Investigation of the Roles of Microtubules, Microtubule-Organizing Centers, and Viral Factors on Zika Virus Viroplasm Development

Zika virus (ZIKV) became a global health concern in 2016 due to its links to congenital microcephaly and other birth defects. ZIKV is part of the genus flavivirus, a class of positive(+)- sense single-stranded RNA viruses. Flavivirus infection results in a reorganization of the endoplasmic reticulum (ER) to form a viroplasm, a subcellular structure where viral RNA replicates and virus particles are assembled. The overall goal of this research project is to better understand how the ZIKV viroplasm forms and organizes. Microtubules (MTs) and microtubule-organizing centers (MTOCs) coordinate structural and trafficking functions in the cell, and MTs also support the infection cycles of flaviviruses. In the first part (Chapter 2), we investigated the roles of MTs and the cell's MTOCs on ZIKV viroplasm organization and virus production. We show that a toroidal-shaped viroplasm forms upon ZIKV infection, and MTs are organized at the viroplasm core and surrounding the viroplasm. We show that MTs are necessary for viroplasm organization and impact infectious virus production. In addition, the centrosome and the Golgi MTOC are closely associated with the viroplasm, and the centrosome coordinates the organization of the ZIKV viroplasm toroidal structure. Surprisingly, viroplasm formation and virus production are not significantly impaired when infected cells have no centrosomes and impaired Golgi MTOC, and we show that MTs are anchored to the viroplasm surface in these cells. We propose that the viroplasm is a site of MT organization, and the MTs organized at the viroplasm are sufficient for efficient virus production. Supplementary movies (Appendix A) for Chapter 2 are available through the online version of this dissertation published on ProQuest. Viral proteins interact with host proteins to cause subcellular rearrangements during infection. In the second part (Chapter 3), we investigated how individual viral proteins contribute towards the formation and organization of the ZIKV viroplasm. We found that expression of NS3 alone was sufficient to form a structure that resembles the ZIKV viroplasm. NS3 has protease and helicase domains, and we further found expression of the NS3 helicase alone was sufficient to form a viroplasm-like structure. In addition, we found that the centrosome and the Golgi associate with this structure. Besides localization and formation of the viroplasm-like structure, NS3 associates with the centrosome and forms filamentous structures. We propose that NS3 may be playing a role in viroplasm organization during ZIKV infection.