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3D Stimulated Emission Depletion super resolution microscopy reveals novel association between replication factories and organized smooth tubular matrices during Zika virus infection Long, Rory Kirk Mackenzie

Abstract

The Zika virus (ZIKV) is a positive-sense RNA flavivirus that has been determined as a causative agent of severe neurological diseases including Guillain-Barré syndrome and microcephaly. A common characteristic of flavivirus infection is the re-organization of host cell membranes, usually endoplasmic reticulum (ER) derived, to promote viral replication. As ZIKV-induced ER structures, being vesicular replication factories (RF) and convoluted membranes (CM), are smaller than the diffraction limit of light, studies to characterize these structures and their protein compositions have been limited. In this thesis, Stimulated Emission Depletion (STED) super resolution microscopy is utilized to study the whole cell organization of ZIKV-induced ER morphologies and several viral proteins involved in replication. Generally in cells, the ER can be divided into two regions, central ER sheets and peripheral ER tubules. Here we report the formation of an ZIKV-induced, organized crescent-shaped, dense central ER region of ER tubules. ZIKV RFs localize to this dense, central ER region in a similar crescent-shaped organization. Computational 3D reconstruction of these 3D STED imaged ZIKV-induced ER structures, along with validation by electron microscopy of ZIKV infected cerebral organoids, revealed that these dense ER regions are composed of novel smooth ER tubular matrices. Moreover, we have found that ZIKV NS4B, a viral encoded integral membrane protein predicted to play a role in ER membrane re-organization during ZIKV replication, is enriched in ZIKV-induced tubular matrices. Additionally, a subpopulation of ZIKV NS4B localizes to active RFs suggesting that NS4B plays a role in the formation of both ZIKV-induced tubular matrices and RFs among other functions. Overall, the application of super resolution microscopy to study ZIKV replication has led to the identification of novel ZIKV-induced, RF-containing tubular matrices and characterization of a viral protein involved in this process.

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