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Stress granules (SGs) are highly dynamic cytoplasmic foci that form in response to activation of the integrated stress response (ISR) that results in eIF2α phosphorylation and global translation shutdown. Stress granules, which are largely nucleated by G3BP1, serve as hubs for mRNA triage, but there is mounting evidence that they also perform cell signaling functions that are vital to cell survival, particularly during viral infection. We previously showed that SG formation leads to NFκB activation and JNK signaling and that this association may be due in part to G3BP1-dependent recruitment of PKR to SGs. Others have reported close associations between G3BP1 and various innate immune PRRs of the type 1 interferon signaling system, including RIG-I. We also reported SG assembly dynamics is dependent on the arginine-methylation status of G3BP1. Another protein that rapidly localizes to SGs, TDRD3, is a methyl reader protein that performs transcriptional activation and adaptor functions within the nucleus, but neither the mechanism nor its function in SGs is clear. Here, we present evidence that TDRD3 localizes to SGs partly based upon methylation potential of G3BP1. We also characterize granules that TDRD3 forms during overexpression and show that these granules can form in the absence of G3BP but also contain translation components found in canonical SGs. We also show for the first time that SGs recruit additional interferon effectors IRF3, IRF7, TBK1, and Sting, and provide evidence that TDRD3 may play a role in recruitment of these factors. We also present evidence that TDRD3 is a novel antiviral protein that is cleaved by enteroviral 2A proteinase. G3BP1 and TDRD3 knockdown in cells results in altered transcriptional regulation of numerous IFN effectors in complex modulatory patterns that are distinctive for G3BP1 and TDRD3. Overall, we describe a novel role of TDRD3 in innate immunity in which G3BP1 and TDRD3 may coordinate to play important roles in regulation of innate antiviral defenses.

Abstract Background Both vector borne and sexual transmission of Zika virus (ZIKV) involve infection of epithelial cells in the initial stages of infection. Epithelial cells are unique in their ability to form polarized monolayers and their barrier function. Cell polarity induces an asymmetry in the epithelial monolayer, which is maintained by tight junctions and specialized sorting machinery. This differential localization can have a potential impact of virus infection. Asymmetrical distribution of a viral receptor can restrict virus entry to a particular membrane while polarized sorting can lead to a directional release of virions. The present study examined the impact of cell polarity on ZIKV infection and release. Methods A polarized Caco-2 cell model we described previously was used to assess ZIKV infection. Transepithelial resistance (TEER) was used to assess epithelial cell polarity, and virus infection was measured by immunofluorescence microscopy and qRT-PCR. Cell permeability was measured using a fluorescein leakage assay. Statistical significance was calculated using one-way ANOVA and significance was set at p

Abstract Background Currently, no FDA-approved vaccines or treatments are available for Ebola virus disease (EVD), and therapy remains largely supportive. Ebola virus (EBOV) has broad tissue tropism and can infect a variety of cells including epithelial cells. Epithelial cells differ from most other cell types by their polarized phenotype and barrier function. In polarized cells, the apical and basolateral membrane domains are demarcated by tight junctions, and specialized sorting machinery, which results in a difference in composition between the two membrane domains. These specialized sorting functions can have important consequences for viral infections. Differential localization of a viral receptor can restrict virus entry to a particular membrane while polarized sorting can lead to a vectorial virus release. The present study investigated the impact of cell polarity on EBOV infection. Methods Characteristics of EBOV infection in polarized cells were evaluated in the polarized Caco-2 model grown on semipermeable transwells. Transepithelial resistance (TEER), which is a function of tight junctions, was used to assess epithelial cell polarization. EBOV infection was assessed with immunofluorescence microscopy and qPCR. Statistical significance was calculated using one-way ANOVA and significance was set at p

Abstract A monkey model of Zika virus (ZIKV) infection is urgently needed to better understand transmission and pathogenesis, given its proven association with fetal brain defects in pregnant women and acute neurological illness. Here we experimentally infected 4 male marmosets with ZIKV (prototype 1947 African strain) and monitored them clinically with sampling of various body fluids and tissues for nearly 3 months. We show that the course of acute infection with ZIKV in these New World monkeys resembles the human illness in many respects, including (1) lack of apparent clinical symptoms in most cases, (2) persistence of the virus in body fluids such as semen and saliva for longer periods of time than in serum, and (3) generation of neutralizing antibodies as well as an antiviral immunological host response. Importantly, ZIKV-infected saliva samples (in addition to serum) were found to be infectious, suggesting potential capacity for viral transmission by the oral route. Re-challenge of a previously infected marmoset with a contemporary outbreak strain SPH2015 from Brazil resulted in continued protection against infection, no viral shedding, and boosting of the immune response. Given the key similarities to human infection, a marmoset model of ZIKV infection may be useful for testing of new drugs and vaccines.