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 (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.

Keywords: G3BP1; HuR; Zika virus; stress granule.

FIG 1

ZIKV restricts the assembly of cellular stress granules. Huh7 cells were either mock infected or infected with the Cambodia ZIKV strain (160310) at an MOI of 5. Twenty-four h postinfection, cells were mock treated or incubated with 1 mM sodium arsenite for 30 min, fixed, permeabilized, and analyzed using a 63× oil immersion objective on a Zeiss LSM710 laser scanning confocal microscope. ZIKV-infected cells were detected with mouse-anti-dsRNA antibody (green). Shown is the distribution of dsRNA (green) and the SG marker TIA-1 (red) in mock- and ZIKV-infected cells left untreated (A) or treated (B) with sodium arsenite. The white arrows highlight stress granules. (C and D) Quantification of SGs containing Ataxin-2, G3BP1, eIF3B, HuR, TIA-1, TIAR, and YB1 proteins in mock- and ZIKV (Cambodia 160310)-infected cells which were either not treated (C) or treated (D) with sodium arsenite. (E and F) The number of cells with G3BP1-, eIF3B-, and TIA-1-containing SGs in mock-infected cells or cells infected with a Puerto Rican isolate of ZIKV at an MOI of 5 and either not treated (E) or treated (F) with sodium arsenite. (G and H) SGs in mock-infected cells and cells infected with the original Ugandan isolate of ZIKV at an MOI of 5 were quantified. The number of cells containing G3BP1, eIF3B, and TIA-1-SG was quantified from cells that were untreated (G) or incubated with sodium arsenite (H). Note that in the original immunofluorescence images, TIA-1 was visualized using an anti-donkey Alexa Fluor 647 (magenta) anti-goat IgG secondary antibody. In panels A and B, TIA-1 has been pseudocolored red. The immunofluorescence images are representative of at least three independent experiments. The errors bars shown on the SG quantification data (C, D, E, F, G, and H) are means ± standard deviations (SD). Significance was determined by two-tailed Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; N.S., not significant).

FIG 2

ZIKV infection changes the nuclear and cytoplasmic distribution of stress granule proteins. (A) Huh7 cells infected with the Cambodia strain of ZIKV at an MOI of 1 or 5 were harvested at 1, 2, and 3 days postinfection. Detection of ZIKV capsid protein by Western blotting confirmed viral infection. The abundance and integrity of Ataxin-2, G3BP1, HuR, TIA-1, and TIAR proteins were also examined at each time point. The Western blot shown is representative of at least three independent experiments. (B) Western blot analysis of SG proteins in MR766- and PRVABC59-infected Huh7 cells 3 days postinfection. (C) Cytoplasmic and nuclear distribution of Ataxin-2, G3BP1, HuR, TIA-1, TIAR, and YB1. Huh7 cells were mock infected or infected with the Cambodia strain of ZIKV at an MOI of 5, and 1 day postinfection the cytoplasmic and nuclear fractions were isolated. The distribution of ZIKV NS5, Ataxin-2, G3BP1, HuR, TIA-1, TIAR, YB1, GRP78, and Fibrillarin was analyzed by Western blotting. The Western blot shown is representative of the three independent experiments performed. (D and E) Quantification of the distribution of Ataxin-2, G3BP1, HuR, TIA-1, TIAR, and YB1 in the cytoplasmic (D) and nuclear (E) subcellular fractions. The abundance of each SG protein in the cytoplasmic or nuclear fraction was standardized against GRP78 or Fibrillarin, respectively. These values were then normalized against the abundance of the specific SG protein in mock-infected cells, which was assigned an arbitrary unit (a.u.) of 1 (denoted by dashed lines on each graph). The data presented in panels D and E were calculated from three independent infections, fractionations, and immunoblots. (F) Quantification of fluorescence intensity signal of the localization of HuR in the nucleus. Fluorescence intensity was derived in ImageJ using a freehand selection of the nucleus in mock- and ZIKV-infected cells. Values obtained for HuR were divided by the nucleus signal (Hoechst) within mock- and ZIKV-infected cells. Fluorescence intensity of the infected cells was standardized against that of mock-infected cells and arbitrarily assigned a value of 1. The relative fluorescence intensity signal was calculated from three independent experiments, and nine cells were counted per experiment. Two-tailed Student t tests were performed to calculate significance. *, P < 0.05; ***, P < 0.001; N.S., not significant.

FIG 3

Stress granule proteins promote and limit ZIKV gene expression. Ataxin-2, G3BP1, HuR, TIA-1, TIAR, and YB1 proteins were depleted in Huh7 cells using target-specific siRNAs and then infected 24 h later at an MOI of 5 with the Cambodia strain of ZIKV (160310). An siRNA targeting Gaussia luciferase (siGL2) was used as a control, nontargeting siRNA. Protein, RNA, and media were harvested 48 h postinfection. (A) Western blot shows expression of ZIKV capsid and depletion of each stress granule protein. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, and the Western blot shown is representative of at least three independent experiments. The Northern blot shows the effect of depleting each stress granule protein on the abundance of the ZIKV genomic (gZIKV) and subgenomic flaviviral (sfZIKV) RNA. Actin mRNA levels were evaluated as a loading control. The Northern blot shown is representative of at least three independent experiments. (B) Quantification of ZIKV gRNA and sfRNA. ZIKV RNA levels were normalized to actin mRNA and then represented relative to the siGL2 control. (C) Viral titers in the extracellular media were determined by plaque assay, and relative viral titers were calculated by normalizing titers relative to those of siGL2-transfected cells. (D) A representative CellTiter-Glo assay. Arbitrary light units (a.u.) quantify the effect of siRNA depletion of the six SG proteins on cell viability. (E) Quantification of ZIKV RNA levels following overexpression of G3BP1-Flag and HuR-Flag. Huh7 cells were transfected with plasmids expressing 3×Flag-bacterial alkaline phosphatase (BAP; control), G3BP1-Flag, or HuR-Flag and then infected with ZIKV (Cambodia 160310) 24 h posttransfection and then harvested 48 h postinfection. The effect of overexpressing G3BP1-Flag and HuR-Flag on gZIKV and sfZIKV RNA was evaluated by Northern blotting. (F) Effect of overexpression of G3BP1-Flag and HuR-Flag on viral titers. Data presented are from at least three independent experiments. Error bars show means ± SD, and statistical significance was determined using a two-tailed Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; N.S., not significant).

FIG 4

G3BP1 and HuR modulate ZIKV replication. (A) Schematic of the ZIKV MR766 Gaussia Luciferase reporter genome within the pCDNA6.2 MR766 clGLuc Intron3127 HDVr plasmid showing the 5ʹ and 3ʹ UTRs, the mature viral proteins within the single open reading frame, and the position of the Gaussia Luciferase gene within the MR766 genome. Two elements within the plasmid, namely, the CMV promoter and HDVr, which creates an authentic 3ʹ UTR in the genome, are also denoted. (B and D) Huh7 cells were first transfected with either the control or target-specific siRNAs and then transfected with the same siRNAs and the pCDNA6.2 MR766 clGLuc Intron3127 HDVr WT replication-competent or Pol(−) replication-deficient plasmid. GLuc activity in the medium of transfected cells was assayed at 6, 24, 48, and 96 h posttransfection. (C and E) Huh7 cells were transfected with p3×Flag-BAP, pG3BP1-Flag, pHuR-Flag, and WT and Pol(−) pCDNA6.2 MR766 clGLuc Intron3127 HDVr. At 6, 24, 48, and 96 h posttransfection, medium from the transfected cells was collected and GLuc activity measured. (B) Effect of G3BP1 knockdown on ZIKV-GLuc genome expression. (C) Effect on ZIKV-GLuc reporter genome expression following overexpression of 3×Flag-BAP (control) and G3BP1-Flag. (D) Effect of depleting HuR on ZIKV-GLuc genome expression. (E) Effect of overexpressing 3×Flag-BAP and HuR-Flag on ZIKV-GLuc genome expression. The data shown are from a single experiment and are representative of at least three independent experiments. Error bars indicate means ± SD.

FIG 5

G3BP1 and HuR localize with ZIKV replication complexes. (A and B) Huh7 cells were either mock infected or infected with the Cambodia ZIKV strain at an MOI of 5. One day postinfection cells were fixed, permeabilized, and prepared for confocal imaging. Immunofluorescence images are representative of at least three independent experiments. (A) Immunofluorescence image showing G3BP1 (red) localization in mock-infected cells and with viral replication sites as visualized by staining with antibodies to dsRNA and NS5 (green) in ZIKV-infected cells. (B) Localization of HuR (red) in mock-infected cells and with dsRNA and NS5 (green) in ZIKV-infected cells. (C) Western blot analysis of ZIKV NS5, Ataxin-2, G3BP1, HuR, TIA-1, TIAR, YB1, and calnexin associated with replication complexes isolated from mock- and ZIKV-infected Huh7 cells. The Western blot is representative of three independent experiments.

FIG 6

G3BP1 colocalizes with ZIKV E protein. One day postinfection, Huh7 cells infected with ZIKV at an MOI of 5 were fixed, permeabilized, and prepared for confocal imaging. (A) Localization of ZIKV E protein with HuR and G3BP1 following infection with the Cambodia strain of ZIKV. (B) Localization of ZIKV E protein with G3BP1 following infection with the Puerto Rican (PRVABC59) and Ugandan (MR766) strains of ZIKV. The immunofluorescence images shown are representative of at least three independent experiments.