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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.
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.
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.
1034 Autophagy limits CHIKV-induced apoptosis | Joubert et al.
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.
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
1036 Autophagy limits CHIKV-induced apoptosis | Joubert et al.
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
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.
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.
1040 Autophagy limits CHIKV-induced apoptosis | Joubert et al.
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).
1042 Autophagy limits CHIKV-induced apoptosis | Joubert et al.
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-