Feline Calicivirus infection disrupts the assembly of cytoplasmic ...

FCV infection impairs stress granules assembly

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FCV infection impairs stress granules assembly 20 21

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JVI Accepted Manuscript Posted Online 4 May 2016J. Virol. doi:10.1128/JVI.00647-16Copyright © 2016 Humoud et al.This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Caliciviridae29

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α35

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Caliciviridae85

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Vesivirus93

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Caliciviridae100

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α111

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μ135

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g °183

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α252

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Picornaviridae325

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FCV - + - +

GAPDH

NS6/7

2 hpi 6 hpi

p-eIF2(S52)

eIF2

SA - + mock

CRFK cells were mock infected or infected with FCV Urbana at an MOI of 1 for 2 or 6h as indicated. As a control CRFK cells were treated with 0.5 mM sodium arsenite (SA) for 30 min or mock treated (+ and -, respectively). Following treatments, cell extracts were prepared and analysed by SDS-PAGE and immunoblotting using the antibodies indicated on the left side of the panels.


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A BNS6/7 G3BP1 merge

+SA

-SA

2 hpi

4 hpi

6 hpi

8 hpi

Figure 2. SGs accumulation during FCV infection. (A) CRFK cells were mock infected or infected with FCV Urbana at an MOI of 1. Infected cells were fixed at the times indicated post-infection (hpi) and stained with either mouse monoclonal antibodies specific for G3BP1 and rabbit polyclonal antiserum specific for FCV NS6/7 (A) or mouse polyclonal antibodies specific for VP1 and rabbit polyclonal antiserum specific for eIF4G (B). This was followed by staining with Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-mouse IgG secondary antibodies. Nuclei were stained with ToPro3. Stained cells were examined by fluorescence microscopy, and representative images are shown. (C) The percentage of cells containing SGs out of the total number of cells was calculated and the mean and standard deviations of three experimental replicates are shown. Results were analysed by two-way ANOVA with Bonferroni corrections: ***p<0.001 (GraphPad Prism 6).

hours post-infection

Cel

ls d

ispl

ayin

g st

ress

gra

nule

s (%

)

-SA +SA 2 hpi 4 hpi 6 hpi 8 hpi0

20

40

60

80

100G3BP1eIF4G

C

eIF4GVP1 merge

+SA

-SA

2 hpi

4 hpi

6 hpi

8 hpi

*** ***


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0

20

40

60

80

100

Cel

ls d

ispl

ayin

g st

ress

gra

nule

s (%

)

**ns

A B

- SA

+ SA

- SA

+ SA

NS6/7 G3BP1 merge

- SA

+ SA

- FC

V+

FCV

+ FC

V(U

V)

- SA + SA - SA + SA - SA + SA

- FCV + FCV + FCV(UV)

Figure 3. SGs accumulation following arsenite treatment in FCV-infected cells. (A) CRFK cells were mock infected or infected with FCV Urbana at an MOI of 1 for 6h. Following infection, cells were treated with 0.5 mM SA for 30 min. The cells were fixed and stained with mouse monoclonal antibodies specific for G3BP1 and rabbit polyclonal antiserum specific for FCV NS6/7. This was followed by staining with Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-mouse IgG secondary antibodies. Nuclei were stained with ToPro3. Stained cells were examined by fluorescence microscopy, and representative images are shown. (B) The percentage of cells containing SGs out of the total number of cells was calculated and the mean and standard deviations of three experimental replicates are shown. Results were analysed by two-way ANOVA with Bonferroni corrections: **p<0.01; ns not significant (GraphPad Prism 6). (C) CRFK cells were infected with FCV or UV-inactivated FCV at an MOI of 1.The cells were incubated for 12 h and the viral titer was estimated by a TCID50 assay. Three separate experiments were analyzed by standard t test: **p<0.01 (GraphPad Prism 6).

0

1

2

3

4

5

6

7

log

(TC

ID50

/mL)

**

- UV + UV

FCV

NS6/7 G3BP1 merge

C


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H2O2 - + - +

GAPDH

4E-BP1

mock

p-4EBP1(T37/46)p-4EBP1(S65)

FCV

0

20

40

60

80

100

Cel

ls d

ispl

ayin

g st

ress

gra

nule

s (%

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***

- H2O2 + H2O2 - H2O2 + H2O2

A B

C

- H2O2

+ H2O2

- H2O2

+ H2O2

NS6/7 G3BP1 merge

Figure 4. SGs accumulation following hydrogen peroxide treatment in FCV-infected cells. (A) CRFK cells were mock infected or infected with FCV Urbana at an MOI of 1 for 6h and treated with 1 mM hydrogen peroxide (H2O2) for 1h or mock treated (+ and -, respectively). Following treatments, cell extracts were prepared and analysed by SDS-PAGE and immunoblotting using the antibodies indicated on the right side of the panels. (B) CRFK cells were mock infected or infected with FCV Urbana at an MOI of 1 for 6h. Following infection, cells were treated with 1 mM H2O2 for 1h. The cells were fixed and stained with mouse monoclonal antibodies specific for G3BP1 and rabbit polyclonal antiserum specific for FCV NS6/7. This was followed by staining with Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-mouse IgG secondary antibodies. Nuclei were stained with ToPro3. Stained cells were examined by fluorescence microscopy, and representative images are shown. (C) The percentage of cells containing SGs out of the total number of cells was calculated and the mean and standard deviations of three experimental replicates are shown. Results were analysed by two-way ANOVA with Bonferroni corrections: ***p<0.001 (GraphPad Prism 6).

+ FC

V- F

CV

+ FCV- FCV


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Figure 5. G3BP1 cleavage during FCV infection. CRFK (A) or FEA (B) cells were mock infected or infected with FCV Urbana at an MOI of 1 for 6h. Cell extracts were prepared and analysed by SDS-PAGE and immunoblotting using the antibodies indicated on the right side of the panels. The arrows denote the position of cleavage product and the molecular mass standards (kDa) are shown on the left.

FCV - + - +

GAPDH

NS6/7

2 hpi

G3BP1

PABP

3 hpi

- + - +4 hpi 5 hpi

- + 6 hpi

7558

46

32

100

75

75

58

G3BP2

46

58

A B

GAPDH

NS6/7

PABP

G3BP1

46

32

100

75

75

58

7558

FCV - + - +2 hpi 6 hpi


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EGFP

Flag

EGFP

Tubulin

Flag

EGFP

Tubulin

Flag-G3BP1 Flag-G3BP2

FCV NS6P

ro

MNV NS6P

ro

PV 3CPro

EGFPFCV N

S6Pro

MNV NS6P

ro

PV 3CProG3BP1

G3BP2

Tubulin

EGFPFCV N

S6Pro

MNV NS6P

ro

PV 3CPro

70

40

55

70

40

55

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70

40

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55

35

40

55

M. musculus

H. sapiens

F. catus

AG322 EQGDVE DKSE LD K DF FQ S YGNVV ELR IN329 352 372 DDSEPVQ K VLS386 396 FR GEVR L N401 409

AG322 EQGD I E DKSE LD K DF FQ S YGNVV ELR IN329 352 372 DDSEPVQ K VLS386 396 FR GEVR L N401 409

AG322 E PGDVE DKSE LD K DF FQ NYGNVV ELR IN329 352 372 DDSEPVQ K VLS386 396 FR GAVR L N401 409

A

PV 3CPro

A A A A A A

FCV NS6Pro

A B

C

D

Flag

Tubulin

Flag

Tubulin5555

70

40

55

70

40

55

wt

EGFP-FCV NS6Pro

Q325A

E355AQ36

1A

EGFP

wt E389A

Q392A

E405A

E368A

wt

EGFP-PV 3CPro

Q325A

E355AQ36

1A

EGFP

wt E389AQ39

2A

E405A

E368A

Figure 6. G3BP1 and G3BP2 cleavage by FCV NS6Pro. (A) EGFP, EGFP FCV NS6Pro, EGFP MNV NS6Pro or EGFP PV 3CPro were expressed in 293T cells. Cell extracts were prepared and analysed by SDS-PAGE and immunoblotting using the antibodies indicated on the right side of the panels. The arrows denote the position of cleavage products and the molecular mass standards (kDa) are shown on the left. (B) EGFP, EGFP FCV NS6Pro, EGFP MNV NS6Pro or EGFP PV 3CPro were co-expressed with Flag-G3BP1 or Flag-G3BP2 in 293T cells, as indicated. Cell extracts were prepared and analysed by SDS-PAGE and immunoblotting using the antibodies indicated on the right side of the panels. The arrows denote the position of cleavage products and the molecular mass standards (kDa) are shown on the left. (C) Comparison of the C-terminal sequences of human, feline and murine G3BP1. The positions of the viral proteinase cleavage sites, identified in this study and previously, are indicated above the sequences boxed in black. The positions where G3BP1 alanine mutants have been introduced are highlighted in black and grey. (D) EGFP, EGFP FCV NS6Pro or EGFP PV 3CPro were co-expressed with wild type (wt) or mutant Flag-G3BP1 in 293T cells, as indicated. Cell extracts were prepared and analysed by SDS-PAGE and immunoblotting using the antibodies indicated on the right side of the panels. The arrows denote the position of cleavage products and the molecular mass standards (kDa) are shown on the left.

35

40 EGFP


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G3BP1wt

G3BP1E405A

G3BP1wt

G3BP1E405A

GFP eIF3 merge

Figure 7. FCV NS6Pro-mediated G3BP1 cleavage impairs SGs assembly. (A) HeLa-R19-G3BP1KO cells were co-transfected with Flag-G3BP1 or Flag-G3BP1-E405A expression plasmids together with EGFP or EGFP-FCV-NS6Pro

stained with goat polyclonal antibodies specific for eIF3

(B) Cell extracts were prepared from HeLa (wt) and HeLa-R19-G3BP1KO cells (G3BP1KO), and analysed by

+ EG

FP-N

S6Pr

o+

EGFP

G3BP1

A B

G3BP2

G3BP1

55

7055

7055

wt

G3B

P1K

O


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8 hpi+ SA

8 hpi- SA

Mock+ SA

Mock - SA

G3BP1 NS3

16 hpi- SA

16 hpi+ SA

Figure 8. SGs accumulation during MNV infection. (A) J774 cells were mock infected or infected with MNV1 at an MOI of 10, or treated with sodium arsenite (SA) for 45 min. Cell extracts were prepared and analysed by SDS-PAGE and immunoblotting using the antibodies indicated on the left side of the panels. The molecular mass standards (kDa) are shown on the right. (B) J774 cells were mock infected or infected with MNV1 at an MOI of 10. Following infection cells were treated with 0.5 mM arsenite for 45 min. Cells were fixed at the times indicated post-infection and stained with rabbit polyclonal antibodies specific for NS3 and mouse monoclonal antibodies specific for G3BP1. This was followed by staining with species-matched AlexaFluor-conjugated secondary antibodies. Nuclei were stained with DAPI. Stained cells were examined by fluorescence microscopy, and representative images are shown.

Mock 8 hpi 16 hpi- + - + - +SA

p-eIF2(S52)

eIF2

G3BP1

NS7

GAPDH

MNV1

75

50

37

37

37

5037

25

20

15

75

A BMerge + DAPI


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Feline Calicivirus infection disrupts the assembly of cytoplasmic ...

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