J Exp Med-2012-Joubert-1029-47

7/31/2019 J Exp Med-2012-Joubert-1029-47

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J. Exp. Med. Vol. 209 No. 5 1029-1047

www.jem.org/cgi/doi/10.1084/jem.20110996

1029

Chikungunya virus (CHIKV) is the causative

agent or Chikungunya ever, an arbovira dis-

ease transmitted by mosquitoes. CHIKV was

rst isoated in 1953 during an epidemic in

Tanzania, East Arica (Mason and Haddow,

1957) and has recenty emerged in isands o

the Indian Ocean in 2005 (Enserink, 2006).

La Reunion, an isand in the Indian Ocean with

a popuation o amost 785,000, was the most

aected region, with an estimation o 300,000cumuative cases in 2005–2006 (Schuenecker

et a., 2006; Simon et a., 2006; Gérardin et a.,

2008). The epidemic invoved India, where

estimates approach six miion inected peope

(Watson, 2007). It has aso emerged in Itay,

southern France, and Austraia, and ongoing in-

ections exist in Southeast Asia (Ng et a., 2009;

Manimunda et a., 2011). CHIKV is a member

o the Togaviridae amiy, genus Alphavirus,

which are characterized by being enveoped,

singe-stranded positive poarity RNA viruses

(Inoue et a., 2003; Sourisseau et a., 2007;

Schwartz and Abert, 2010). In human, CHIKV

typicay induces symptoms 2–7 d ater inection

and is characterized by a rapid onset o ever,

severe arthragia, and myagia, oowed by

constitutiona symptoms (headache, photopho-

bia, nausea, and abdomina pain) and a rash

(Bodenmann et a., 2007; Borgherini et a.,

2007). In vivo, CHIKV mainy targets the con-nective tissue (i.e., primariy brobast ces),

but inection has aso been reported in the

iver, musce, and brain in human and mice

(Ozden et a., 2007). In vitro, CHIKV is abe to

inect a wide range o adherent ces, incuding

CORRESPONDENCEMatthew Albert:[email protected]

Abbreviations used: BDI,

bright detai intensity; CHIKV,

Chikungunya virus; CPE,

cytopathic eect; MOI,

mutipicity o inection; PE,

phosphatidyethanoamine;

RNS, reactive nitrogen species;

ROS, reactive oxygen species.

Chikungunya virus–induced autophagy

delays caspase-dependent cell death

Pierre-Emmanuel Joubert,1,5

Scott W. Werneke,6

Claire de la Calle,2

Florence Guivel-Benhassine,4 Alessandra Giodini,1,5 Lucie Peduto,3 Beth Levine,7,8 Olivier Schwartz,4 Deborah J. Lenschow,6 and Matthew L. Albert1,2,5

1Unité Immunobiologie des Cellules Dendritiques, 2Centre d’Immunologie Humaine, and 3Lymphoid Tissue Development Unit,Department o Immunology, and 4Unité de recherche Virus et Immunité, Institut Pasteur, 75724 Paris, Cedex 15, France

5INSERM U818, 75724 Paris, France6Department o Pathology and Immunology, Department o Medicine, Washington University School o Medicine, St. Louis, MO 631107Department o Internal Medicine and 8Howard Hughes Medical Institute, University o Texas Southwestern Medical Center,Dallas, TX 75390

Autophagy is an important survival pathway and can participate in the host response to inec-

tion. Studying Chikungunya virus (CHIKV), the causative agent o a major epidemic in India,Southeast Asia, and southern Europe, we reveal a novel mechanism by which autophagy limitscell death and mortality ater inection. We use biochemical studies and single cell multispectralassays to demonstrate that direct inection triggers both apoptosis and autophagy. CHIKV-

induced autophagy is mediated by the independent induction o endoplasmic reticulum andoxidative stress pathways. These cellular responses delay apoptotic cell death by inducing the

IRE1–XBP-1 pathway in conjunction with ROS-mediated mTOR inhibition. Silencing o autoph-agy genes resulted in enhanced intrinsic and extrinsic apoptosis, avoring viral propagation

in cultured cells. Providing in vivo evidence or the relevance o our fndings, Atg16L HM mice,which display reduced levels o autophagy, exhibited increased lethality and showed a highersensitivity to CHIKV-induced apoptosis. Based on kinetic studies and the observation that ea-

tures o apoptosis and autophagy were mutually exclusive, we conclude that autophagy inhibitscaspase-dependent cell death but is ultimately overwhelmed by viral replication. Our study

suggests that inducers o autophagy may limit the pathogenesis o acute Chikungunya disease.

© 2012 Joubert et al. This article is distributed under the terms o an Attribution–Noncommercial–Share Alike–No Mirror Sites license or the rst six monthsater the publication date (see http://www.rupress.org/terms). Ater six monthsit is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

Published April 16, 2012

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1030 Autophagy limits CHIKV-induced apoptosis | Joubert et al.

autophagic processes to enhance their repication or transmis-

sion. Exampes incude Shigella exnerii and Listeria monocyto-

genes, which express viruence actors capabe o inhibiting

autophagosome ormation, herpes simpex 1, which expresses

ICP-34.5 or inhibition o initiation step in the autophagic

process, and inuenza A, which encodes M2 or inhibition o

autophagosome/ysosome usion (Deretic and Levine, 2009).

Some viruses manage to be even more subversive, using

mechanisms o autophagy or enhanced repication and vira

reease, incuding hepatitis C virus, poiovirus, and HIV-1

(Deretic and Levine, 2009; Banchet et a., 2010).

Recent data suggest cross tak between autophagic and

apoptotic pathways; or exampe, evidence supports inter-

actions between Bc-2 and Becin-1 (aso known as Atg6), and

both may be engaged ater induction o simiar ce stress path-

ways (e.g., ER or oxidative stress; Thorburn, 2008; Djavaheri-

Mergny et a., 2010; Kang et a., 2011). Herein, we characterized

the roe o apoptosis and autophagy in the context o CHIKV

pathogenesis and report that host mechanisms, incuding ER

and oxidative stress, imit the cytopathic eect (CPE) o

CHIKV through the induction o autophagy. Moreover, in

the absence o autophagic genes, we observe higher eves o

apoptosis, both in vitro and in vivo. With respect to the atter,

Atg16LHM mice were more sensitive to CHIKV-mediated

ethaity. These resuts dene autophagy as a host deense

mechanism that imits CHIKV pathogenesis.

RESULTSCHIKV inection induces autophagosome ormationin inected cellsDuring autophagy, Becin-1–PI3K-III compex activation

resuts in isoation membrane ormation that surrounds its

substrates to generate an autophagic vesice characterized bya doube membrane, which is caed an autophagosome

(Mehrpour et a., 2010). Two ubiquitin-ike systems have

been shown to be essentia or autophagosome ormation. In

the rst, Atg12 (autophagy-reated gene 12) is conjugated to

Atg5, together orming a compex with Atg16L1, which

decorates the outer membrane o the isoation membrane.

Microtubue-associated protein 1 LC3 (ight chain 3, aso

known as Atg8) constitutes the second ubiquitin-ike system:

LC3 conjugates phosphatidyethanoamine (PE) at the outer

and inner autophagosoma membrane. Unike the Atg12–

Atg5–Atg16L1 compex that is recyced, the LC3-PE

(reerred to as LC3-II) remains associated with the inner

membrane o autophagosome, making it a useu marker o autophagosomes (Kroemer et a., 2010; Mehrpour et a.,

2010). To dene the reationship between CHIKV inection

and autophagy, we rst examined autophagosome ormation.

Mouse embryonic brobasts (MEFs) were inected and LC3

puncta or LC3-II ormation was measured by immunouor-

escence or Western bot, respectivey. Compared with unin-

ected MEFs, CHIKV-inected ces showed increased

numbers o LC3 puncta (Fig. 1, A–C) and stronger staining

or LC3-II by Western botting (Fig. 1 D). Autophagosome

ormation coud be rst observed ater 5 h and peaked at 9 h

macrophages, brobasts, and HeLa ces (Sourisseau et a., 2007;

Schwartz and Abert, 2010). As observed or other aphaviruses

(e.g., SINV and Ross River), CHIKV inection has the capac-

ity to induce apoptosis, which has been suggested to be associ-

ated with pathogenesis. Notaby, apoptotic bebs have been

shown to contain CHIKV, which may represent a mechanism

o ce-to-ce vira spread (Krejbich-Trotot et a., 2011a).

Apoptosis (or programmed ce death type I) is an energy-

dependent process that is reguated by a preormed cascade

o proteases caed caspases (Dania and Korsmeyer, 2004;

Kurokawa and Kornbuth, 2009). Two main pathways are

invoved in apoptosis: (1) the intrinsic pathway, in which ac-

tivation o Bak and Bax resuts in mitochondria outer mem-

brane permeabiization inducing apoptosome ormation and

activation o caspase-9 (Kroemer et a., 2007); and (2) the ex-

trinsic pathway that is initiated by death receptor oigomer-

ization and ceavage o caspase-8 (or caspase-10; Wison

et a., 2009; Kurokawa and Kornbuth, 2010). Both pathways

resut in the activation o executioner caspases (e.g., caspase-3,

-6, and -7), eading to the morphoogica and biochemica mod-

ications recognized as apoptosis (Kurokawa and Kornbuth,

2009). Apoptosis is considered a rst ine o deense against

vira inection, where engagement o intraceuar stress path-

ways may trigger ce death, serving to imit vira repication

(Grifn and Hardwick, 1997; Everett and McFadden, 1999; Li

and Stoar, 2004). Many viruses have evoved strategies to

escape or deay apoptosis, urther pointing toward the roe o

ce death as an antivira mechanism (Teodoro and Branton,

1997; Kepp et a., 2009).

The autophagic pathway is a buk degradation system,

which contros the cearance and recycing o intraceuar

constituents or the maintenance o ceuar surviva (Deretic

and Levine, 2009). Autophagy consists o at east three path-ways: microautophagy, macroautophagy, and chaperone-

mediated autophagy (Crotzer and Bum, 2010; Li et a., 2011).

O those, macroautophagy is best characterized and has been

impicated in both innate and adaptive immunity (Deretic and

Levine, 2009). For the purpose o this study we wi reer to

macroautophagy as autophagy. Athough the autophagic path-

way was originay identied as a process induced by ceuar

starvation, there has been a strong interest in characterizing

the roe o autophagy as a mechanism o host deense (Deretic

and Levine, 2009; Crotzer and Bum, 2010; Joubert et a.,

2011). In vitro, autophagy is abe to isoate and degrade Strep-

tococcus pyogenes ater it enters in the cytoso (Nakagawa et a.,

2004; Joubert et a., 2009). Other in vitro and in vivo exam-pes incude the contro o Mycobacterium tuberculosis and Listeria

monocytogenes (Deretic and Levine, 2009). Some vira proteins

are aso targeted by autophagy (e.g., Sindbis or Tobacco

Mosaic virus; Deretic and Levine, 2009; Orvedah et a., 2010,

2011). Other roes or autophagy in the host response incudes

the enhancement o type I IFNs, or the processing and pre-

sentation o antigen or MHC I or MHC II presentation and

T ce priming (Dengje et a., 2005; Engish et a., 2009; Uh

et a., 2009; Crotzer and Bum, 2010). There aso exist exampes

o microbes that are capabe o abrogating and/or expoiting


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Figure 1. Autophagosome induction within CHIKV-inected cells. (A and B) WT MEFs were incubated or 24 h in control media (uninected), innutrient-deprived media (starvation), or in the presence o live or UV-B–treated CHIKV (MOI = 1). (A) Immunofuorescence was perormed using anti-LC3

antibody and DAPI. Bars, 10 µm. (B) The number o LC3 puncta (autophagosomes) per cell is depicted. Data shown represent mean ± SEM or triplicatesamples o >100 cells per experimental condition. Similar results were observed in three independent experiments. (C) GFP-LC3 transected cells were

inected with CHIKV and the number o GFP dots is represented at dierent times ater inection. Similar results were observed in two independent ex-periments. (D) WT or Atg5 / MEFs were inected or not with CHIKV or 24 h and Western blotting was perormed using anti-LC3 antibody to distinguishunconjugated LC3-I rom PE-ylated LC3-II, and GAPDH to control or protein loading. Band intensity was calculated using ImageJ sotware and the ratio

o LC3-II/GAPDH expression was normalized across the experiment and values are indicated. Similar results were observed in three independent experi-ments. (E and F) WT or Atg5 / MEFs were transected with Atg5 cDNA plasmid. Cells were inected with CHIKV and autophagy was monitored as inA and B. Data shown represent mean ± SEM or triplicate samples o at >100 cells per experimental condition. Similar results were observed in two inde-

pendent experiments. Bars, 10 µm. (G) WT or Bcl2AAA MEFs were incubated or 24 h in control media (uninected), in nutrient-deprived media (starvation),or in the presence o CHIKV (MOI = 1). Immunofuorescence was perormed using anti-LC3 antibody, and the number o LC3 puncta (autophagosomes)

per cell is depicted. Data shown represent mean ± SEM or triplicate samples o >100 cells per experimental condition. Similar results were observed inthree independent experiments. (H–L) WT MEFs were inected or 24 h with GFP-expressing recombinant CHIKV (MOI=1) and stained with anti-LC3 anti-body. Cells were analyzed using an ImageStreamX multispectral cytometer allowing gating o uninected (R1) and inected cells (R2) according to GFP

intensity (H) while simultaneously assessing the LC3 puncta based on BDI o LC3 staining (I). The mean BDI was marked. Representative uninected (J) andinected (K) cells are shown. (L) The percentage o BDIhi cells was calculated and plotted or comparison across experiments. More than 10,000 cells percondition were collected. Error bars indicate the SEM. Similar results were observed in three independent experiments. Student’s t test: **, P < 0.05.


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ater inection (Fig. 1 C), suggesting the requirement or

CHIKV repication as the trigger or autophagy induction.

Repication-deective CHIKV (achieved by UV-B irradia-

tion) aied to induce autophagosome ormation, thus sup-

porting the requirement or vira repication (Fig. 1, A and B).

Serum starvation served as a positive contro or autophagy

induction (Fig. 1, A and B; and not depicted).

To evauate i the induction o autophagosomes was oc-

curring via a cassica macroautophagy pathway, we evauated

CHIKV inection using MEFs decient in the key autophagy

gene Atg5 . As predicted, Atg5 / MEFs showed no evidence

o LC3-II conversion upon CHIKV inection (Fig. 1 D).

Simiary, immunouorescence studies demonstrated that

LC3 puncta observed during CHIKV inection were depen-

dent on Atg5 expression (Fig. 1, E and F). Restoration o

Atg5 expression, achieved by cDNA transection, rescued the

ces’ abiity to induce autophagy (Fig. 1, E and F). We ob-

served simiar resuts using ces decient or Atg7 , another

key autophagy gene (data not depicted). Furthermore, we

investigated the impication o Becin-1 in CHIKV-induced

autophagy using MEFs that express a mutant orm o Bc-2

(MEF-Bc2AAA). Notaby, Bc-2 directy reguates the acti-

vation o Becin-1 and aanine substitution o the three

phosphoryation sites (T69A/S70A/S87A) in Bc-2 prevents

dissociation o the Bc-2–Becin-1 compex, seectivey in-

hibiting autophagy induction by Becin-1 without aecting

the antiapoptotic roe o Bc-2 (Wei et a., 2008; He et a.,

2012). As expected, MEF-Bc2AAA ces were not abe to in-

duce autophagy under starvation conditions (Fig. 1 G). Simi-

ary, CHIKV-induced autophagy was abrogated in Bc-2AAA

MEFs, indicating that CHIKV-induced autophagy occurs via

a Becin-1–dependent mechanism. To conrm that CHIKV

inection induced autophagosome ormation in other cetypes, we aso investigated the appearance o autophagosomes

in HeLa ces and human oreskin brobasts. Siencing o

Atg5 or Atg7 genes using sma intererence RNA (siRNA)

conrmed data shown using MEFs (unpubished data).

To anayze whether autophagosome ormation was de-

pendent on direct vira inection, we marked active repication

using GFP-expressing recombinant CHIKV (Vanandingham

et a., 2005) and anayzed LC3 puncta using ImageStreamX.

In brie, mutispectra cytometric anaysis enabes the capture

o high-resoution images o ces in ow (up to 500 ces/s)

and permits anaysis o LC3 puncta (de a Cae et a., 2011).

24 h ater inection, GFP-expressing ces were gated (Fig. 1 H,

R2), and LC3 bright detai intensity (BDI) was integrated or each ce as a measure o autophagosome ormation. For

comparison, GFP-negative ces were gated (Fig. 1, H [R1]

and I [bue ine]), and histogram pots representing LC3

puncta indicate that CHIKV-inected ces (Fig. 1 I, red ine)

have higher LC3 BDI. Representative ImageStreamX images

with median intensity eves o BDI are shown or CHIKV-

inected and uninected ce popuations (Fig. 1, J and K),

conrming that LC3 puncta (scored based on high BDI) cor-

reated with the presence o both vira-encoded GFP and

robust autophagosome accumuation. Using this method, we

quantied the percentage o LC3-positive ces (BDIhi) when

buk cutures are segregated or CHIKV inection (Fig. 1 L,

R2 vs. R1, P < 0.05). Starved ces were used as positive

contro or autophagy induction (unpubished data). Based

on these data, we concude that autophagy induction occurs

via a Becin-1–dependent mechanism in a ce-intrinsic man-

ner; in other words, vira repication within the ce, as op-

posed to secreted actors produced by neighboring inected

ce, is the stimuus or autophagy induction.

Autophagosome/lysosome usion remains intactduring CHIKV inectionUpon maturation, autophagosomes use with ate endosomes

and ysosomes, which resuts in the ormation o a degradative

compartment reerred to as autoysosomes (Deretic and Levine,

2009). Some viruses encode inhibitors o this event (e.g., inu-

enza virus), and as a resut enhanced numbers o LC3 puncta

coud be a reection o basa autophagy accumuation and not

de novo autophagosome ormation (Gannagé et a., 2009). To

discriminate between these two possibiities, we anayzed auto-

phagy in the presence o ysosoma inhibitors eupeptin and

E64D in transected GFP-LC3 HeLa ces (Fig. 2, A and B). As

expected, inhibition o autophagosome/ysosome usion re-

suted in an increased number o LC3 puncta in contro (basa

autophagy ux) and starved (induced autophagy) HeLa ces.

The presence o eupeptin and E64D aso enhanced the num-

ber o autophagosomes in inected ces, aowing us to con-

cude that CHIKV inection induces de novo autophagosome

ormation (Fig. 2 B). To conrm this nding, we directy

assessed coocaization o LC3 and the ysosoma-associated

membrane protein LAMP-1 (Fig. 2 C). Optica sectioning o

ces was perormed and LC3+, LAMP-1+, and doube-abeed

vesices were enumerated on a per ce basis. Approximatey45% o the LC3+ puncta coocaized with LAMP-1+ ysosomes

in CHIKV-inected ces (Fig. 2 D). Line scan anaysis con-

rmed coocaization and indicated that the ysosoma struc-

tures were discrete vesices within the ce (Fig. 2 E).

As a na measure o autophagic ux, we transected MEF

ces with a construct encoding an RFP-GFP-LC3 poypro-

tein. In this way, it is possibe to distinguish autophagosomes

(RFP+ GFP+ puncta) and autoysosomes (RFP+ GFP vesi-

ces), with the atter being GFP negative as a resut o the

quenching o signa by the acidic microenvironment o the

ysosome. CHIKV inection increased the number o both

autophagosomes and autoysosomes compared with unin-

ected ces (Fig. 2, F and G). Moreover, an equivaent ratioo autophagosomes and autoysosomes was measured in in-

ected ces and starved ces (Fig. 2 G). Together, the data

presented in Figs. 1 and 2 provide evidence or CHIKV in-

ection inducing de novo autophagosome ormation without

inhibition o autophagosome maturation.

ER stress serves as a trigger or autophagyduring CHIKV inectionThe ER serves as an important sensor o ceuar stress. It de-

tects changes in ce homeostasis and responds by triggering


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Figure 2. CHIKV induces autophagic fux. (A and B) GFP-LC3–expressing HeLa cells were starved (starvation) or inected with CHIKV (MOI = 1) alone

or in the presence o leupeptin and E64D (L+E) or 24 h. The numbers o GFP+ vesicles per cell were enumerated. Data shown represent mean ± SEM ortriplicate samples o at least 100 cells per sample. Similar results were observed in our independent experiments. Bars, 5 µm. (C–E) WT MEFs were in-ected with CHIKV or 24 h. Immunofuorescence was perormed using anti-LC3, anti-Lamp-1, and DAPI to assess autophagosome and autolysosome

ormation. The number o LC3 puncta (green), Lamp-1+ vesicles (red), or costained vesicles (yellow) are depicted (C and D). Data represent mean ± SEM ortriplicate samples o at least 100 cells per sample. Similar results were observed in two independent experiments. Bars, 10 µm. (E) Line scale bar analysis

o a representative cell indicated in C is depicted. LC3 proles (green curve), Lamp-1 proles (red curve), and DAPI staining (blue curve) are shown. Theblack vertical line indicates colocalization o LC3 and Lamp-1 staining. (F and G) WT MEF cells were transected with a plasmid encoding a dual-labeledLC3 probe, RFP-GFP-LC3. Cells were again starved (starvation) or inected with CHIKV or 24 h. Representative images are shown and the number o total

autophagic vacuoles (GFP+RFP+ + GFP-RFP+), autophagosomes (GFP+RFP+), and autolysosomes (GFPRFP+) were enumerated. The inset permits visualiza-tion o yellow autophagosome (yellow arrow) and red autolysosomes (red arrow). Data represent mean ± SEM or three independents experiments o atleast 100 cells per sample. Similar results were observed in three independent experiments. Bars, 5 µm. Student’s test: **, P < 0.05.


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pathways reerred to as the unoded protein response (UPR;

Lee et a., 2003; McGuckin et a., 2010). Vira inection has

been shown to activate UPR as a resut o the accumuation

o vira proteins. At east three dierent pathways may be acti-

vated during ER stress, which are reguated by the signaing

moecues eIF2, IRE1, and ATF6, respectivey (McGuckin

et a., 2010). We screened a three pathways (unpubished

data) and identied a critica roe or IRE1. A roe or IRE1

activation during CHIKV inection was rst demonstrated

by anayzing the phosphoryation o IRE1 (p-IRE-1) at

Figure 3. CHIKV-induced autophagy is regu-lated by ER and oxidative stress. (A–C) WT MEFswere inected with CHIKV at indicated time pointsand Western blotting was perormed to detect phos-

phorylation o IRE1 (p-IRE1) and JNK (p-JNK) aswell as the ormation o spliced orm o XBP1 (XBP1s)

and the conjugation o LC3 (LC3-II). (A) IRE1 andGAPDH were also ollowed to control protein expres-sion and loading. Similar results were observed in two

independent experiments. (B) Immunofuorescencewas perormed using anti-pIRE1 and anti-E3 anti-body. Bars, 15 µm. (C) The number o cells positive or

pIRE1 in the E3+ (inected cells) and E3 (uninectedcells) populations is depicted. Data shown representmean ± SEM or triplicate samples o >100 cells per

experimental condition. Similar results were observedin three independent experiments. (D and E) WT MEFs

were pretreated with control siRNA or siRNA againstIRE1 or 3 d ollowed by inection with CHIKV or24 h at MOI 1. The number o LC3 punctas per cell

and the amount o LC3-II are depicted. Data shown

represent mean ± SEM or triplicate samples o >100cells per experimental condition. Similar results were

observed in three independent experiments. (F and G)WT MEFs were incubated or 24 h in control media (ø)

or in the presence o CHIKV (MOI = 1). (F) Immuno-fuorescence was perormed using an ROS/RNS de-tection kit that specically stains oxygen species and

ree NO. As positive controls or ROS or RNS induc-tion, WT MEFs were incubated or 5 h with pyco-

cyanin and l-arginine, respectively. Bars, 10 µm.(G) Percentage o cells containing ROS or NO amonginected with CHIKV and/or pretreated with specic

inhibitor o ROS and RNS as indicated is depicted.Data shown represent mean ± SEM or triplicate

samples o >100 cells per experimental condition.Similar results were observed in two independentexperiments. (H) WT MEFs or cells pretreated with

siRNA against IRE1 or 3 d were inected with CHIKV or 24 h in presence o a ROS inhibitor. The number o LC3 punctas per cell and the amount o LC3-II are

depicted. Data shown represent mean ± SEM ortriplicate samples o >100 cells per experimentalcondition. Similar results were observed in three inde-

pendent experiments. (I) WT MEFs were inected withCHIKV at indicated time points and Western blotting

was perormed to detect phosphorylation o mTOR(p-mTOR), S6KI (p-S6K1), and AMPK (p-AMPK). mTOR,S6K1, AMPK, and GAPDH were also ollowed to con-

trol protein expression and loading. As control to ROS

implication, similar experiments were perormed incells pretreated with ROS inducer and/or ROS inhibi-

tor. Black lines indicate that intervening lanes werespliced out. Similar results were observed in three

independent experiments. Student’s test: **, P < 0.05.


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RNS scavenger cross-inhibited ROS production, highighting

the interconnectivity between the ROS and RNS pathways.

This phenomenon coud be expained by NO reacting with

O2 to orm the oxidant peroxynitrite (ONOO-; Djavaheri-

Mergny et a., 2007; Novo and Paroa, 2008).

To conrm the impication o ROS/RNS production in

CHIKV-induced autophagy, we investigated LC3+ staining and

the conversion o LC3-I to LC3-II in inected MEFs pretreated

with N -acety-l-cysteine (Fig. 3 H). A signicant decrease in

LC3 puncta was observed, demonstrating the importance o

ROS production in CHIKV-induced autophagy. We urther

evauated potentia overap with the ER stress pathway by assess-

ing autophagy in ces sienced or IRE1 mRNA and treated

with N -acety-l-cysteine. Strikingy, we observed an additive

inhibitory eect that reduced the number o autophagosomes

per ce to near baseine eves (Fig. 3 H). This data suggests that

ER stress and oxidative stress act via independent mechanisms to

induce autophagy during CHIKV inection.

Athough oxidative stress is known to induce autophagy

upon microbia inection, the precise mechanism remains

poory documented. Based on recenty estabished inks be-

tween ROS and mTORC1 inhibition, which appears to be

dependent on TSC2 (tuberous scerosis compex 2), itse regu-

ated by the AMP-activated protein kinase (AMPK; Aexander

et a., 2010a,b), we investigate the reguation o this compex

during CHIKV inection. Importanty, phosphoryated mTOR

can be integrated into two dierent compexes, caed mTORC1

and mTORC2, depending on its interactions with Raptor and

Rictor, respectivey (Zoncu et a., 2011). Athough both com-

pexes are impicated in protein synthesis, ony mTORC1 is

inked to autophagy (Thomson et a., 2009). To discriminate

the activation o mTORC1 rom mTORC2, we anayzed

both mTOR phosphoryation and the induction o p-S6K1, aspecic substrate o mTORC1. As shown, we observed a

diminished eve o both p-mTOR and p-S6K1 24 h ater in-

ection (Fig. 3 I). This inhibition was transient, and 2–3 d ater

inection, a strong induction o the mTOR–S6K1 pathway

coud be detected. The kinetics o mTOR–S6K1 inhibition

correated with conversion o LC3-I to LC3-II, suggesting a

roe or mTORC1 as a mediator o CHIKV-induced auto-

phagy. To dene how CHIKV-induced ROS is capabe o in-

hibiting mTORC1, we next evauated the activation o AMPK.

Strikingy, the active orm o AMPK was detected 24 h ater

inection, coincident with the inhibition o mTORC1 (Fig. 3 I).

Moreover, the impication o the AMPK pathway in ROS-

mediated inhibition o mTORC1 coud be conrmed usingN -acety-l-cysteine (Fig. 3 I). Together, these data provide a

mechanistic understanding o CHIKV-induced autophagy.

Autophagy is a prosurvival mechanismthat limits CHIKV-induced cell deathIn addition to autophagy, other orms o ce stress may be

triggered as a resut o vira inection, incuding activation o

ce death pathways. Increasing evidence suggests that ce

stress pathways intersect and in some instances cross-inhibit

each other (Thorburn, 2008). As CHIKV inection triggers a

dierent time points ater inection. Western botting and

immunouorescence indicated that p-IRE1 was observed

during the eary phase o inection (Fig. 3, A and B) and coud

be detected ony in inected ces (as evauated based on E3

coocaization; Fig. 3, B and C). These data suggested that

CHIKV eads to an intrinsic activation o ER stress. Impor-

tanty, p-IRE1 was no onger detected 3 d ater inection.

This shutdown seems to be the resut o a decreased eve o

IRE1 protein, as indicated by Western bot anaysis (Fig. 3 A).

Remarkaby, the kinetics o IRE1 phosphoryation correated

with the conversion o LC3-I to LC3-II (Fig. 3 A).

To dene the moecuar events triggered by p-IRE1, we

investigated the activation o XBP1 and c-Jun amino-termina

kinases (JNK; McGuckin et a., 2010). Activation o XBP1 is

reguated by a dierentia spice variant o XBP1 mRNA,

which may be evauated based on the expression o a protein

o higher moecuar weight and is reerred to as XBP1s (or

spiced XBP1; Yoshida et a., 2001). This pathway has been

shown to avor ce surviva (McGuckin et a., 2010). In con-

trast, IRE1-induced phosphoryation o JNK is considered a

ink between ce stress and apoptosis (Urano et a., 2000).

During CHIKV inection, we observed an induced expres-

sion o XBP1s, but did not detect enhanced phosphoryation

o JNK (p-JNK; Fig. 3 A). To examine the unctiona ink

between IRE1 in CHIKV-induced autophagy, we sienced

expression o IRE1 using siRNA and anayzed CHIKV-

induced LC3 puncta as we as LC3-II conversion (Fig. 3,

D and E). Reduced IRE1 gene expression was conrmed by

Western bot, and shown to resut in ewer CHIKV-induced

autophagosomes. These data dene a roe or CHIKV activa-

tion o ER stress, which induces autophagy via an IRE1-

and XBP1s-mediated signaing pathway.

CHIKV-induced oxidative stress avors autophagosomeproduction through the inhibition o mTORC1Oxidative stress, primariy caused by increased eves o reac-

tive oxygen species (ROS) and reactive nitrogen species

(RNS), is a eature o the host response to vira inections

(Catadi, 2010). O2 and NO are considered to be the most

important mediators among ROS and RNS, respectivey. Free

oxidative agents are known to induce autophagy and can aso

ead to ce death during strong and proonged stimuation

(Djavaheri-Mergny et a., 2007; Fiomeni et a., 2010; Guo

et a., 2010). To assess the impact o ROS/RNS production in

CHIKV-induced autophagy, we rst investigated whether

CHIKV inection induces ROS and/or RNS production. Weinected WT MEF or 24 h and monitored the presence o

oxygen species and ree NO. As positive contro, we used pyco-

cyanin and l-arginine, inducers o ROS and NO, respectivey

(Fig. 3 F). As expected, pycocyanin increased the percentage o

ces that produced ROS, in the absence o NO production,

whereas l-arginine induced ree NO but not ROS. Interest-

ingy, we observed that CHIKV inection ed to increased pro-

duction o both ROS and NO (Fig. 3 F), which coud be

inhibited using the ROS inhibitor N -acety-l-cysteine or the

RNS scavenger c-PTIO (Fig. 3 G). O note, exposure to a


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was most prominent at ow mutipicity o inection

(MOI), suggesting a ink between ce death and

vira propagation throughout the cutured MEFs.

Simiar resuts were obtained by siencing expression

o autophagy genes in WT MEFs (Fig. 4, E and G).

To expore aternative ce death pathways, autoph-

agy genes were sienced in the Bax/ Bak/ MEFs.

Importanty, the absence o both autophagy andapoptosis pathways did not urther sensitize the ces

to aternative orms o ce death (Fig. 4 F). Finay,

we tested the importance o vira repication or ce

death induction. WT MEFs were inected with ive

or UVB-inactivated CHIKV, and ce death was

evauated. As shown, repication deective CHIKV

ais to induce ce death (Fig. 4 H).

To dene the orm o programmed ce death re-

sponsibe or the CPE in WT and Atg5 / MEFs, we

used pharmacoogica inhibitors o apoptosis (z-VAD,

a broad spectrum caspase inhibitor) or necroptosis

(necrostatin-1, an inhibitor o RIPK1). Inected ces

treated with necrostatin-1 exhibited a simiar eve o ce deathas compared with inected contro ces, whereas z-VAD rescued

both the WT and Atg5 / MEFs rom CHIKV-induced CPE.

(Fig. 4, I and J). These data, as we as the absence o CPE in

Bax/ Bak/ MEFs, demonstrate that the principe orm o

ce death induced by CHIKV is caspase-mediated apoptosis.

Autophagy delays both intrinsicand extrinsic apoptosis pathwaysTo dene the interaction between autophagy and apoptosis at

the singe ce eve, WT and Atg5 / or Bax/ Bak/ ces

Figure 4. Enhanced CHIKV-induced cell death in theabsence o autophagy. (A–D) WT, Bax / Bak / , or Atg5 / MEFs were inected with CHIKV at indicated doses and timepoints. Percentage o cell death was measured using a mem-

brane-permeable fuorescent probe and assessed by cytometry.Death curves over the 3 d ater inection are shown or a rep-

resentative experiment (A and C). The induction o cell deathwas also evaluated as a unction o the MOI (B and D). In allconditions ≥10,000 cells were acquired. Similar results were

observed in ve independent experiments. (E and F) WT orBax / Bak / MEFs were pretreated with the indicated siRNAor 3 d, ollowed by inection with CHIKV or 24 h at the indi-

cated doses. Cells death is analyzed as described or A–D.Similar results were observed in three independent experi-ments. (G) WT MEFs were treated with si-Atg5 or si-Atg7, and

Western blotting was perormed using anti-Atg5 or anti-Atg7antibodies. Similar results were observed in two independent

experiments. (H) WT MEFs were inected with CHIKV or UVB-treated CHIKV at indicated doses. Percentage o cell death wasmeasured using a membrane permeable fuorescent probe and

assessed by cytometry. Similar results were observed in two

independent experiments. (I and J) WT or Atg5 / MEFs werepretreated with z-VAD or necrostatin-1 (Nec-1) beore inec-tion by CHIKV. Percentage o cell death is depicted. Error barsindicate mean values ± SD rom three independent experi-

ments. Student’s test: **, P < 0.05.

pronounced CPE (Sourisseau et a., 2007), it was important toinvestigate the unction o autophagy on CHIKV-induced

ce death. Atg5 / MEFs and ces unabe to engage the

intrinsic apoptosis pathway (Bax/ Bak/ MEFs) were

inected with CHIKV and oss o membrane integrity was

anayzed. Whereas CHIKV inection triggered ce death in

WT ces in a time- and dose-dependant manner, Bax/

Bak/ MEFs remained reractory, showing ony minima ev-

idence o CPE at day 3 (Fig. 4, A and B). In contrast, Atg5 /

MEFs showed a dramatic increase in ce death compared with

its WT contro (Fig. 4, C and D). Notaby, the enhanced CPE


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Data are represented in Fig. 5 A with each dot indicating a

singe ce. Regions were estabished as detaied previousy

(de a Cae et a., 2011), and the percentage o autophagic

ces (LC3 BDIhi, ceaved caspase-3o; dened by R1), apoptotic

were inected with CHIKV. As previousy, LC3 BDI was

used as a measure or autophagy induction, and apoptosis ac-

tivity was characterized by abeing with antibodies specic or

the active, ceaved orm o caspase-3 (de a Cae et a., 2011).

Figure 5. Autophagy limits CHIKV-induced apoptosis. (A–D) WT, Bax / Bak / , or Atg5 / MEFs were inected with CHIKV (MOI = 1) or 24 h and

were stained or LC3 and activated caspase-3. (A) Representative ImageStreamX dot plots rom WT, Bax / Bak / , or Atg5 / inected MEFs are depictedand the gating strategy is indicated. (B–D) The relative percentage o autophagic cells (LC3 BDIhi, cleaved caspase-3lo; dened by R1), apoptotic cells (LC3

BDIlo, cleaved caspase-3hi; dened by R2), or cells with evidence or both processes (LC3 BDIhi, cleaved caspase-3hi; dened by R3) is shown. Error barsindicate mean ± SD o three independent experiments. (E and F) WT, Bax / Bak / , or Atg5 / MEFs were inected with CHIKV (MOI = 1) at the indicated

time points, and activated caspase-3 (a-CASP3), activated caspase-9 (a-CASP9), or activated caspase-8 (a-CASP8) were stained rom parallel cultures.Representative microscopic images are shown (E), and the percentage o positive cells were determined or >100 cells per condition (F). Error bars showmean ± SD o three independent experiments. Bars, 25 µm. (G and H) WT, Bax / Bak / , or Atg5 / MEFs were inected with GFP-expressing recombi-nant CHIKV (MOI = 1) and stained with anti–active capase-3, -8, and -9 antibody. Cells were gated as uninected (R1) and inected (R2) cells according to

GFP intensity (G) while simultaneously assessing the active capase-3, -8, and -9 staining (H). Similar results were observed in three independent experi-ments. (I) HFFs were inected with CHIKV (MOI = 1) or 24 h, and the percentage o activated caspase-3 cells was determined or >100 cells per condition.

Error bars show mean ± SD o three independent experiments.


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uninected ces. These data suggest that CHIKV inection

induces apoptosis through both intra- and extraceuar ac-

tors (Fig. 5 H) and that autophagy, acting in a ce-intrinsic

manner, preerentiay protects inected ces rom apoptosis.

CHIKV-induced apoptosis is independento ER and oxidative stressAs introduced in the previous sections, both ER and oxida-

tive stress may trigger proapoptotic pathways (McGuckin

et a., 2010). To determine the reative contribution o stress

pathway induction on autophagy versus apoptosis, we again

used Atg5 / MEFs. We rst conrmed the antiapoptotic

eect o autophagy by investigating the ceavage o pro–

caspase-3 in WT and Atg5 / MEFs (Fig. 6, A and B).

Whereas pro–capase-3 was ceaved ony ater 48 h o inec-

tion in WT ces, the active orm o caspase-3 was detected

24 h ater inection in Atg5 / MEFs. Moreover, the ratio o

active caspase-3/GAPDH was increased in Atg5 / ce dur-

ing a time points investigated (Fig. 6 B).

We next investigated the activation o IRE1 and inhibi-

tion o mTOR in Atg5 / MEFs. Interestingy, both path-

ways were simiary reguated by CHIKV inection as

compared with WT MEFs (Fig. 6 C compared with Fig. 3,

A and I). To ascertain the impact o these pathways on apop-

tosis, we evauated by immunouorescence the percentage o

active caspase-3–positive ces in WT or Atg5 / MEFs in

which ER and oxidative stress had been inhibited (Fig. 6 D).

IRE1 and/or ROS inhibition increased the percentage o

WT ces with active caspase-3. Importanty, no change in

active caspase-3 expression was observed in Atg5 / MEFs.

These data suggest that ER and oxidative stress occur beore

the roe o Atg5 in aciitating autophagy and are not critica

or the induction o CHIKV-induced apoptosis.Together, the data presented in Figs. 1–6 demonstrate

that during the eary phase o CHIKV inection, autophagy is

induced via the activation o ER stress and the inhibition o

mTOR by ROS production. By triggering autophagy,

CHIKV-induced ce stress indirecty imits apoptotic ce

death. Nevertheess, ater 48 h o inection, ER stress is

bunted, in part secondary to IRE1 degradation, and mTOR

becomes hyperphosphoryated. These events resut in de-

creased autophagic tone and, via a sti undened mechanism,

apoptosis pathways dominate, in turn resuting in pronounced

CHIKV-induced CPE.

Regulation o apoptosis and in vitro CHIKV propagationBoth autophagy and apoptosis have been reated to the regu-

ation o vira repication and/or propagation. Athough au-

tophagy can resut in the degradation o vira proteins (e.g.,

Sindbis) without signicanty aecting vira inection, it has

aso been reported to enhance vira repication (e.g., HCV;

Dreux and Chisari, 2009). Simiary, apoptosis has been im-

picated in both pro- and antivira responses (Li and Stoar,

2004). As both pathways are engaged by CHIKV inection, it

was important to evauate the eect o autophagy and apop-

tosis on vira propagation. To achieve this, we measured vira

ces (LC3 BDIo, ceaved caspase-3hi; dened by R2), or ces

with evidence or both processes (LC3 BDIhi, ceaved caspase-

3hi; dened by R3) were enumerated and represented graphi-

cay (Fig. 5, B–D). Supporting immunouorescence resuts

(Fig. 1), CHIKV inection triggered an increase in LC3

puncta in WT and Bax/ Bak/ MEFs, but not in Atg5 /

MEFs (Fig. 5 B). Strikingy, the number o ces exhibiting

active caspase-3 was increased upon CHIKV inection as

compared with uninected ces, shown at 0 h (Fig. 5 C). The

number o LC3+ ces was dramaticay reduced in WT ces 3

d ater inection. This observation correated with high eve

o apoptosis and suggests that autophagy and apoptosis are

mutuay excusive processes (Fig. 5, compare B with C). This

observation was urther supported by the absence o doube-

positive ces (region R3; Fig. 5 D).

Two important observations emerged rom the study o

the knockout MEFs. The rst intriguing nding concerned a

marked increase in active caspase-3+ ces when Atg5 is absent

(Fig. 5 C). These data suggested an important roe or

autophagy in the reguation o CHIKV-induced apoptosis.

The second discovery concerned the possibiity o the Bax/

Bak/ ces to activate caspase-3; the timing o apoptosis on-

set showed interexperimenta variation with ces showing

detectabe eves o ceaved caspase-3 between 48 and 72 h

(Fig. 5, C, E, and F). Evidence or caspase-3 activation in

Bax/ Bak/ ces indicated activation o the extrinsic

apoptosis pathway, which may be induced independenty o

mitochondria outer membrane permeabiization.

We then investigated whether autophagy is abe to regu-

ate apoptotic ce death in human brobasts ces (HFF),

which are known ce targets o CHIKV inection (Sourisseau

et a., 2007). We down-reguated the expression o Atg5 and

Atg7 genes in HFF by an siRNA strategy and anayzed theactivation o caspase-3 ater 24 h o inection with dierent

vira inputs (Fig. 5 I). Inhibition o both Atg5 and Atg7 dra-

maticay increased the percentage o ceaved caspase-3–positive

ces according to vira doses, demonstrating that the antiapop-

totic unction o autophagy is aso observed in human ces.

To distinguish the roe o autophagy in imiting the dis-

tinct apoptosis pathways, we evauated ceaved caspase-9

(a marker o the intrinsic pathway) and ceaved caspase-8

(indicative o activation o the extrinsic pathway) during

CHIKV inection (Fig. 5, E and F). Eary during the kinetics

o vira inection (16 h), caspase-9 activation was evident in

WT ces in the absence o detectabe eves o ceaved

caspase-8. In comparison, Atg5 / MEFs dispayed a twoodincrease in the percentage o active caspase-9–positive ces, as

we as eary evidence or ceaved caspase-8. By 40 h ater in-

ection, both the intrinsic and extrinsic pathways were engaged

in the WT ces and, again, Atg5 / ces demonstrated higher

eves o activation or both ce death pathways (Fig. 5 F).

Using GFP-expressing CHIKV, we next evauated the

reationship between inection and activation o caspase-3,

-9, and -8 (Fig. 5, G and H). Whereas the intrinsic apoptotic

pathway was engaged primariy in CHIKV+ ces, the extrin-

sic pathway was detectabe in both inected and bystander


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decient ces expressed simiar expression intensity to

CHIKV E3 proteins as compared with its WT contro, indi-

cating that autophagy had minima impact on viray encoded

GFP expression within the ce (Fig. 7 D). These data were

urther conrmed in a secondary cuture–based assay; ce

supernatants rom the respective ce ines were exposed to

uninected WT MEFs and GFP expression was scored ater

an additiona 24-h incubation, indicating higher eves o in-

ectious virus in the absence o autophagic ux and ower

eves in apoptosis-decient ces (unpubished data). To-

gether, these data suggested that autophagy coud reguate

vira propagation by imiting the reease o virus induced by

apoptotic ce death.

To support this observation, we estabished an image

anaysis script using the ImageStreamX, which integrated ce

area (unpubished data), thereby permitting quantication o

ces in the na stages o apoptosis based on their being sma

and pyknotic. Importanty, anaysis o the pyknotic ces indi-

cated that 80% expressed vira capsid protein as compared

with arger, ess dead ces, o which 45% stained positive

or E3 (Fig. 7, E and F), suggesting that ingestion o apoptotic

bodies coud contribute to inection o phagocytic neighbor-

ing ces (Krejbich-Trotot et a., 2011a). Apoptotic body ac-

cumuation upon CHIKV inection was next determined in

WT or Atg5 / MEFs (Fig. 7 G, R1). We aso conrmed

that accumuation o sma area events was dependent on an

apoptotic process, as use o z-VAD eiminated this popuation

oad in the supernatant o WT, Atg5 / , or Bax/ Bak/

MEFs. CHIKV titers seemed enhanced in supernatant o

Atg5 / ces, suggesting that autophagy coud restrict vira

reease (Fig. 7 A). However, modest vira titers were ower in

Bax/ Bak/ MEFs as compared with WT contros at day 1

ater inection, thereby suggesting a roe or apoptotic ce

death in vira reease during eary phase inection (Fig. 7 B).

Simiar resuts were obtained in HeLa ces using siRNA spe-

cic or Atg5 or Atg7 (unpubished data).

To urther anayze the unction o autophagy and apop-

tosis in vira inection, we assessed the percentage o inected

ces ater 24 h o inection by using GFP-expressing recom-

binant CHIKV as a marker o active inection (Fig. 7 C). At

ow vira dose (MOI = 0.1), Atg5 / MEFs showed greater

inection than WT contros. This dierence is a resut o en-

hanced inection and not dierentia vira entry, as suggested

by a kinetic study o inected ces (data not depicted). Resuts

or Bax/ Bak/ MEFs were even more striking as ony a

minority o ces were GFP+ ces, supporting that apoptotic

ce death inuences CHIKV propagation (Fig. 7 C). Cyto-

metric assessment o the Bax/ Bak/ MEFs indicated that

athough ewer ces were inected, those ces that were in-

ected showed higher expression o CHIKV E3 proteins as

compared with its WT contro (Fig. 7 D). This suggested that

a deay in ce death aowed or greater per ce vira repica-

tion, but aiure to undergo rapid ce death resuted in ewer

inected ces at the popuation eve. In contrast, autophagy-

Figure 6. ER and oxidative stress don’t enhance in CHIKV-induced apoptosis. (A and B) WT or Atg5/ MEFs were inected with CHIKV at theindicated time points. (A) Western blotting was perormed using anti-LC3 and anti–caspase-3 (Casp3) to discriminate pro–caspase-3 and cleavedcaspase-3, and GAPDH to control or protein loading. Black lines indicate that intervening lanes were spliced out. (B) Band intensity was calculated using

ImageJ sotware and the ratio o pro–caspase-3/GAPDH and cleaved-caspase-3/GAPDH was represented by graphs. Similar results were observed inthree independent experiments. (C) Atg5/ MEFs were inected with CHIKV at indicated time points and Western blotting was perormed to detectedphosphorylation o IRE1 (p-IRE1) and mTOR (p-mTOR). IRE1, mTOR, and GAPDH were ollowed to control protein expression and loading. Similar

results were observed in two independent experiments. (D) WT and Atg5/ MEFs were pretreated or not with siRNA against IRE1 or 3 d and were theninected by CHIKV or 48 h in control media or in the presence o ROS inhibitor. Activated caspase-3 was stained and the percentage o positive cells was

determined. Error bars show mean ± SD o three independent experiments. Student’s test: **, P < 0.05.


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Figure 7. Regulation o apoptotic cell death limits CHIKV propagation in vitro. (A and B) WT, Bax / Bak / , or Atg5 / MEFs were inected withCHIKV at indicated MOI and extracellular viral titers were determined during the 3 d ater inection. Results were expressed as TCID50/ml. Error bars indi-

cate mean viral titer ± SD o our independent experiments. (C) WT, Bax / Bak / , or Atg5 / MEFs were inected with GFP-expressing recombinantCHIKV (MOI = 0.1). ImageStreamX dot plots based on GFP intensity are shown. Similar results were observed in three independent experiments. (D) WT,

Atg5 / , or Bax / Bak / MEFs were inected at MOI = 1 and viral proteins were stained using anti-E3 Ab and analyzed by cytometry. Similar results were

observed in three independent experiments. (E and F) WT MEFs were inected with GFP-CHIKV (MOI = 0.1) or 48 h and arealo events were gated (R1 in G).


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propagation. In vivo data in Atg16LHM mice support an anti-

apoptotic eect or autophagy and indicate that in the ab-

sence o autophagic genes, there is an increased susceptibiity

to severe orms o Chikungunya disease.The roe or autophagy in host deense has been docu-

mented or bacteria and severa viruses (Deretic and Levine,

2009); however, the resuts rom our current study identiy a

previousy uncharacterized roe or autophagy as a mecha-

nism o imiting disease pathogenesis. Importanty, we dem-

onstrate that autophagy is triggered in a ce-intrinsic manner

by direct CHIKV inection, which eads to the induction o

both ER and oxidative stress (Figs. 1 and 3). Specicay,

among the ER stress pathways that are induced during vira

inection, ony IRE1/XBP1s is invoved in autophagic

DISCUSSIONThe recent CHIKV outbreak has exposed how itte we un-

derstand about the pathogenesis o this virus or the abiity to

harness host responses to enhance the contro o arbovirainection. Immunoogica studies have suggested that eary

events o vira–host ce interactions determine whether

CHIKV achieves disseminated inection, or i repication is

imited and controed by innate deense mechanisms. In our

prior studies, we evauated the roe o pattern recognition re-

ceptors and type I IFNs as mediators o vira contro (Schite

et a., 2010). Here, we examined an aternative stress response

pathway that is activated upon inection, making the exciting

discovery that engagement o the autophagy pathway imits

vira-induced ce death as a mechanism or sowing vira

Figure 8. Autophagy limits apoptotic induction in CHIKV inected tissues and delays lethality o mice. (A–F) WT (n = 43) and Atg16LHM mice(n = 34) were inected at 9 d o age with 4 × 105 PFU CHIKV subcutaneously. (A) Mice were monitored or lethality or 21 d with data displayed asKaplan-Meier curves. (B) Skin, muscle, and serum were collected ater days 1 (n = 3), 2 (n = 3), 5 (n = 4), and 9 (n = 5) o inection, homogenized, and viral

titers were determined by standard plaque assay. Median values or WT (black bars) or Atg16LHM (blue bars) mice are depicted. (C) Muscle, skin, liver, brain,BM, and spleen were collected rom WT inected mice at day 5. Tissues were xed in PFA, rozen in OCT blocks, and stained or cleaved capsase-3 (green)and E3 (red). Similar results were observed in two independent experiments. Bars: (muscle) 50 µm; (other tissues) 20 µm. (D–F) Muscle rom inected WT

or Atg16LHM mice was collected at days 1, 2, and 5 ater inection and stained or cleaved caspase-3, E3, or DAPI or nucleus staining (D). The percentageo cleaved capsase-3–positive cells was numerated in all population (E) or in E3+ and E3 cells (F). Error bars mean ± SD o three independent experi-

ments. Bars, 60 µm. Student’s test: **, P < 0.05.


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pathogens (Deretic and Levine, 2009). Physica degradation

o the microbe or microbia components may be achieved

through their isoation in autophagic vesices that in turn use

with ysosomes, which is reerred to as xenophagy (Orvedah

and Levine, 2008). A seective mechanism that eads to their

capture is reguated, in part, by the poy-ubiquitin binding

adaptor proteins p62 and NDP52. Components o the SinV

are aso sequestered by autophagy in a p62-dependant man-

ner (Orvedah et a., 2010). In vivo, genetic inactivation o

Atg5 in viray inected mouse neurons resuted in deayed

cearance o SinV capsid, the accumuation o p62 aggregates,

increased neurona apoptosis, and rapid mortaity, a in the

apparent absence diering vira oad.

A second mechanism by which autophagy may partici-

pate in the host response to inection reates to the cross tak

between autophagy genes and pattern recognition receptors.

Lee et a. (2007) demonstrated in a semina study that

autophagy sequesters cytosoic vira genome and/or host

RNA during VSV inection. This aowed igand to access

the umina domain o TLR7 in pDCs and enhanced produc-

tion o IFN-/ and other proinammatory cytokines. A-

though this pathway is unikey to contribute to the contro

o CHIKV during inection o brobast or epitheia ces, as

a resut o their ack o TLR7 expression, it is possibe that

autophagy my impact RIG-I signaing, athough as discussed

previousy, the intersection between Atg5 and RIG-I resuts

in a bunting o the antivira response (Jounai et a., 2007).

However, we did not observe atered IFN- production in

ces acking Atg5 or atered IFN- induction in mice hypo-

morphic or Atg16L (unpubished data), suggesting that this

mechanism o reguation did not account or autophagy-

mediated suppression o CHIKV repication in brobasts

and epitheia ces.Our study provides mechanistic inormation or another

strategy by which autophagy participates in the host response

to imit ce mortaity ater vira inection, thus controing

the reease o inectious vira partices secondary to apoptotic

ce death. We demonstrate that autophagy promotes ce sur-

viva by imiting apoptotic ce death. Importanty, autophagy

induction is dependent on CHIKV repication; consequenty,

the deay in apoptosis occurs in a ce-intrinsic manner. In

other words, neighboring uninected ces remain sensitive

to putative extraceuar inducers o the extrinsic pathway

(Fig. 5 H). The mechanism accounting or autophagy induc-

tion is inked to the activation o host stress responses during

acute inection, arguing that autophagy is induced by ces toreguate these stress responses. Regarding the oxidative stress,

autophagy has been described to restrict ROS production,

or exampe by eiminating damaged mitochondria. Indeed,

this is occurring in the context o CHIKV inection, as indi-

cated by their being higher eves o ROS production in

inected Atg5 / ces (unpubished data). Nonetheess, au-

tophagy did not succeed to competey contro the high eves

o oxidative stress induced by vira inection and repication.

For both ER and oxidative stress, it has been suggested

that their induction may resut in autophagy and apoptosis,

reguation (Fig. 3, A–D; and not depicted). Oxygen species

produced during CHIKV inection are abe to independenty

enhance autophagy via the inhibition o mTORC1 (Fig. 3,

E–I). The autophagy pathway induced is dependent on

Becin-1 and proceeds unimpeded, with utimate usion be-

tween autophagosomes and degradative ysosomes (Figs. 1 G

and 2). Using nove, singe ce assays that permitted simuta-

neous assessment o autophagy and apoptosis, we were abe

to demonstrate that at a time points studied, the two bio-

ogica processes are mutuay excusive (Fig. 5, A–D). That

said, kinetic studies, as we as assessment o vira protein ex-

pression in dying ces, suggest that as vira inection pro-

gresses, the autophagy pathway is overwhemed and ces

began to undergo apoptosis (Fig. 5).

Role o apoptosis in acute viral inectionApoptosis ater vira inection is considered to be an important

mechanism o host deense, as autodestruction may imit vira

repication (Everett and McFadden, 1999). RNA viruses re-

sponsibe or acute inection may have a distinct agenda, and

growing evidence suggests that a subset o agents may have

evoved to activey trigger programmed ce death (Yatim and

Abert, 2011). Members o the aphavirus amiy have been

shown to activey induce programmed ce death. For exam-

pe, Sindbis virus (SinV) may trigger both intrinsic and extrin-

sic apoptosis (Li and Stoar, 2004). Athough transection o

SinV nsp2 or the E1, pE2, and E3 structura proteins may

trigger ce death, the precise mechanism remains unknown

(Zhang et a., 1997; Li and Stoar, 2004). Mathematica mod-

es argued that a virus wi evove toward increasing cytopath-

icity when the mean ietime o a ce is high and the rate o

vira budding is ow (Krakauer and Payne, 1997). Indeed,

CHIKV inects primariy brobasts and other ong-ived stro-ma ces (Powers and Logue, 2007). In accordance with this

prediction, it was recenty demonstrated by Krejbich-Trotot

et a. (2011a) that apoptotic ce death is required or efcient

CHIKV propagation. Our ndings support this observation,

as mutispectra anaysis o CHIKV-inected ces indicated the

eary engagement o the intrinsic ce death pathway (Fig. 5 F).

Simiar to SinV, CHIKV inection aso resuted in caspase-8

activation (Fig. 5 F), suggesting a possibe roe or TNFSF re-

ceptor engagement (e.g., TRAIL-R). Interestingy, in con-

trast to caspase-9 activation, there was evidence or uninected

ces with active caspase-8 (Fig. 5 H).

Using z-VAD or gene siencing o caspase-3, we urther

showed that inhibition o apoptosis signicanty inhibitedCHIKV propagation (Fig. 7, B, C, and I; and not depicted).

Simiar resuts were observed in ces decient or Bax/

Bak/ , abeit with a ess dramatic eect as a resut o the act

that the extrinsic pathway remains intact in these ces (Fig. 7).

Thus, we suggest that both the intrinsic and extrinsic path-

ways participate in enhancing CHIKV propagation.

Autophagy as an antiviral mechanismThe autophagy pathway and/or autophagy proteins have been

recenty shown to participate in the host response to intraceuar


7/31/2019 J Exp Med-2012-Joubert-1029-47



or deaying apoptosis and protecting the host rom acute

aphavira inections. Integration o these data in the context

o ER and oxidative stress may revea nove mechanisms by

which viruses moduate host pathways. Our data characteriz-

ing ate phases o CHIKV inection (e.g., 48–72 h) identied

mTOR overexpression and IRE1 degradation as possibe

cues or how these stress pathways may be perturbed.

In sum, we have provided evidence or a nove mecha-

nism by which autophagy participates in host deense. In the

context o acute inection by CHIKV, apoptotic ce death is

important or vira propagation, and autophagy, in a ce-

intrinsic manner, protects ces rom apoptosis and may imit

vira reease. These data suggest that inducers o autophagy

may be useu therapeutics or imiting the pathogenesis o

acute Chikungunya disease or protecting neonates rom

mother-to-chid transmission.

MATERIALS AND METHODS

Cells and mice. WT and Bax/ Bak/ MEF ces rom the Korsmeyer

aboratory (Dana-Farber Cancer Institute, Boston, MA) were derived romC57BL/6 mice (H-2b). Atg5 / ces rom the Kroemer Laboratory

(NSERM U848, Institut Gustave Roussy, Viejui, France) were derived

rom C57BL/6 mice. GFP-LC3-HeLa ces were obtained ater transection

with peGFP-LC3 pasmid (peGFP-LC3, Addgene; rom the Yoshimori

aboratory, Okazaki, Japan) using Lipoectamine 2000 reagent (Invitrogen).

A stabe GFP-LC3-HeLa cone was estabished by imited diution in

0.5 mM geneticin-seective medium. A ce ines were maintained at 37°C

in humidied atmosphere containing 5% CO2 in medium suppemented

with 10% heat inactivated eta ca serum, 100 µg/m peniciin (Invitrogen),

100 U/m streptomycin (Invitrogen), and 1× MEM nonessentia amino acid

(Invitrogen). HFFs were obtained rom American Type Cuture Coection.

WT mice were obtained rom Chares River or The Jackson Laboratory.

Atg16LHM mice on the C57BL/6 background, uy backcrossed by speed

congenics, were generated by H. Virgin (Washington University Schoo o

Medicine, St. Louis, MO) and have been previousy described (Cadwe

et a., 2008). These mice were received into an enhanced barrier aciity with

speciaized eatures as previousy described (Cadwe et a., 2010). Mouse

studies at the Institut Pasteur and at Washington University were perormed

in strict accordance with the recommendations in the Guide or the Care

and Use o Laboratory Animas o the Nationa Institutes o Heath and

according to the Internationa Guiding Principes or Biomedica Research

Invoving Animas. The protocos were approved by the Anima Studies

Committee at Washington University (#20090287) and the Institutiona

Committees on Anima Weare o the Institut Pasteur (OLAW assurance

# A5476-01). A eorts were made to minimize suering.

siRNA treatment. SMARTpoo siRNA targeting Atg5 , Atg7 , or caspase-3

and contro siRNA were obtained rom Thermo Fisher Scientic. 0.106 HeLa

or MEF ces were cutured in 6-we pates or 1 d in OptiMEM (Invitrogen)

containing 10% FCS and transected with 30 nM o indicated siRNA using

Lipoectamine RNAiMAX (Invitrogen). In some experiments, Atg5 or Atg7 cDNA was transected ater 2 d o siRNA treatment. For a experiments,

CHIKV inection was perormed ater 3 d o siRNA incubation.

CHIKV inection and UV inactivation. The preparation o CHIKV

rom cinica sampes has been previousy described (Schuenecker et a.,

2006). CHIKV-21 strain was propagated in C6/36 ces and supernatants

were harvested and rozen at 80°C beore titration and urther use. MEF or

HeLa ces (pate at50% conuence in 24- or 48-wes pates) were exposed

to the indicated viruses or 1–2 h at 37°C, extensivey washed with PBS, and

cutivated or various periods o time beore urther anaysis. The MOI was

dened as the amount o CHIKV inectious units (cacuated on BHK ces as

PFU) per target ce. 3CHIKV-GFP was generated using a u-ength inection

depending on the signa intensity or duration o the stress in-

ducer. In the case o IRE1, we demonstrate that during the

eary phase o CHIKV inection, IRE1 avors XBP1 mRNA

spicing and autophagy, yet does not ead to proapoptotic

JNK phosphoryation. Future studies wi be required to as-

certain i this signaing event is dictated by viray encoded

proteins or a reection o the concentration o unoded pro-

teins during CHIKV repication. It wi aso be vauabe to

estabish a better understanding o how autophagy is termi-

nated, with an absoute switch to an apoptotic phenotype as

suggested by our singe ce anaysis (Fig. 5 A).

Cross talk between autophagy and apoptosisOur study provides an interesting ink between autophagy,

apoptosis, and vira propagation, aso highighting the active

investigation into cross tak between ce stress and ce death.

Severa mechanisms may account or autophagy imiting

apoptotic ce death. For exampe, degradation o protein ag-

gregates and damaged mitochondria that resut rom vira in-

ection may deay induction o apoptosis (Kroemer et a.,

2010). Additionay, it has been suggested that reease o bc-2

and FLIP rom Atg3 protein compexes may activey inhibit

the intrinsic and extrinsic pathways o apoptosis (Thorburn,

2008; Kroemer et a., 2010). Conversey, apoptosis may in-

hibit autophagy as capain has been shown to ceave Atg5 and

caspase-3 ceaves Atg4D, an enzyme impicated in the deipi-

dation o Atg protein (Kang et a., 2011). Many moecuar

detais must sti be dened, yet the avaiabe resuts have ed

to the concept that autophagy and apoptosis are antagonistic

events that are cross inhibitory. That said, prior studies evau-

ating these antagonistic mechanisms used buk assays such as

Western botting and thus coud not discern activation o

both pathways in a singe ce.One o the unique aspects presented here is the use o mu-

tispectra images cytometer, which permitted unbiased me-

dium throughput and simutaneous assessment o vira protein

expression, LC3 puncta, and caspase activation at the singe

ce eve. Consistent with the prevaiing dogma, but dierent

rom other vira inections such as inuenza A (de a Cae

et a., 2011), we demonstrate the absence o ces showing

eatures o both autophagy and apoptosis. Even in situations

where both pathways are active in the buk popuation o

ces, the two pathways were mutuay excusive. This resut

suggests that vira inection may trigger either autophagy or

apoptosis and that the moecuar decision is a proxima event.

One obvious candidate is Becin-1 (Atg6), which interactswith bc-2, reguating both pathways. In resting ces, Becin-

1–bc-2 compexes imit autophagy without atering the anti-

apoptotic eects o bc-2. Nutrient starvation, ER stress, or

oxidative damage may disrupt these interactions, avoring the

association o Becin-1 with PI3K-III and the induction o

autophagy (Kang et a., 2011). I, however, apoptosis pre-

cedes autophagy, caspase ceavage o Becin-1 wi prevent its

induction (Kang et a., 2011). Interestingy, Becin-1 has

been shown to imit SinV propagation (Liang et a., 1998),

suggesting a more genera roe or autophagy as a mechanism


7/31/2019 J Exp Med-2012-Joubert-1029-47


JEM Vol. 209, No. 5

Article

1045

with Cytox/Cytoperm beore abeing with anti-capsid (rom the Schwartz

aboratory). To detect inected ces, MEFs were inected with CHIKV-GFP

and GFP intensity was anayzed according to ImageStreamX procedure.

In vivo inection, titration, and staining. WT ittermate contros were

used or a experiments (C57BL/6). 9-d-od C57BL/6 WT (n = 43) and

Atg16LHM mice (n = 34) were inected with 4 × 105 PFU CHIKV s.c. in the

upper chest and were oowed or ethaity or 25 d ater inection. Litters

were weight matched at the start o the experiments. For vira titration

(n = 3), tissues were coected, homogenized, and vira sampes were titrated

by standard paque assay on BHK ces. For histoogy bocks, skin (n = 3) and

musce (n = 3) rom the site o inection was xed in PFA and rozen in OCT

bocks. Tissues were sectioned and stained or activated caspase-3 and CHIKV

E3 protein and image anaysis was perormed as previousy described.

The authors would like to acknowledge Herbert Virgin III or providing Atg16LHM

mice, and the Centre d’Immunologie Humaine at Institut Pasteur or support

o experimental study. We also thank F. Rey, M. Lecuit, and members o the

Immunobiologie des Cellules Dendritiques laboratory or their helpul advice and

review o the paper.

This work was supported by the Institut Pasteur, Institut National de la Santé et

de la Recherche Médicale, Agence Nationale de Recherches sur le Sida et les hépatites

virales, the European Research Council, and the French National Research Agency (ANR).None o the authors have competing nancial interests.

Submitted: 17 May 2011

Accepted: 19 March 2012

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