ZIKVassociationwithcongenitalmicrocephalybecameevidentduringtherecentepidemicinSouthandCentral America,whichhasalreadyresultedinalmost3,000suchcases.Despitethisglobalhealthburden,littleisknown aboutthemechanismsofZIKV-inducedmicrocephaly,whichoftenpresentswithseizures.Cellularandmolecular 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 infectionsofNPC.Themajorfindingsofourpreliminarystudiesarethat:1)ZIKVinfectionofhumanNPCinduces 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 mitoticcatastrophe(MC),and3)PNKPinhibitorsinhibitZIKVreplication.PNKPisacriticalDNAdamagerepair 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 microcephalyinthatsinglegenemutations,ofwhichoneinducescytoplasmicPNKPlocalization,produce(rare) recessive genetic syndromes characterized by congenital microcephaly with seizures (MCSZ). MCSZ- associatedPNKPmutationsresultinaccumulationofDNAdamageandactivationofDNAdamageresponses (DDR)andaffectthedevelopinghumanormousebrain.Basedontheliteratureandourownpreliminaryresults, 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,leadingtoMC,impairedcellproliferationanddeath.WewilltestwhetherPNKPrelocalizationandMCare specificforNPCorcontemporaryZIKVstrains,characterizetheconsequencesforDNArepairandthecellcycle of the cytoplasmic PNKP localization, and identify the mechanisms whereby ZIKV induces this PNKP relocalization.Wewillusestandardtraditionalmethodologiesandnewertechniquessuchasinduciblehuman stemcells,high-resolutionconfocalmicroscopy,nextgenerationsequencing,andmassspectrometrytostudy DNAdamage,DDRandthecellcycleinZIKV-infectedNPC.WewillchallengethehypothesisthatZIKVinduced 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 therolesofPNKP,acellularproteinknowntobeinvolvedinmicrocephaly,inZIKVpathogenesis.Wewillfilla majorgapintheunderstandingofZIKV-inducedmicrocephaly,themostsignificantZIKVburden.
WhilecongenitalmicrocephalyisthemostsignificantburdenofZIKVinfection,leadingtopregnancylosesand lifetime consequences for live born infants, the mechanisms responsible for the fetal neurotoxicity are largely unknown.ThisresearchisdesignedaroundourfindingthatZIKVinfectiondisruptstheintracellularlocalization of a cellular enzyme that is directly linked to inherited primary microcephalies, ?PNKP?. By using neuronal progenitorstemcellsandavarietyofconventionalandadvancedmolecularandcellulartechniquestotestPNKP, DNA damage, the ensuing signaling pathways, and their influence on proliferation and survival of neuronal progenitor cells, we will push forward the understanding of the molecular mechanisms of ZIKV-induced microcephaly,openingnewavenuestoexploretowarddiagnosticsorperhapseventreatments.
