Flaviviruses limit the cell stress response by preventing the formation of stress granules (SGs) and modulate viral gene expression by subverting different proteins involved in the stress granule pathway. In this study, we investigated the formation of stress granules during Zika virus (ZIKV) infection and the role stress granule proteins play during the viral life cycle. Using immunofluorescence and confocal microscopy, we determined that ZIKV disrupted the formation of arsenite-induced stress granules and changed the subcellular distribution, but not the abundance or integrity, of stress granule proteins. We also investigated the role of different stress granule proteins in ZIKV infection by using target-specific short interfering RNAs to deplete Ataxin2, G3BP1, HuR, TIA-1, TIAR, and YB1. Knockdown of TIA-1 and TIAR affected ZIKV protein and RNA levels but not viral titers. Conversely, depletion of Ataxin2 and YB1 decreased virion production despite having only a small effect on ZIKV protein expression. Notably, however, depletion of G3BP1 and HuR decreased and increased ZIKV gene expression and virion production, respectively. Using an MR766 Gaussia Luciferase reporter genome together with knockdown and overexpression assays, G3BP1 and HuR were found to modulate ZIKV replication. These data indicate that ZIKV disrupts the formation of stress granules by sequestering stress granule proteins required for replication, where G3BP1 functions to promote ZIKV infection while HuR exhibits an antiviral effect. The results of ZIKV relocalizing and subverting select stress granule proteins might have broader consequences on cellular RNA homeostasis and contribute to cellular gene dysregulation and ZIKV pathogenesis. IMPORTANCE Many viruses inhibit SGs. In this study, we observed that ZIKV restricts SG assembly, likely by relocalizing and subverting specific SG proteins to modulate ZIKV replication. This ZIKV-SG protein interaction is interesting, as many SG proteins are also known to function in neuronal granules, which are critical in neural development and function. Moreover, dysregulation of different SG proteins in neurons has been shown to play a role in the progression of neurodegenerative diseases. The likely consequences of ZIKV modulating SG assembly and subverting specific SG proteins are alterations to cellular mRNA transcription, splicing, stability, and translation. Such changes in cellular ribostasis could profoundly affect neural development and contribute to the devastating developmental and neurological anomalies observed following intrauterine ZIKV infection. Our study provides new insights into virus-host interactions and the identification of the SG proteins that may contribute to the unusual pathogenesis associated with this reemerging arbovirus.

Introduction: 37 Zika virus (ZIKV) is an enveloped, single-stranded positive-sense RNA virus belonging to the 38 Flaviviridae family, which includes Dengue virus (DENV), Yellow fever virus (YFV), and West Nile 39 virus (WNV) (1). While ZIKV was discovered in Uganda in 1947 (2) , the virus garnered renewed 40 interest during the 2015-2016 outbreak in the Americas (3), in particular because of intrauterine 41 infections and resulting developmental abnormalities such as severe microcephaly, decreased 42 brain tissue, macular scarring, congenital contractures, and hypertonia (4-9). Additionally, adults 43 infected with ZIKV were reported to develop Guillain-Barré syndrome, a debilitating disorder 44 affecting the peripheral nerves (10-13). Similar to other flaviviruses, ZIKV is transmitted by the 45 Aedes aegypti and Aedes albopictus mosquitoes, although recent evidence has shown ZIKV is 46 also capable of sexual and vertical transmission (14-17). While half a century has passed since 47 65 particular, the presence of double-stranded RNA (dsRNA) during viral infection activates protein 66 kinase R (PKR) (19); the accumulation of unfolded proteins in the endoplasmic reticulum (ER) 67 and resulting stress activates PKR-like endoplasmic reticulum kinase (PERK) (20); amino acid 68 starvation activates general control non-repressed 2 (GCN2) (21); and oxidative stress activates 69 5 heme-regulated inhibitor kinase (HRI) (22). Phosphorylation of the a subunit of eIF2 by one of 70 the four stress response kinases results in the stalling of translation initiation, and disassembly of 71 polysomes. Stalled translation initiation complexes bound to mRNA are recognized by several 72 RNA binding proteins, which aggregate to form RNA-protein macromolecular complexes called 73 stress granules (SGs) (23). Once the stressor is abated, eIF2a is dephosphorylated by protein 74 phosphatase 1 (PPI) and the PPI cofactor growth arrest and DNA-damage-inducible 34 75 (GADD34), allowing for the return of sequestered mRNA transcripts to active translation (23). 76 77 SGs are dynamic nonmembrane-bound cytoplasmic structures that can rapidly assemble in 78 response to stress and disassemble once the stress has been alleviated (23). SGs typically 79 contain mRNAs, stalled translation initiation complexes, and numerous RNA binding proteins. 80 Indeed, SGs may contain upwards of 260 different proteins (24), and ~50% of these are proposed 81 to be RNA-binding proteins (25). Of these Ras-GTPase activating binding protein 1 (or GAP SH3 82 domain binding protein 1 [G3BP1]), Caprin1, T-cell internal antigen 1 (TIA-1) and TIA-1 related 83 protein (TIAR) are proposed to be key nucleators of SG assembly (26-29). In addition to 84 translation repression and mRNA sorting, SGs also amplify the innate immune response by 85 aggregating critical antiviral factors (23). Because translation is a critical first step in the flavivirus 86 life cycle, the formation of SGs presents an immediate obstacle to infection. Notably, however, 87 during infection w...