ZIKV association with congenital microcephaly became evident during the recent epidemic in South and Central America, which has already resulted in almost 3,000 such cases. Despite this global health burden, little is known about the mechanisms of ZIKV-­induced microcephaly, which often presents with seizures. Cellular and molecular studies support different mechanisms, including direct cytotoxicity to neural progenitor cells (NPC), placental insufficiency, and immune responses. ZIKV congenital syndrome thus likely results from a combination of mechanisms, none of which is yet fully understood. We have focused on direct cytotoxicity, studying ZIKV infections of NPC. The major findings of our preliminary studies are that: 1) ZIKV infection of human NPC induces relocalization of protein kinase 3’phosphatase (PNKP) to the cytoplasm where it co-­localizes with ZIKV non-structural protein 1 (NS1), 2) infected NPC show obvious morphological nuclear abnormalities consistent with mitotic catastrophe (MC), and 3) PNKP inhibitors inhibit ZIKV replication. PNKP is a critical DNA damage repair enzyme, restoring the 3’-­OH and 5’-­P DNA termini at single and double-­stranded DNA breaks. It normally localizes to the nucleus and mitochondria, localization that is disrupted by ZIKV. PNKP has a direct link to microcephaly in that single gene mutations, of which one induces cytoplasmic PNKP localization, produce (rare) recessive genetic syndromes characterized by congenital microcephaly with seizures (MCSZ). MCSZ- associated PNKP mutations result in accumulation of DNA damage and activation of DNA damage responses (DDR) and affect the developing human or mouse brain. Based on the literature and our own preliminary results, we developed the hypothesis that ZIKV-­induced cytoplasmic PNKP relocalization causes functional (nuclear) PNKP depletion, DNA damage accumulation, and DDR dysregulation. NPC then enter mitosis with damaged DNA, leading to MC, impaired cell proliferation and death. We will test whether PNKP relocalization and MC are specific for NPC or contemporary ZIKV strains, characterize the consequences for DNA repair and the cell cycle of the cytoplasmic PNKP localization, and identify the mechanisms whereby ZIKV induces this PNKP relocalization. We will use standard traditional methodologies and newer techniques such as inducible human stem cells, high­resolution confocal microscopy, next generation sequencing, and mass spectrometry to study DNA damage, DDR and the cell cycle in ZIKV-­infected NPC. We will challenge the hypothesis that ZIKV induced microcephaly is caused by targeting PNKP, an essential DNA damage repair enzyme directly linked to microcephaly. We will learn the roles of DNA damage accumulation due to inefficient PNKP-­dependent DNA repair and dysregulated DDR in impaired proliferation, MC, and death of ZIKV-­infected NPC. We will address the roles of PNKP, a cellular protein known to be involved in microcephaly, in ZIKV pathogenesis. We will fill a major gap in the understanding of ZIKV-­induced microcephaly, the most significant ZIKV burden.