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Mismatches in the Influenza A Virus RNA Panhandle Prevent

Retinoic Acid-Inducible Gene I (RIG-I) Sensing by Impairing RNA/

RIG-I Complex Formation

Stéphanie Anchisi, Jessica Guerra, Geneviève Mottet-Osman, Dominique Garcin

Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland

Influenza virus RNA (vRNA) promoter panhandle structures are believed to be sensed by retinoic acid-inducible gene I (RIG-I). The

occurrence of mismatches in this double-stranded RNA structure raises questions about their effect on innate sensing. Our results

sug-gest that mismatches in vRNA promoters decrease binding to RIG-I

in vivo

, affecting RNA/RIG-I complex formation and preventing

RIG-I activation. These results can be inferred to apply to other viruses and suggest that mismatches may represent a general viral

strategy to escape RIG-I sensing.

D

etection of viral infections at the cellular level is crucial for the

establishment of an innate immune response. Accordingly,

viruses have developed strategies to circumvent this response.

They can actively block the pathways involved or prevent the

for-mation of viral molecular patterns sensed by specific cellular

re-ceptors, such as retinoic acid-inducible gene I (RIG-I) and

mela-noma differentiation-associated protein 5, that recognize viral

RNA patterns. For RIG-I, this pattern consists of double-stranded

RNA (dsRNA) structures of various lengths, with 5=

tri- or

diphos-phate (5=

ppp or 5=

pp, respectively) base-paired ribonucleotides

(

1

,

2

). For segmented negative-strand RNA viruses, dsRNA

struc-tures are found in panhandles formed by base pairing of conserved

and complementary 5=

and 3=

genome ends, which activate RIG-I

Received29 June 2015 Accepted5 October 2015

Accepted manuscript posted online7 October 2015

CitationAnchisi S, Guerra J, Mottet-Osman G, Garcin D. 2016. Mismatches in the influenza A virus RNA panhandle prevent retinoic acid-inducible gene I (RIG-I) sensing by impairing RNA/RIG-I complex formation. J Virol 90:586 –590.

doi:10.1128/JVI.01671-15.

Editor:A. García-Sastre

Address correspondence to Dominique Garcin, Dominique.Garcin@unige.ch. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Input - RNA

ssRNA C perf. C mis.

RNA Pull Down

ATP+Mg

RIG-I +

-+ + -+ + -+ + -+ +

-+ + -+

ssRNA C perf. C mis. 1 RIG-I

2 RIG-I

unbound RNA

Rel. A

T

Pase

activity

- RNA 20r

ssRNA C perf. C mis.

** *

0 2 4 6 8

B A

D

C

E

20r 5’ GCGCACCGGGGAACCAAGGCGAACACGGACA3’ CGCGUGGCCCCUUGGUUCCGU ...

5’ GGCAAAAGCAGGGAGACAAAGACAAAAAGGC ssRNA

5’ GGCAAAAGCAGGGAGACAAAGACAAAAAGGC 3’ CCGUUUUCGUCCCUCUGUUUCUGUUUUUCCG C perf.

5’ GG A AAGCAGGGAGACAAAGACAAAAAGGC

3’ CC U UUUGUCCCUCUGUUUCUGUUUUUCCGU C

C A C mis.

0 10 20 30 40

IFNß fold induction

Mock 20r C perf. C mis.

FIG 1Mismatches do not affect RIG-I binding and ATPase activity but prevent IFN-␤activation. (A) Sequences of the RNAs used to mimic the influenza virus cRNA panhandle structures with (mis.) or without (perf.) mismatches. (B) RIG-I binding. RNA pulldown assays were performed with 13 pmol of biotinylated

RNA ligands (as previously described [12]) and 13 pmol of His–RIG-I (16). Reaction products were analyzed by Western blotting with antihistidine antibody

(1:2,000, H1029; Sigma). (C) RIG-I oligomerization was monitored by EMSA. Radiolabeled RNAs (25 pmol) were incubated with purified His–RIG-I (50 pmol

of RIG-I). Reaction products were analyzed on native gradient acrylamide gel and revealed by phosphorimaging (Typhoon; GE Healthcare Life Sciences) (12).

(D) Relative (Rel.) RIG-I ATPase activity. Purified RIG-I was incubated at 37°C with [␥-32P]ATP in the presence of various RNA ligands as indicated. Data are

presented as the mean⫾the standard error of the mean (n⫽4). Statistical significance: NS,P⬎0.05; *, 0.01ⱕP⬍0.05; **,P⬍0.001 (12). (E) IFN-␤promoter

activation. A549 cells were transfected with luciferase-based reporter gene plasmids (p␤-IFN-fl-lucter, which carries the firefly luciferase gene driven by the

human IFN-␤promoter [17], and pTK-rl-lucter, which carries theRenillaluciferase-encoding gene [Promega] driven by the herpes simplex virus thymidine

kinase promoter) and increasing amounts (200, 400, and 800 ng) of the RNAs indicated. Relative luciferase activation (fold IFN-␤induction) was calculated by

normalization to the mock-treated control and is represented as the mean⫾the standard deviation (n⫽2) (12).

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(

3

,

4

). To avoid detection, viruses have evolved strategies to

pre-vent their formation. Some arenaviruses and bunyaviruses have

unusual ways to initiate genome replication, leading to the

forma-tion of panhandles with a non-base-paired 5=

pppN overhang or

with a 5=

monophosphate end, respectively (

5–7

). These

struc-tures did not activate RIG-I (

7

,

8

) and, in the case of arenaviruses,

were seen as a viral decoy strategy to prevent RIG-I activation

from

bona fide

ligands (

9

).

Influenza virus genome promoters are contained within their

mostly complementary genome ends, which can form 5=

ppp

blunt-ended dsRNAs with conserved mismatches (see

Fig. 1A

and

3A

). When these promoter sequences are bound to their

polymer-ase, the complementary genome ends cannot form dsRNA, except

for a region relatively distant from the 5=

ppp and 3=

OH ends (

10

,

11

). However, a fraction of the influenza virus genome promoters

is likely not bound to the polymerase during infection. As residues

close to the 5=

ppp blunt end of RNA duplexes are critical for RIG-I

ATPase activity (

12

), the presence of mismatches in influenza

vi-rus panhandles raises the possibility that, in addition to their

pri-mary role in RNA synthesis, mismatches represent a viral strategy

to minimize detection by RIG-I.

To study the effect of these mismatches on RIG-I activation, we

designed synthetic dsRNAs based on the H1N1 Taiwan NS

seg-ment that mimic the influenza virus panhandles, with or without

mismatches (see

Fig. 1A

and

3A

). To obtain a 5=

ppp RNA, the top

strand was made

in vitro

by using a T7 polymerase. For this

pur-pose, the initial AG nucleotides were changed to GG. The length of

these panhandle-like structures was extended to 30 bp (see

Fig. 1

,

2

, and

4

) to circumvent stability problems and to monitor only the

effects of the mismatches. The 5=

ppp RNA made

in vitro

was used

as a single-stranded RNA (ssRNA) control, and a dsRNA of 20 bp

(20r) known to activate RIG-I was used as a positive control (

Fig.

1A

). We first studied the binding of these dsRNAs to RIG-I.

Pull-down experiments showed that RIG-I binds equally to dsRNAs

mimicking influenza virus cRNA panhandle structures with

(Cmis.) or without (Cperf.) mismatches (

Fig. 1B

). Using a

differ-ent approach, Liu et al. have shown recdiffer-ently that the panhandle

structure without mismatches exhibited higher affinities for RIG-I

(

3

). In addition, the presence of mismatches did not influence the

ability of RIG-I to form dimers (2 RIG-I) on these dsRNAs, as

shown by electrophoretic mobility shift assays (EMSA) (

Fig. 1C

,

Cperf. and Cmis. versus ssRNA). In EMSA, addition of ATP and

MgCl

2

, which allows RIG-I ATPase activity, had no effect on

RIG-I dimerization, as previously observed (

12

). Interestingly,

al-though these mismatches are positioned in key residues for RIG-I

ATPase activity (

12

), their presence had no effect on RIG-I

ATPase activity

in vitro, which was higher than that of 20r because

of their greater dsRNA length (30 versus 20 bp) (

Fig. 1D

).

Never-theless, when these dsRNAs were tested for the ability to activate

the beta interferon (IFN-

) promoter in a luciferase-based

re-porter gene assay, the presence of mismatches completely

abro-gated activation (

Fig. 1E

). Here again, Cperf. was more active than

20r, very likely because of its greater dsRNA length. To rule out the

hypothesis of selective RNA degradation, A549 cells engineered to

express the green fluorescent protein (GFP)-encoding gene under

the control of the IFN-

promoter (

13

) were transfected with

dsRNAs labeled with Cy5-fluorescent CTP. The cells were then

analyzed by flow cytometry for GFP expression (IFN-

activa-tion) and Cy5 fluorescence (RNA stability) at 9 and 24 h

post-transfection. The results confirmed that, contrary to Cperf., Cmis.

did not induce IFN-

under conditions where no difference in

Cy5 fluorescence could be detected (

Fig. 2A

and

B

), ruling out

RNA degradation as an explanation for the lack of IFN induction.

We further tested the effects of individual mismatches (

Fig. 3A

,

Cmis.3 and Cmis.5). As shown in

Fig. 1D

for the

double-mis-match construct (Cmis.), the two single-misdouble-mis-match dsRNAs

in-duced RIG-I ATPase as efficiently as the perfect dsRNA (

Fig. 3B

).

When tested for the ability to induce the IFN-

promoter in a

reporter gene assay, the duplex with the mismatch at position 3

(Cmis.3) exhibited no stimulatory activity, while Cmis.5 retained

some stimulatory activity (

Fig. 3C

, black bars). We have

previ-A

B

Mock 20r C perf. C mis.

WL

GFP

20 40 60

10

0

% GFP

+ cells

%GFP

+ cells

0 20 40 60

Mock ssRNA C perf

.

C mis. MockssRNA C perf .

C mis. 24hrs 9hrs 9hrs

24hrs

20 40 80 60 100

%Cy5

+ cells

0

Mock

GFP

Cy5

ssRNA C perf. C mis.

24hrs 9hrs

FIG 2Mismatches do not affect RNA stability. (A, B) IFN-␤promoter

acti-vation. A549/pr(IFN-␤).GFP reporter cells (13) were transfected with 400 ng

of the RNAs indicated for 24 h before fluorescence-activated cell sorter (FACS) analysis. (A) At the top are white light (WL) and fluorescence (GFP) micro-scope pictures. At the bottom are flow cytometry data. Results are plotted as

percentages of GFP-positive cells (xaxis). Data are presented as means⫾

standard deviations (n⫽2). (B) RNA stability. A549/pr(IFN-␤).GFP reporter

cells were transfected with Cy5-labeled RNAs, and Cy5 fluorescence was mon-itored over time by FACS analysis. Results are plotted as percentages of Cy5- or

GFP-positive cells. Data are presented as means⫾standard deviations (n⫽2).

At the bottom are representative plots of the FACS data. Results are plotted as

percentages of Cy5 (xaxis)- and GFP (yaxis)-positive cells.

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ously shown that IFN-

promoter activation can be enhanced by

pretreating cells (priming) with IFN-

(

12

). Under these

condi-tions, the inactive dsRNAs with single or double mismatches now

activated the IFN-

promoter, confirming that these RNAs are

not degraded (

Fig. 3C

, gray bars). The observed increased RIG-I

levels upon priming (

Fig. 3C

) could promote the formation of an

active tetrameric RIG-I complex (

14

), especially for unstable RNA/

RIG-I complexes, as seen for dsRNAs shorter than 13 bp (

12

). When

short dsRNAs mimicking the precise length of influenza vRNA

pan-handles (13 bp) with or without mismatches, G-U bonds, and an AU

tail mimicking the loop were used (

Fig. 3A

, Vperf. and Vmis.), similar

results in ATPase activity and RIG-I activation in cells and primed

cells were obtained, with the exception of Vmis., which remained

inactive even in IFN-primed cells (

Fig. 3D

and

E

). Taken together,

these results suggested that these mismatches might act by lowering

the formation of the RNA/RIG-I complex.

To monitor the formation of the RNA/RIG-I complex, we

per-formed

in vivo

RNA pulldown experiments after transfection of

biotinylated dsRNAs in cells overexpressing RIG-I. Results

pre-sented in

Fig. 4A

show that significantly less RIG-I was found

associated with dsRNAs with mismatches than with perfect

dsRNAs (65% versus 100%,

Fig. 4A

). In order to further test this

complex formation

in vitro, competition assays were performed in

RNA pulldown experiments using an excess of nonbiotinylated

C

20 40 60 80

0 100

Mock C perf.

-IFNß +IFNß

C mis. C mis. 3

C mis.5

% GFP

+ cells

Mock C perf. C mis. C mis.

3

C mis. 5

- + - + - + - + - +

IFNß RIG-I

Rel. A

T

Pase

activity

20r

C perf. C mis.

0 2 4 6 8

C mis.3 C mis. 5

B

5’ GG AAAAGCAGGGAGACAAAGACAAAAAGGC 3’ CC UUUUU UGUCCCUCUGUUUCUGUUUUUCCG

C

C mis.3

5’ GGCA AAGCAGGGAGACAAAGACAAAAAGGC 3’ CCGU UUC UGUCCCUCUGUUUCUGUUUUUCCG

A

C mis.5

5’ GGGAGAAACAAGGGCGGCAACAACCAACAAA 3’ CCCUCUUUGUUCC

V perf.

5’ GGGAGAA CAAGGGCGGCAACAACCAACAAA 3’ CCUUUUU GUCCCC

A

V mis.

A

E

5 10 15

0 20

Mock ssRNA

-IFNß +IFNß

C perf. C mis. V perf .

% GFP

+ cells

V mis.

D

Rel. A

T

Pase

activity

C perf. C mis.

0 2 4

V perf. V mis.

NS

NS NS

AUUUUAA

AUUUUAA

FIG 3A single mismatch on dsRNA is enough to limit IFN-␤induction. (A) Sequences of the RNAs used. (B) RIG-I ATPase activity. Purified RIG-I was

incubated at 37°C with [␥-32P]ATP in the presence of various RNA ligands as indicated. Data are presented as the meanthe standard error of the mean (n

4). Statistical significance: NS,P⬎0.05; *, 0.01ⱕP⬍0.05; **,P⬍0.001 (12). (C) IFN-␤promoter activation. A549/pr(IFN-␤).GFP reporter cells were

transfected with 500 ng of the RNAs indicated for 24 h with or without IFN-␤treatment 6 h prior to transfection. At the top are the percentages of GFP-positive

cells measured by FACS analysis. Data are presented as the mean⫾the standard deviation (n⫽2). At the bottom are the intracellular levels of RIG-I analyzed

by Western blotting. As a loading control, the membrane was stained with Coomassie blue after immunoblotting. (D) RNA-induced ATPase activity of RIG-I.

Purified RIG-I was incubated at room temperature with [␥-32P]ATP in the presence of various RNA ligands as indicated. Data are presented as the meanthe

standard error of the mean (n⫽5). Statistical significance: NS,P⬎0.05; **,P⬍0.001. (E) IFN-␤promoter activation. A549/pr(IFN-␤).GFP reporter cells were

transfected with 500 ng of the RNAs indicated for 24 h with or without IFN-␤treatment 6 h prior to transfection. Results are plotted as the percentages of

GFP-positive cells measured by flow cytometry. Data are presented as the mean⫾the standard deviation (n⫽2).

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dsRNAs with (Cmis.) or without (Cperf.) mismatches under

con-ditions where RIG-I ATPase activity and related RNA recycling

are allowed (

12

).

Figure 4B

shows that more RIG-I is associated

with the biotinylated RNA when the competitor has mismatches.

This indicates that the rate of RIG-I exchange is lower with a

competitor carrying mismatches (50% versus 22% after 6 min of

incubation), consistent with lower stability of the RNA/RIG-I

complex in this case. As shown in

Fig. 4C

, the rate of RIG-I

ex-change and related RNA/RIG-I complex stability is dependent on

RIG-I ATPase activity since no exchange is observed in the

ab-sence of ATP and MgCl

2

. In conclusion, mismatches as found in

the panhandle structures of segmented negative-strand RNA

vi-ruses may prevent RIG-I activation by impairing the formation of

stable RNA/RIG-I complexes. As mismatches can be observed in

the panhandle structure of other segmented negative-strand

vi-ruses (

15

), the results presented here can be inferred to apply to

other viruses and suggest that mismatches could represent a

gen-eral viral strategy to escape RIG-I sensing.

ACKNOWLEDGMENTS

This work was supported by Swiss National Science Foundation grant 31003A_135467.

Thanks to Steve Goodbourn and Richard Randall for providing GFP–

IFN-␤reporter A549 cells. We also thank Daniel Kolakofsky, Laurent

Roux, and Mirco Schmolke for their precious help discussing the project and critically reading the manuscript.

FUNDING INFORMATION

Swiss National Science Foundation (SNSF) provided funding to Domin-ique GARCIN under grant number 31003A_135467/2.

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Figure

FIG 1 Mismatches do not affect RIG-I binding and ATPase activity but prevent IFN-cRNA panhandle structures with (mis.) or without (perf.) mismatches
FIG 2 Mismatches do not affect RNA stability. (A, B) IFN-At the bottom are representative plots of the FACS data
FIG 3 A single mismatch on dsRNA is enough to limit IFN-GFP-positive cells measured by flow cytometry
FIG 4 Mismatches destabilize RNA/RIG-I complex formation. (A)quantifications were performed with ImageJ version 1.44p (National Institutes of Health, Bethesda, MD;nonbiotinylated perfect (Cperf.) or mismatched (Cmis.) cRNA for 1, 3, or 6 min at 37°C in the

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