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

我们的研究计划的广泛，长期目标是推进病毒与宿主细胞相互作用的知识，这与疾病预防有关。我们研究的病毒是正链RNA病毒，如登革病毒（DENV）、丙型肝炎病毒（HCV）和寨卡病毒（ZIKV）。目前的提案重点关注ZIKV，它已在全球范围内重新出现，并对人类健康构成重大威胁。了解ZIKV如何与宿主细胞相互作用对于开发抗病毒药物和预防性疫苗至关重要，这两者目前都缺乏。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工作，我们提出解开ZIKV阻碍人类皮质神经祖细胞（hNPC）生长并导致皮质发育缺陷的机制。我们的初步数据表明，细胞周期停滞的部分机制是ZIKV诱导的DNA损伤反应（DDR），其阻断DNA复制并导致S期停滞。出乎意料的是，通常用于处理S期DNA复制应激的ATR/Chk 1 DNA检查点途径没有被激活，表明ZIKV在DNA复制期间抑制ATR/Chk 1激活。增加DNA复制应激而同时抑制ATR应答的能力构成了一种有效的“一击二击”，其加剧复制缺陷并最终导致细胞周期停滞。我们将进行实验以研究ZIKV实现这些壮举的机制，并确定病毒蛋白质反应。我们还将用大脑类器官和寨卡/登革热嵌合病毒的感染性克隆交叉验证我们的结果。我们期望清楚地了解ZIKV扰乱细胞周期以实现其对hNPC介导的体外神经发育的病理作用的机制。我们还期望揭示ZIKV的毒性决定因素，其对大脑发育的影响至关重要。这些结果不仅将从根本上推进我们对这种重要的人类病原体的理解，而且还将对开发治疗ZIKV感染及其相关疾病的有效疗法的使命产生直接和直接的影响。

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.