Zika Virus Subverts Stress Granules to Promote and Restrict Viral Gene Expression

Abstract

Flaviviruses limit the cell stress response by preventing the formation of stress granules and utilize different proteins involved in the stress granule pathway to modulate viral gene expression. 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. To investigate the role of different stress granule proteins in ZIKV infection we used target-specific siRNAs to deplete six proteins, namely Ataxin2, G3BP1, HuR, TIA-1, TIAR and YB-1. Depletion of TIA-1 and TIAR affected ZIKV protein and RNA levels, but viral titers did not change. Conversely, depletion of Ataxin2 and YB-1 decreased virion production despite having only a small effect on ZIKV protein and 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, which is where G3BP1 functions to promote ZIKV infection, while HuR exhibits an antiviral effect. The consequence of ZIKV re-localizing and subverting select stress granule proteins might have broader consequences on cellular RNA homeostasis contributing to cellular gene dysregulation and ZIKV pathogenesis.\n\nImportanceIn response to viral infection, cellular translation is stalled, and translation initiation complexes, cellular mRNAs, and RNA binding proteins aggregate in stress granules. Because the assembly of stress granules antagonize translation of viral proteins, which is a critical step for single-stranded positive-sense RNA viruses to replicate the viral genome, many viruses have developed strategies to inhibit stress granule formation. In this study, we observed that Zika virus restricts the formation of stress granules likely by re-localizing specific stress granule proteins during infection. We also determined that specific stress granule proteins function to facilitate and limit Zika virus replication. This interaction of Zika virus with stress granule proteins is interesting, as many stress granule proteins are also known to function in neuronal granules, which are critical in neural development and function. Moreover, dysregulation of different stress granule proteins in neurons has been shown to play a role in the progression of neurodegenerative diseases. The likely consequences of Zika virus modulating stress granule assembly and subverting specific stress granule proteins are alterations to cellular mRNA transcription, splicing, RNA stability, and translation. Such changes in cellular ribostasis could have profound effects on neural development and contribute to the devastating developmental and neurological anomalies observed following intrauterine Zika virus infection. Our study provides new insights into virus-host interactions and the identification of the stress granule proteins that may contribute to the unusual pathogenesis associated with this reemerging arbovirus.