Mechanisms of tunneling nanotube formation by ZIKV NS1 in viral host interactions at the maternal-fetal interface - PROJECT SUMMARY Although primarily transmitted by mosquitoes, the Zika virus (ZIKV) has the unique ability among flaviviruses to be transmitted horizontally through sexual contact, or vertically from mother to fetus. The vertical mode of transmission has led to devastating pregnancy and fetal development outcomes that came to light in 2015-16, as a ZIKV epidemic emerged in the Western hemisphere and a public health emergency of international concern was declared. The threat of future ZIKV epidemics persists and many nations where it has become endemic continue to struggle with the adverse effects of infection. Meanwhile, scientists have struggled to understand the mechanisms that underlie maternal-fetal transmission of ZIKV on a cellular and molecular level. This proposal presents preliminary data for the first time that supports a novel hypothesis that transmission of ZIKV at the maternal-fetal interface is facilitated by the formation of tunneling nanotubes (TNTs), which are actin-based intercellular transport conduits that have been shown to play a role in the spread of other viruses. Among other things, the preliminary data indicates that, 1) ZIKV infection induces the formation of TNTs in various cell types, including human placental trophoblast cells, 2) the ZIKV non-structural protein 1 (NS1) is alone sufficient to induce TNT formation, whereas NS1 proteins of several other flaviviruses tested do not appear to have this ability, with the exception of WNV to a small degree, and 3) ZIKV-induced TNTs transport mitochondria in trophoblast cells in a manner that may be exploited for the benefit of virus propagation and transmission. Building on these observations, and using the tools the PIs have so far developed, they propose a formal collaboration and rigorous set of experiments designed to test this hypothesis. First, using a library of labeled ZIKV constructs, confocal imaging, and an advanced affinity purification-mass spectrometry approach, the role of NS1 in inducing TNT formation without affecting virus replication and assembly will be established in human trophoblast cells; then specific regions of this protein will be identified that may participate in signaling or interaction with host proteins; and the question of whether TNTs transport ZIKV RNA and/or whole virions will be evaluated. Second, mouse models of ZIKV infection during pregnancy that the PIs have developed in prior work will be used to investigate the role of TNT formation in ZIKV pathogenesis and vertical transmission; the impact of TNT formation on ZIKV infection pathogenesis in the placenta will be evaluated; and spatial transcriptomics paired with tissue profiling will be used to identify genomic pathways in the placenta that are key for TNT formation. And thirdly, the nature of mitochondria transport via TNTs will be investigated to determine the role of any interactions with ZIKV virions or NS1, and the effects this has on cell metabolism and viral transmission. If successful, the resulting findings will establish an important and undescribed cellular pathway for transmission of the ZIKV that will serve as a likely target for future therapeutic interventions aimed at preventing microcephaly and other effects of ZIKV infection during pregnancy.
