Defining the molecular interactions required for flavivirus genome packaging and virus assembly

Arthropod-borne flaviviruses such as Zika, dengue, West Nile, and Powassan viruses cause hemorrhagic fever, congenital diseases, and fatal encephalitis in humans. Zika virus (ZIKV) has the unique ability of human-to- human transmission vertically from mother to fetus and horizontally through sexual contact. In the current proposal, we identify a previously undefined cellular mechanism of ZIKV assembly in live placental cells and aim to characterize the underlying mechanisms of formation and intracellular trafficking of modified membrane structures that facilitate maturation and release. We present preliminary data supporting our novel hypothesis that aptamer tagging of genomic RNA and using fluorescent-protein tagged capsid and nonstructural protein 2A (NS2A), we can visualize and understand mechanisms of virus assembly and virus-host interactions in live cells. By creating a library of mutant ZIKV for live imaging, we identified critical amino acids in capsid protein and NS2A coordinating virus assembly. Using tagged NS2A ZIKV, we present the first viral and host interactome of NS2A from infected cells that have thus far evaded mass spectrometry approaches and identified NS2A-interacting host proteins associated with microcephaly, RNA trafficking, and ER modifications. Using confocal and transmission electron microscopy, we found that ZIKV NS4B modifies the canonical secretory pathway and mediates homotypic fusion of vesicles originating from the ER exit sites forming large perinuclear structures near the Golgi apparatus. Building on our preliminary data and using unique ZIKV infectious clones and tools we have so far developed; we propose a rigorous set of experiments designed to test our hypothesis that flaviviruses modify the ER and host secretory pathways to form large vesicular structures that facilitate assembly and intracellular trafficking. First, using a library of labeled viral RNA and protein constructs with confocal imaging, we will confirm the fate of viral RNA, identify the specific regions of C, NS2A, and RNA that may participate in virus budding and interaction with viral and host proteins, and evaluate whether RNA interacting proteins facilitate assembly (Aim 1). Second, using an advanced affinity purification-mass spectrometry and siRNA and CRISPR knockdown approach, we will determine the genomic pathways in the ER that are key for virus production and cellular membrane modification (Aim 2). And third, we will investigate the ER exit of assembled viruses, NS4B mediated large vesicle formation, and vesicular retrograde transport via microtubules and determine its effect on the dynamics of ZIKV maturation (Aim 3). Our findings will establish a previously undescribed and essential cellular mechanism for the assembly of ZIKV and identify critical targets for intervention in ZIKV infections and other related viruses, addressing a significant global public health need and contributing to the NIAID mission to understand, treat, and prevent infectious diseases. This knowledge can generate new ER-associated targets for therapeutic intervention to mitigate viral transmission at the maternal-fetal interface.