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.