Atlastin Endoplasmic Reticulum-Shaping Proteins Facilitate Zika Virus Replication

Abstract

The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by "implosive" cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication.IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.

Keywords: Zika; atlastin; endoplasmic reticulum.

FIG 1

Silencing of ATL impairs ZIKV replication. (A) HeLa cells were transfected with siRNAs targeting a control scrambled RNA (siSCR), dolichyl-diphosphooligosaccharide–protein glycosyltransferase RNA (siDDOST), or targeting ATL1, ATL2, and ATL3 (siATL1/2/3). The efficiency of the silencing was checked by RT-qPCR at 3 days posttransfection. The relative expression of each RNA compared to GAPDH is shown. (B) Cell viability was assessed by flow cytometry after 4 days of siRNA treatment using forward- and side-scatter parameters. (C) Cells were infected with ZIKV HD78 (at the indicated MOI), and the percent E-positive cells was determined by flow cytometry at 48 h p.i. using 4G2 antibody. (Upper panel) Representative experiment (MOI of 1 at 48 h p.i.) showing fluorescence-activated cell sorting (FACS) dot plots. (Left panel) Representative experiment showing kinetics (MOI of 1). (Right panel) Means ± the standard errors of the mean (SEM) of five independent experiments at 48 h p.i. (D) Supernatants from infected cells (at 24 h p.i.) were used to infect fresh HeLa cells. The percent E-positive cells was determined at 24 h p.i. The means ± the SEM of three independent experiments are shown. Statistical significance was determined by using analysis of variance (ANOVA) and Bonferroni posttests. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 2

Silencing of ATL impairs ZIKV-induced vacuoles. (A) HeLa siSCR or siATL1/2/3 cells infected or not with ZIKV (MOI of 10) were observed by light microscopy at 24 h p.i. to visualize virus-induced vacuoles. (B) Cells infected with ZIKV were observed by light microscopy at 24 h p.i., and the percentage of vacuole-positive cells was calculated. (C) HeLa siSCR or siATL1/2/3 cells infected or not with ZIKV (MOI of 10) were observed by transmission electron microscopy at 24 h p.i. to visualize virus-induced vacuoles. (Left) Percent vacuoles and cells quantified at 24 h p.i. at the indicated MOI. (Right) Percent vacuoles and cells at the indicated times (MOI of 1). The means ± the SEM of three independent experiments are shown. Statistical significance was determined by using ANOVA and Bonferroni posttests. ***, P < 0.001.

FIG 3

Ultrastructural analysis of ZIKV-infected HeLa cells. HeLa siSCR or siATL1/2/3 cells infected with ZIKV (MOI of 10) were observed by electron microscopy at 24 h p.i. Two representative pictures are shown for each condition. White arrows, examples of ZIKV-induced spherules (vRNA replication); white asterisks, examples of viral particles.

FIG 4

Silencing of ATL impairs ZIKV replication in primary adult human dermal fibroblast (HDFa) cells. (A) HDFa cells were transfected with siRNAs targeting a control scrambled RNA (siSCR) or targeting ATL1, ATL2, and ATL3 (siATL1/2/3). The efficiency of the silencing was checked by RT-qPCR at 3 days posttransfection. The relative expression of each RNA compared to GAPDH is shown. (B) Cell viability was assessed by flow cytometry after 4 days of siRNA treatment using forward- and side-scatter parameters. (C) Cells were infected with ZIKV HD78 (at the indicated MOI), and the percent E-positive cells was determined by flow cytometry at 48 h p.i. using 4G2 antibody. (Left) Representative experiment (MOI of 1). (Right) Means ± the SEM of three independent experiments at 48 h p.i. The statistical significance was determined by using ANOVA and Bonferroni posttests. *, P < 0.05. (D) Cells infected or not with ZIKV (MOI of 1) were observed by light microscopy at 24 h p.i. to visualize virus-induced vacuoles.

FIG 5

Characterization of ATL KO HeLa cells. (A) HeLa cells were knocked out for ATL2 and ATL3 genes (dKO) using CRISPR. HeLa WT or dKO cells were then transduced to express exogenous ATL3-myc, ATL3 WT, or ATL3 mutated in the GTPase activity domain (ATL3KA). ATL3 expression was assessed by Western blotting. Actin was used as a loading control. (B) HeLa WT or dKO cells transduced with control (ctrl), ATL3 WT, or ATL3KA vectors and expressing an ER-tracker dsRed2 were examined by confocal microscopy to observe the structure of the peripheral ER tubular network. Three-way junctions were quantified as previously described (42). At least three cells per condition per experiment from three independent experiments were analyzed and plotted. Statistical significance was determined by using a one-way ANOVA test and a Dunnett posttest. ***, P < 0.001.

FIG 6

Knockout of ATL impairs ZIKV replication. (A) HeLa WT or dKO cells, transduced with control (ctrl), ATL3 WT, or ATL3KA vectors were infected with ZIKV (MOI of 0.1). The percentage of E⁺ cells was monitored over time by flow cytometry using 4G2 antibody. (Upper panels) Representative FACS dot plots. (Lower left panel) A representative experiment. (Lower right panel) Means ± the SEM of five independent experiments at 48 h p.i. (B) Percent vacuoles and cells quantified at 24 h p.i. (MOI of 1). The means ± the SEM of three independent experiments are shown. (C) The percent dead cells were quantified at 72 h p.i. (MOI of 0.1) by Live/Dead staining and flow cytometry. The means ± the SEM of three independent experiments are shown. (D) HeLa WT or dKO cells transduced with control (ctrl) or ATL3 WT vectors were infected with ZIKV PF13, NC14, or ZIKV FG16 (MOI of 0.01), and the percent E⁺ cells was determined by flow cytometry at 48 h p.i. The means ± the SEM of three independent experiments are shown. Statistical significance was determined by using a t test in comparison to the WT+Ctrl condition. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 7

Impact of ATL on ZIKV-induced ER relocation. HeLa WT or dKO cells expressing a fluorescent Sec61β-mEmmerald ER protein (green) were infected with ZIKV (MOI of 1). (A) The ER structure was observed by time-lapse microscopy for 24 h. Still images extracted from Video S1 in the supplemental material are shown for transmitted light and Sec61β at the specified time points. White arrows indicate ZIKV-induced ER relocation. (B) HeLa WT or HeLa cells expressing Sec61β-mEmmerald (green) were infected or not for 24 h with ZIKV (MOI of 1). Cells were fixed and stained for the viral E protein (red) and observed by super-resolution imaging (SIM). A representative image is shown.

FIG 8

ATL3 is recruited to the viral replication site. HeLa cells expressing ATL3-myc were infected or not with ZIKV (MOI of 1) for 24 h. The cells were then fixed and stained for ATL3-myc (green) and viral E protein (white) and analyzed by confocal microscopy. A representative image is shown.

FIG 9

ATLs facilitate ZIKV replication after viral entry. (A) HeLa WT or dKO cells were exposed to ZIKV for 1 h at 4°C to allow viral binding. Viral input was washed out, and bound viral particles were quantified by RT-qPCR. The data are means ± the SEM of three independent experiments. (B) HeLa WT or dKO cells were exposed to ZIKV for 1 h at 4°C, washed out, and treated with trypsin to remove the bound viral particles. The cells were incubated at 37°C for 2 h to allow endocytosis and/or viral fusion. Intracellular viral RNA was quantified by RT-qPCR. Means ± the SEM of three independent experiments are shown. (C) HeLa WT or dKO cells were transfected with RNA from a ZIKV infectious clone expressing luciferase. The results are expressed in relative light units (RLU). The means ± the SEM of at least three independent experiments are shown. Statistical significance was determined using a t test, and each group was compared to the WT+ctrl condition. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 10

ATL3 colocalizes and interacts with ZIKV NS proteins. (A) HeLa cells expressing ATL3-myc were transfected with plasmids coding for individual NS-HA tagged proteins. NS proteins were eluted with an anti-HA antibody, and elution fractions were stained with an anti-myc antibody to detect coimmunoprecipitated ATL3. A representative blot from three independent experiments is shown. (B) HeLa cells expressing ATL3-myc were transfected with plasmids coding for HA-tagged NS1 or NS2B3 ZIKV proteins. The cells were fixed, stained for ATL3-myc (green) and NS-HA (red), and observed by confocal microscopy. One field from a representative experiment is shown. Colocalization (Pearson’s coefficient) was determined after deconvolution with Imaris software. At least 10 cells per condition (from three independent experiments) were plotted. Statistical significance was determined by using a t test ***, P < 0.001.

FIG 11

ATL3 colocalizes with NS3 in ZIKV-infected cells. HeLa dKO cells transduced with a control vector (dKO+ctrl) or stably expressing ATL3-myc (dKO+ATL3) were infected with ZIKV (MOI of 1) for 24 h. The cells were then fixed and stained for ATL3-myc (green) and NS3 (red). One field from a representative experiment is shown. Colocalization (Pearson’s coefficient) was determined after deconvolution with Imaris software. At least 10 cells were plotted from three independent experiments. Statistical significance was determined using a t test. ****, P < 0.0001.