Perturbation of Host DNA Replication and Cell Cycle Progression by Zika Virus

我们的研究计划的长远目标是推进与疾病预防相关的病毒-宿主细胞相互作用的知识。我们研究的病毒是阳性链RNA病毒，如登革热病毒（DENV）、丙型肝炎病毒（HCV）和寨卡病毒（ZIKV）。目前的建议侧重于寨卡病毒，该病毒已在世界范围内重新出现，并对人类健康构成重大新威胁。了解寨卡病毒如何与宿主细胞相互作用对于开发目前缺乏的抗病毒药物和预防性疫苗至关重要。基于我们最近发表的关于ZIKV对人类神经干细胞的感染和影响的研究，我们提出揭示ZIKV阻碍人类皮层神经祖细胞（hNPCs）生长并导致皮层发育缺陷的机制。我们的初步数据表明，细胞周期阻滞的部分机制是zikv诱导的DNA损伤反应（DDR），它阻断DNA复制并导致s期阻滞。出乎意料的是，通常在s期处理DNA复制应激的ATR/Chk1 DNA检查点通路没有被激活，这表明ZIKV在DNA复制过程中抑制了ATR/Chk1的激活。在增加DNA复制压力的同时抑制ATR反应的能力构成了一个强有力的“组合拳”，加剧了复制缺陷，最终导致细胞周期停滞。我们将开展实验来研究寨卡病毒实现这些壮举的机制，并确定病毒蛋白的反应。我们还将在脑类器官和寨卡/登革热嵌合病毒的传染性克隆中交叉验证我们的结果。我们希望清楚地了解ZIKV通过扰乱细胞周期来实现其对体外hnpc介导的神经发育的病理作用的机制。我们还希望揭示寨卡病毒的毒力决定因素对其对大脑发育的影响至关重要。这些结果不仅将从根本上增进我们对这一重要人类病原体的了解，而且还将对开发治疗寨卡病毒感染及其相关疾病的有效疗法的任务产生直接和即时的影响。

The broad, long-term goal of our research program is to advance knowledge of virus–host cell interactions that are relevant for disease prevention. The viruses that we study are positive- strand RNA viruses such as dengue virus (DENV), hepatitis C virus (HCV), and Zika virus (ZIKV). The current proposal focuses on ZIKV, which has re-emerged worldwide and poses a major emerging threat to human health. Understanding how ZIKV interacts with the host cell is of critical importance to the development of antiviral drugs and a prophylactic vaccine, both of which are currently lacking. Building upon our recently published work on the infection and impact of ZIKV on human neural stem cells, we propose to unravel the mechanism by which ZIKV impedes the growth of human cortical neural progenitor cells (hNPCs) and leads to defects in cortex development. Our preliminary data indicate that part of the mechanism for cell cycle arrest is a ZIKV-induced DNA damage response (DDR) which blocks DNA replication and leads to S-phase arrest. Unexpectedly, the ATR/Chk1 DNA checkpoint pathway that normally functions to deal with DNA replication stress in S-phase was not activated, suggesting that ZIKV suppresses ATR/Chk1 activation during DNA replication. The ability to increase DNA replication stress while simultaneously inhibiting ATR responds constitute a potent “one-two punch” that exacerbates replication defects and ultimately leads to cell cycle arrest. We will carry out experiments to investigate the mechanisms by which ZIKV achieve these feats and identify the viral proteins responses. We will also cross-validate our results with brain organoids and infectious clones of Zika/Dengue chimeric viruses. We expect to clearly understand the mechanisms by which ZIKV perturbs the cell cycle to achieve its pathological effect on hNPC-mediated neural development in vitro. We also expect to reveal the virulent determinant of ZIKV critical for its effect on brain development. These results will not only fundamentally advance our understanding of this important human pathogen but also provide direct and immediate impact on the mission to develop effective therapy to treat ZIKV infection and its associated diseases.