika virus (ZIKV) outbreaks has surprised the world because of its rapid spread through the America and its association with birth defects such as microcephaly in the newborns and other neurologic dysfunctions in adults such as the Guillain-Barré syndrome (GBS). However, we are currently challenged with the fact that we know very little about the ZIKV functionality, nor do we know why has ZIKV suddenly become so pathogenic. Attempts to rapidly assess the ZIKV functionality is further challenged by a wide range of host cells that ZIKV infects. Here, we propose a unique and combined research approach to meet these challenges. Specifically, we plan to use fission yeast as a surrogate system for the rapid functional analysis of the ZIKV genome followed by immediate translations in human neural cells and a ZIKV infection model. Fission yeast is a simple and single cell eukaryote that has been used extensively as a model organism to study human cell biology and virology. It is a very well-tested model to study highly conserved cellular activities such as those described ZIKV cytopathic effects on cellular growth, cell cycle regulation, chromosomal biology, and cell death. Thus, study of these ZIKV effects in fission yeast is not only clinically relevant to the ZIKV diseases but we can also expedite the functional characterization of the ZIKV genome. We are uniquely qualified for the proposed study because we have extensive experiences in functional analyses of viral genomes using fission yeast and in the associated mammalian studies on viral infections. In fact, we were the first to characterize the HIV-1 genome and to develop model systems in fission yeast to study HIV-1 viral protein R (Vpr) and proteases (PRs). Our study is further strengthen by the participation of a multidisciplinary team whose expertise covering the entire spectrum of the proposed study. Therefore, we should have a high likelihood of success in the proposed study. We hypothesize that the ZIKV diseases such as microcephaly or GBS are caused by viral pathogenicity factors that are intrinsic to all ZIKVs. Alternatively, new ZIKV variants have emerged as the results of gene mutations that have led to the adaptation of high pathogenicity. To test this hypothesis, we will focus on two Specific Aims (SAs). The SA1 is to quickly phenotype the ZIKV genome for the identification of pathogenicity factor(s), which will be further compared against genetically distinctive ZIKV viral variants to answer the question of whether the observed ZIKV pathogenicity is an intrinsic viral property or the newly acquired viral function. The SA2 is to develop anti-ZIKV drug testing and screening systems against the viral pathogenicity factor and the ZIKV NS2B/NS3 protease, the latter has already been used as a therapeutic target for other flaviviruses. The successful completion of the proposed experiments will 1) provide a genome-wide description of the ZIKV phenotypes, 2) identify viral pathogenic factors that are linked to individual ZIKV proteins, and 3) develop fission yeast cell-based systems for anti-ZIKV drug testing and screenings.