Dynamic Interaction of Stress Granule, DDX3X and IKK-α...
Transcript of Dynamic Interaction of Stress Granule, DDX3X and IKK-α...
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Dynamic Interaction of Stress Granule, DDX3X and IKK-α Mediates Multiple Functions in
Hepatitis C Virus Infection
Véronique Pène, Qisheng Li#, Catherine Sodroski, Ching-Sheng Hsu, T. Jake Liang#
Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MD, 20892, USA
Running Head: DDX3X Localizations and Functions in HCV infection
#Address correspondence to Qisheng Li, [email protected], or T. Jake Liang,
V.P. and Q.L. contributed equally to this work.
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SUPPLEMENTAL FIGURES
C D
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DDX3X IKK-α DDX3X Mito IKK-α Mito
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Mito DDX3X IKK-α
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DDX3X Mito DDX3X LDs
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DDX3X Catalase DDX3X LDs
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Figure S1. HCV 3’UTR RNA or HCV-induced DDX3X–IKK-α granules do not co-
localize with mitochondria, peroxisomes or autophagosomes. Huh-7.5.1 cells were
transfected with EGFP control (Ctrl) or HCV 3'UTR RNA for 24 h or infected with HCVcc
for 48 h. DDX3X, IKK-α and various cellular compartment markers were stained and
examined by confocal microscopy. (A) DDX3X–IKK-α granules are not localized to
mitochondria (Mito, stained by Mitotracker). (B) DDX3X granules are not localized to
peroxisomes (Catalase as a marker). (C, D) IKK-α granules are not on peroxisomes (Catalase
as a marker)(C) or autophagosomes (LC3B as a marker)(D). (E, F) Upon HCV infection,
DDX3X granules partially co-localize with LDs but not with mitochondria (Mito, stained by
Mitotracker)(E) or peroxisomes (Catalase as a marker)(F). Magnifications of two independent
selected areas are shown in the right panels: small DDX3X granules at the LD surface (orange
area) and big DDX3X granules not associated with LDs (blue area). (A-D) Magnifications of
selected area are shown in the right panels. (A, E, F) Colors in far right pictures were digitally
changed for better visualization: IKK-α (A) and DDX3X (E, F), green instead of magenta.
Scale bars, 20 µm.
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Figure S2. HCV-induced DDX3X–IKK-α–G3BP1 granules co-localize with SG and P-
body markers. (A-C) Huh-7.5.1 cells were infected with JFH-1 HCVcc for 48 h, and then
immunostained for DDX3X, IKK-α and various SG and P-body markers. (A) DDX3X, IKK-α
and G3BP1 co-localize with PABP1 (another SG marker). (B, C) DDX3X, IKK-α and G3BP1
co-localize with P-body markers DDX6 (B) and EDC4 (C). Scale bars, 20 µm. (D) Huh-7.5.1
cells were mock infected or infected with JFH-1 HCVcc for 48 h. Immunoprecipitations (IP)
were performed on whole cell lysate (WCL) with anti-G3BP1, anti-DDX3X or IgG control
antibody, followed by Western blot using anti-G3BP1 or anti-DDX3X antibody. HC, IgG
heavy chain.
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PABP1 DDX3X
PABP1 IKK-α
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Figure S3. Dynamic association of DDX3X with HCV core protein and LDs during HCV
infection. (A) Huh-7.5.1 cells were mock infected or infected with JFH-1 HCVcc for 0.5 to
72 h. DDX3X, HCV core protein and LDs were stained and their subcellular localizations and
A Core LDs DDX3X LDs
DDX3X Core M
ock
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associations were examined. Magnifications of selected areas are shown in the right panels.
Colors in far right panels were modified for better visualization: HCV core protein, green
instead of magenta. Scale bars, 10 µm. (B) Quantification of DDX3X co-localization with
HCV core protein and LDs. Five to ten confocal microscopic pictures from at least three
independent experiments with a total of at least 30 cells were analyzed for co-localization
signals. Percentages of cells without granular structures (light grey striped bars) or presenting
DDX3X structures that are either not co-localized (blue stripped bars) or co-localized with
LDs only (green striped bars) or with both HCV core protein and LDs (green bars) are shown.
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Figure S4. Dynamic redistribution of SGs towards LDs during HCV infection. Huh-7.5.1 cells
were mock infected or infected with JFH-1 HCVcc for 4 to 72 h. SG proteins G3BP1 and
PABP1 and LDs were stained and examined by confocal microscopy. Magnifications of
selected areas are shown in the right panels. Colors in far right pictures were changed for
better visualization: PABP1 (green instead of magenta) and G3BP1 (red). Scale bars, 20 µm.
PABP1 LDs PABP1 G3BP1 G3BP1 LDs M
ock
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G3BP1 LDs PABP1 Merge
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G3BP1 LDs PABP1 Merge
G3BP1 LDs PABP1 Merge
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Figure S5. SGs are not induced in the absence of infectious HCV. Huh-7.5.1 cells were
inoculated for 30 min with culture supernatant of uninfected cells (A) or with heat-inactivated
(10 min at 100°C) JFH-1 HCVcc (B). DDX3X and G3BP1 were then immunostained and
examined by confocal microscopy. Scale bars, 20 µm.
DDX3X G3BP1 Merge
DDX3X G3BP1 Merge
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Figure S6. Effects of DDX3X or SG proteins silencing in cells on productive HCV
infection. Huh-7.5.1 cells were transfected with non-targeting control (siNT) or various
indicated siRNAs for 3 days and then infected with JFH-1 HCVcc (A-C, F-H) or transfected
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siNTsiDDX3X
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with HCV IRES RNA (D) or JFH1-Rluc subgenomic replicon RNA (E, I). (A) DDX3X
siRNA-mediated knockdown efficiency, examined by gene expression assay.
(B) Quantification of HCV RNA levels in cells (intracellular) or supernatant (extracellular) by
RT-qPCR in DDX3X-silenced cells. (C) HCV core protein staining by immunofluorescence
in DDX3X-silenced cells. Nuclei (blue) were stained with DAPI to determine the total cell
numbers and percentage of infected cells. The ratio of infected cells as compared to siNT (as
1.00) was shown on top left corner of each image. (D) HCV IRES-mediated translation assay
in DDX3X-silenced cells, performed at 24 h after transfection of the IRES replicon RNA that
harbours firefly luciferase reporter gene. (E) HCV subgenomic replicon assay in DDX3X-
silenced cells. Renilla luciferase activity was measured 2 days post-tranfection to determine
the level of viral RNA replication. (F) Knockdown efficiencies of various indicated siRNAs
in Huh-7.5.1 cells. (G) Quantification of HCV RNA levels in cell lysate (intracellular) or
supernatant (extracellular) in cells deprived of various indicated SG proteins. (H) G3BP1
depletion suppressed HCV core expression. Scale bars, 20 µm. (I) G3BP1 siRNA reduces
HCV subgenomic replicon activity. Error bars represent ± s.d. of triplicate experiments. * and
**, P < 0.05 and 0.01, respectively.
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Figure S7. Knockdown of various SG-associated proteins exhibited different effects on
SG formation and DDX3X–IKK-α aggregation. Huh-7.5.1 cells were transfected with
indicated siRNAs for 3 days, and then infected with JFH-1 HCVcc for 2 days. DDX3X, IKK-
B
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siIK
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DDX3X G3BP1
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α, SG markers G3BP1 and PABP1 and LDs were detected by immunofluorescence and
examined by confocal microscopy. Magnifications of selected areas are shown in the right
panels. (A) DDX3X–G3BP1 granules in IKK-α-, PABP1-, or PKR-silenced cells. (B) IKK-α–
G3BP1 in PKR-silenced cells. (C) PABP1 in G3BP1-silenced cells. Colors in far right
pictures were changed for better visualization: PABP1 (green instead of magenta) and G3BP1
(red). Scale bars, 20 µm.