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1 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, [email protected]. V.P. and Q.L. contributed equally to this work.

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Page 1: Dynamic Interaction of Stress Granule, DDX3X and IKK-α …jvi.asm.org/content/suppl/2015/04/20/JVI.03197-14.DC... ·  · 2015-04-20Jc1 n 4 h 8 h 24 h 48 h 72 h ! 6! associations

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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,

[email protected].

V.P. and Q.L. contributed equally to this work.

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SUPPLEMENTAL FIGURES

C D

A

B

DDX3X IKK-α DDX3X Mito IKK-α Mito

Ctrl

H

CV

3’U

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Mito DDX3X IKK-α

Mito DDX3X IKK-α

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Merge

Mito DDX3X IKK-α Merge

HC

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H

CV

3’U

TR

Catalase IKK-α

Catalase IKK-α

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IKK-α Catalase Merge

HC

V

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H

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3’U

TR

LC3B IKK-α

LC3B IKK-α

Merge

Merge

IKK-α LC3B Merge

HC

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DDX3X Mito DDX3X LDs

LDs DDX3X Mito

DDX3X Catalase DDX3X LDs

LDs DDX3X Catalase

Ctrl

Catalase DDX3X Merge HC

V 3

’UTR

Catalase DDX3X Merge

E

F

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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.

A

C

PABP1 DDX3X

PABP1 IKK-α

PABP1 G3BP1

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Merge

Merge

DDX6 IKK-α

DDX6 G3BP1

DDX6 DDX3X Merge

Merge

Merge

EDC4 DDX3X

EDC4 G3BP1

EDC4 IKK-α

Merge

Merge

Merge

B

D + _ HCV

anti-IgG

+ _ + _

anti-G3BP1 anti-DDX3X

WB: anti-DDX3X

+ _

WB: anti-G3BP1

WCL

HC

DDX3X

G3BP1

IP:

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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

HC

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.5 h

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DDX3X LDs Core Merge

DDX3X LDs Core Merge

DDX3X LDs Core Merge

DDX3X LDs Core Merge

DDX3X LDs Core Merge

DDX3X LDs Core Merge

DDX3X LDs Core Merge

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0%

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Mock 30 min 4 h 8 h 24 h 48 h 72 h

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No granules DDX3X granules DDX3X granules at LD DDX3X/Core granules at LD

0%

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Ctrl SGR2a

JFH1 Con1

Jc1

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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

HC

V 4

h

HC

V 8

h

HC

V 2

4 h

HC

V 4

8 h

HC

V 7

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G3BP1 LDs PABP1 Merge

G3BP1 LDs PABP1 Merge

G3BP1 LDs PABP1 Merge

G3BP1 LDs PABP1 Merge

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

A

B

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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

A

siNTsiDDX3X

0.0

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DD

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siNTsiG3BP1

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** ** ** G3BP1 TIA1 TIAL1

siNTsiDDX3X

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Core Nuclei C

siD

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siNTsiG3BP1

siTIA1

siTIAL1

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HC

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** *

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siNTsiG3BP1

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*

I F G

siG

3BP

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Core DDX3X

Core DDX3X

Core G3BP1

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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

A

DDX3X G3BP1

DDX3X G3BP1

DDX3X G3BP1

siIK

K-α

si

PAB

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G3BP1 LDs

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DDX3X G3BP1

IKKα G3BP1

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KR

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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.