The Atg5–Atg12 conjugate associates with innate
antiviral immune responses
Nao Jounai*, Fumihiko Takeshita*†, Kouji Kobiyama*, Asako Sawano‡, Atsushi Miyawaki‡, Ke-Qin Xin*, Ken J. Ishii§¶,
Taro Kawai§储, Shizuo Akira§储, Koichi Suzuki**, and Kenji Okuda**Department of Molecular Biodefense Research, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan; ‡Laboratory for Cell
Function Dynamics, Advanced Technology Development Group, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), Saitama
351-0198, Japan; §Exploratory Research for Advanced Technology, Akira Innate Immunity Program, Japan Science and Technology Agency, Osaka 565-0871,
Japan; Departments of ¶Molecular Protozoology and 储Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; and**Department of Bioregulation, Leprosy Research Center, National Institute of Infectious Diseases, Tokyo 189-0002, Japan
Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved July 11, 2007 (received for review May 1, 2007)

Autophagy is an essential process for physiological homeostasis, but
its role in viral infection is only beginning to be elucidated. We show
here that the Atg5–Atg12 conjugate, a key regulator of the autophagic process, plays an important role in innate antiviral immune
responses. Atg5-deficient mouse embryonic fibroblasts (MEFs) wereresistant to vesicular stomatitis virus replication, which was largely
due to hyperproduction of type I interferons in response to immunostimulatory RNA (isRNA), such as virus-derived, double-stranded,or 5ⴕ-phosphorylated RNA. Similar hyperresponse to isRNA was also
observed in Atg7-deficient MEFs, in which Atg5–Atg12 conjugation is
impaired. Overexpression of Atg5 or Atg12 resulted in Atg5–Atg12
conjugate formation and suppression of isRNA-mediated signaling.
Molecular interaction studies indicated that the Atg5–Atg12 conjugate negatively regulates the type I IFN production pathway by direct
association with the retinoic acid-inducible gene I (RIG-I) and IFN-␤
promoter stimulator 1 (IPS-1) through the caspase recruitment domains (CARDs). Thus, in contrast to its role in promoting the bactericidal process, a component of the autophagic machinery appears toblock innate antiviral immune responses, thereby contributing to RNA
virus replication in host cells.
innate immunity 兩 signal transduction 兩 type I interferonAutophagy mediates a nonspecific bulk degradation/recycling
pathway essential for turnover of stable macromolecules, themaintenance of an amino acid pool upon starvation, and the cellularresponse to a variety of hormonal stimuli (1). In addition to its
physiological functions, this pathway plays crucial roles in theelimination of certain intracellular bacteria, such as invading group
A Streptococcus, Mycobacterium tuberculosis, and Shigella flexneri(2–4). However, some bacteria have evolved and adapted themselves to this bactericidal process for survival or replication within
the host’s cells. The autophagic process also seems to be engaged
during the host’s antiviral responses against herpesvirus infection
and replication of plant tobacco mosaic virus and Sindbis virus,
while having no effect on the replication of drosophia picornavirus
and vaccinia virus (1, 5–8). In contrast, components of the autophagic machinery seem to have been subverted to promote replication of RNA viruses, such as coronavirus [mouse hepatitis virus(MHV)], poliovirus, and rhinoviruses 2 and 14, by serving as themembrane scaffold for RNA replication (9, 10). Infection withRNA viruses induces the generation of double-membraned cytoplasmic vesicles, in which the viral RNA replication complex
accumulates and initiates replication of the viral genome. Several
studies have shown that autophagy-related Atg family members,
including LC3, Atg5, and Atg12, colocalize with such vesicles andviral replication complexes and that MHV growth is decreased in
autophagy-deficient cells, suggesting that autophagosome-like vesicles support RNA virus replication (10).
Accumulating evidence indicates that host’s innate immune
system has several sensors specific for RNA virus infection. During
RNA virus replication, double-stranded or 5⬘-phosphorylated immunostimulatory RNA (isRNA) is generated and triggers the host14050 –14055 兩 PNAS 兩 August 28, 2007 兩 vol. 104 兩 no. 35

innate antiviral immune signaling, leading to type I IFN production(11–14). Such virus-derived isRNA is directly recognized by
DExD/H box RNA helicases containing caspase recruitment domains (CARDs), i.e., retinoic acid-inducible gene I (RIG-I) andmelanoma differentiation associated gene 5 (MDA5) in a variety ofcell types (15, 16). Although these RNA helicases discriminate
among different classes of virus-derived RNA structure, both
sensors associate with a crucial adaptor molecule, IFN-␤ promoter
stimulator 1 [IPS-1, also known as mitochondrial antiviral signaling(MAVS), virus-induced signaling adaptor (VISA), or Cardif],through CARD–CARD homotypic interactions (17–20). This association facilitates TRAF family member-associated NF-␬B activator (TANK)-binding kinase 1 (TBK1)- and inducible I␬B kinase(IKKi)-mediated phosphorylation of IFN regulatory factor (IRF) 3
and 7, resulting in type I IFN gene activation (17, 21). This pathway
is indispensable in fibroblasts, because ablation of the IPS-1 gene
results in complete loss of isRNA-mediated type I IFN production(22, 23). In contrast, TLR7- or TLR8-mediated recognition of viral
RNA plays a pivotal role in type I IFN production from plasmacytoid dendritic cells (PDCs), and it was recently shown that the
autophagic process mediates such TLR7 recognition of viral RNA,
particularly in PDCs (24).
To understand the involvement of the autophagic machinery inviral replication mechanisms, we examined the association between
Atg family members regulating the autophagic process and thesignaling molecules involved in innate immune responses. By using
genetic, biochemical, and cell-imaging analysis, we show that the
Atg5–Atg12 conjugate interacts directly with the IPS-1 and RIG-I
through the CARDs. This molecular association results in theinhibition of type I IFN production and permits viral replication
within the cells. Thus, the autophagic machinery does not seem tohave a destructive role during RNA virus infection, but instead
contributes to RNA virus replication by inhibition of the hostantiviral responses.

Author contributions: N.J. and F.T. contributed equally to this work; N.J., F.T., A.M., T.K.,
S.A., and K.O. designed research; N.J., F.T., K.K., A.S., and K.J.I. performed research; N.J.,
K.K., A.S., and K.J.I. analyzed data; and N.J., F.T., K.-Q.X., and K.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: CARD, caspase recruitment domain; CFP, cyan fluorescent protein; HEK,
human embryonic kidney cells; isRNA, immunostimulatory RNA; IPS-1, interferon-␤ promoter stimulator 1; KO, knockout; MAVS, mitochondrial antiviral signaling; MDA5, melanoma differentiation associated gene 5; MEF, mouse embryonic fibroblast; moi, multiplicity of infection; PRD, proline rich domain; RIG-I, retinoic acid-inducible gene I; TMD,
transmembrane domain; VSV, vesicular stomatitis virus; YFP, yellow fluorescent protein.
†To whom correspondence should be addressed at: Department of Molecular Biodefense

Research, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawaku, Yokohama 236-0004, Japan. E-mail: takesita@yokohama-cu.ac.jp.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0704014104/DC1.
© 2007 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0704014104

Atg5 KO MEFs. As shown in Fig. 1C, the levels of viral titer in theculture supernatants were significantly lower in Atg5 KO MEFs
compared with those in WT MEFs, suggesting that the autophagicmachinery, at least Atg5, regulates the VSV replication. The
cytopathic effect of VSV was also examined by neutral red staining,
and the result showed that the number of plaque formation was
lower in Atg5 KO MEFs compared with WT MEFs [supporting
information (SI) Fig. 6]. As a control, the efficiency of humanherpes simplex virus (HSV) replication in Atg5 KO MEFs wascomparable with that in WT MEFs (Fig. 1C). Interestingly, although the type I IFN and IFN-stimulated gene (ISG) production
was elicited by VSV infection, the levels of IFN-␤ and IP-10 mRNA
were significantly higher in Atg5 KO MEFs relative to WT MEFs
(Fig. 1D). In contrast, the induction of these mRNA in response to
HSV infection was impaired in Atg5 KO MEFs (Fig. 1D). We also
examined the activation of the master transcription regulator of thetype I IFN gene, IRF-3, after viral infection. Although the nuclear
transition of phosphorylated IRF-3 was induced by VSV infection,
the levels of such transition were higher in Atg5 KO MEFs
compared with WT MEFs (Fig. 1E). Neutralization of IFN-␣ andIFN-␤ reversed the VSV replication efficiency and the differencein viral replication between WT and Atg5 KO MEFs was abolished(Fig. 1F). These results suggest that Atg5-mediated autophagicmachinery regulates VSV replication by repressing type I IFN
production.

Fig. 1. VSV facilitates autophagy for efficient replication. (A) WT or Atg5 KO
MEFs were transfected with the LC3-GFP expression plasmid and were infectedwith or without VSV at moi ⫽ 1.0, and the LC3 signal was analyzed 8 h after
infection by fluorescent deconvolution microscopy. (Scale bar, 10 ␮m.) (B) WT or
Atg5 KO MEFs were infected with VSV at moi ⫽ 1.0, and cell lysates were preparedat 0, 4, or 8 h after infection. Samples were analyzed by immunoblotting tocompare the levels of two different forms of LC3 [LC3-I (18 kDa) and LC3-II (16kDa)]. (C) WT (open bars) or Atg5 KO (filled bars) MEFs were infected with VSV or
HSV at moi ⫽ 0.1 or 1.0. Twenty-four hours after infection, the cells and culture
supernatants were recovered, and the levels of viral titer were examined by theplaque assay. The graph shows the mean ⫾ SD. *, P ⬍ 0.05 by Student’s t test. (Dand E) WT (open bars) or Atg5 KO MEFs (filled bars) were infected with VSV or HSV
at moi ⫽ 1.0. (D) Total RNA was isolated 0, 4, and 8 h after stimulation, mRNA wasreverse-transcribed, and then the levels of IFN-␣4, IFN-␤, or IP-10 cDNA were
quantified by real-time PCR. (E) The nuclear extracts were isolated, and 12-␮gsamples were subjected to SDS/PAGE and immunoblotting analysis for phosphorylated IRF-3 or Sp1. (F) WT (open bars) or Atg5 KO MEFs (filled bars) were infectedwith VSV at moi ⫽ 1.0 in the presence of anti-IFN-␣- and IFN-␤-neutralizing IgG orcontrol rabbit IgG. Twenty-four hours after infection, the culture supernatants
were recovered, and the levels of viral titer were examined by the plaque assay.
The graph shows the mean ⫾ SD. *, P ⬍ 0.05 by Student’s t test.

Results
Involvement of the Atg5-Mediated Autophagic Process in Vesicular
Stomatitis Virus (VSV) Replication. To examine the roles of the

autophagic process in RNA virus replication, mouse embryonic
fibroblasts (MEFs) derived from WT and Atg5-deficient mice[Atg5 knockout (KO)] were infected with VSV. VSV induction of
autophagy was determined by visualizing LC3-GFP expressed
exogenously (25). Eight hours after VSV infection, LC3-positive
vacuole-like signals were prominent in WT, but not in Atg5 KO
MEFs (Fig. 1A). The conversion of LC3 was also characterized after
VSV infection. The amount of the modified form (LC3-II), a
hallmark of the autophagic process, was increased, whereas that of
the free form (LC3-I) was decreased upon VSV infection of WT
MEFs. Such LC3 conversion was completely abrogated in Atg5 KO
MEFs (Fig. 1B). These results confirmed that VSV infection causes
autophagy through a process mediated by Atg5.
We next examined the efficiency of VSV replication in WT and
Jounai et al.

yield of most viral replication is largely dependent on the efficiencyof the replication machinery within the host cell and the activity ofantiviral responses mediated by type I IFNs. Such type I IFN
production is induced mainly by isRNA, a byproduct of RNA virus
replication within the host’s cell (26). To examine the involvementof Atg5 in innate antiviral immune responses, the levels of type I
IFN production were compared between WT and Atg5 KO MEFs
after dsRNA [poly(I:C)] stimulation. As shown in Fig. 2 A and B,
Atg5 KO MEFs produced significantly higher amounts of type I
IFN mRNA than the WT isogenic counterparts in response todsRNA (⬎ 25-fold in IFN-␣ and ⬎10-fold in IFN-␤ at both 4 and8 h after stimulation), which was similar to that of VSV infection.
The results were consistent when culture supernatants were examined for type I IFN protein levels (SI Fig. 7). Similarly, dsRNA
stimulated the higher levels of IL-6 and IP-10 production in Atg5
KO MEFs compared with WT MEFs (Fig. 2 C and D). The
specificity of this hyperresponse to dsRNA stimulation was further
confirmed by using Atg5 KO MEFs complemented with the atg5
gene under the control of the doxycycline-regulated expression
system (m5-7 cells) (27) (SI Fig. 8). The expression levels of Atg5
in m5-7 cells were decreased by increasing concentrations of
doxycycline in the culture media (SI Fig. 8A). Of note, the levels oftype I IFN production from dsRNA-stimulated m5-7 cells wereincreased with decreasing cellular expression of Atg5 (SI Fig. 8 B
and C). Taken together, these results suggest that the autophagic
process mediated by Atg5 is involved in the induction of type I IFN
production upon VSV infection, which seems to play a pivotal rolein the mechanism underlying suppression of viral replication.
The Atg5–Atg12 Conjugate Negatively Regulates IPS-1-Mediated
NF-␬B and Type I IFN Promoters. It has been established that after

binding to isRNA, RIG-I or MDA5 interacts with IPS-1 andtransmits intracellular signaling to activate type I IFN genes (23). To
examine the functional involvement of Atg5 in the IPS-1-mediatedcellular signaling, a reporter gene assay was performed. Overexpression of a constitutively active mutant of RIG-I (a COOH
terminus-truncated mutant, RIG-I ⌬C) resulted in activation of the
NF-␬B-dependent promoter and type I IFN promoters in humanepithelial kidney (HEK)293 cells. This activation was suppressed bycoexpression of Atg5, but not by control LacZ (data not shown),suggesting that Atg5 negatively regulates IPS-1-mediated cellular
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Involvement of Atg5 in isRNA-Mediated Type I IFN Production. The

Fig. 2. Hyperproduction of dsRNA-mediated type I IFNs in Atg5 KO MEF and the
Atg5–Atg12 conjugate negatively regulates
IPS-1-mediated promoter activation of type I
IFNs. (A–D) WT (open bars) or Atg5 KO (filledbars) MEFs were transfected with 10 ␮g/ml
poly(I:C). Total RNA was isolated 0, 2, 4, and
8 h after stimulation, mRNA was reversetranscribed, and then the levels of targeted
cDNAs [IFN-␣4 (A), IFN-␤ (B), IL-6 (C), IP-10 (D)]were quantified by real-time PCR. (E)HEK293 cells were transiently transfectedwith 25 ng of Renilla luciferase reporter plasmid, 25 ng of firefly luciferase reporter plasmid for NF-␬B, IFN-␣4, or IFN-␤ promoter(Prom), 50 ng of expression plasmid for FLAGhRIG-I ⌬C plus 0, 50, 100, or 150 ng of HA
hAtg5, HA hAtg12, or HA hAtg5-K130R expression plasmid or empty vector to give a
constant 250 ng of DNA per transfection.
Forty-eight hours after transfection, cells
were lysed and luciferase activity was measured by using a luminometer. The firefly
luciferase activity was normalized to Renillaluciferase activity in each sample. The same
samples were subjected to immunoblot analysis targeting the FLAG or HA epitope. The
graph shows the mean ⫾ SD. *, P ⬍ 0.05; **,P ⬍ 0.01; ***, P ⬍ 0.001; ****, P ⬍ 0.0001 by
Student’s t test.

signaling (Fig. 2E). Because Atg5 molecules are conjugated with
Atg12 under physiological conditions (27), Atg12 and Atg5-K130R,
a mutant that is incapable of conjugation with Atg12 (28), for theireffects on IPS-1-mediated signaling. Similar suppression was observed with Atg12 alone, but not with Atg5-K130R (Fig. 2E).
Although the expression levels of RIG-I ⌬C were comparable
among samples cotransfected with different doses of the Atg
expression plasmid, exogenously expressed Atg5 or Atg12, but not
Atg5-K130R, was coupled with its endogenous counterpart to form
the Atg5–Atg12 conjugate (Fig. 2E). Thus, these results suggest that
the Atg5–Atg12 conjugate, rather than monomeric Atg5 or Atg12,
is functionally involved in this suppression mechanism. A series ofoverexpression experiments was also performed using MDA5 ⌬C,and similar suppression was observed with Atg5 or Atg12, but notwith Atg5-K130R (SI Fig. 9). Interestingly, MDA5 ⌬C-mediatedIFN-␣4 promoter activation was not suppressed by the Atg5–Atg12
conjugate, suggesting that the pathway for IFN-␣4 promoter activation is distinct from that for IPS-1-dependent signaling (SI Fig. 9).
To further confirm the involvement of the Atg5–Atg12 conjugate
in the suppression of IPS-1-dependent signaling, we examined the
responses of Atg7 KO MEFs to isRNA stimulation. Atg7 has an
E1-like activating activity in the Atg12 conjugation system; deficiency of Atg7 results in a loss of the Atg5–Atg12 conjugate but does
not otherwise affect Atg5 or Atg12 molecules (29). Consistent withthe results obtained from overexpression experiments describedabove, Atg7 KO MEFs produced higher amounts of type I IFNs in
response to dsRNA when compared with the WT isogenic counterpart (SI Fig. 10). Collectively, these data suggest that the
Atg5–Atg12 conjugate, but not monomeric Atg5 or Atg12, acts as
a suppressor for IPS-1-mediated signaling, resulting in the downregulation of innate antiviral immune responses.
Physical Association Between the Atg5–Atg12 Conjugate and CARDContaining Signaling Molecules. To further examine physical asso-

ciation between the Atg5–Atg12 conjugate and isRNA-mediatedsignaling molecules, an immunoprecipitation assay was performed.
When expressed in HEK293 cells, the Atg5–Atg12 conjugate
coprecipitated with RIG-I, MDA5, and IPS-1, but not with GFP
control (Fig. 3A). RIG-I ⌬C bound better to the Atg5–Atg12
14052 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0704014104

conjugate relative to full-length RIG-I, suggesting that one or moreelements present in the COOH terminus weakened RIG-I association with the Atg5–Atg12 conjugate. Monomeric Atg5 and Atg12
each also coprecipitated with RIG-I, MDA5, and IPS-1, but notwith GFP, suggesting that Atg5 or Atg12 monomer is sufficient tointeract with these signaling molecules (Fig. 3A). Monomeric
Atg5-K130R was also coprecipitated with RIG-I and IPS-1 (SI Fig.
11). To confirm the interaction between endogenous Atg5–Atg12
conjugate and IPS-1, cell lysates prepared from HEK293 cells
infected with or without VSV were subjected to immunoprecipitation analysis (Fig. 3B). Endogenous IPS-1 coprecipitated with the
Atg5–Atg12 conjugate under physiological conditions, suggestingthat viral infection is not necessary for interaction between the
Atg5–Atg12 conjugate and IPS-1. However, Atg5–Atg12 conjugate–IPS-1 interaction is further enhanced upon VSV infection(Fig. 3B).
To examine the intracellular localization of these molecules,
endogenous Atg5 and IPS-1 in HeLa cells were stained separately
and analyzed by confocal microscopy. As shown in Fig. 3C, Atg5
was present diffusely within the cytoplasm, whereas most IPS-1
localized to mitochondria, which is consistent with a previous report(19) (yellow signal in Fig. 3C‘‘Merge’’). A portion of Atg5 and IPS-1
colocalized with a signal specific for mitochondria (white signal in
Fig. 3C‘‘Merge’’). The intracellular association between Atg5 and
IPS-1 was further confirmed by FRET assay. As a control, a FRET
signal was observed when cyan fluorescent protein (CFP)–IPS-1(donor) and yellow fluorescent protein (YFP)–Mit (encoding mitochondria localization signal) (acceptor), but not YFP-KDEL(encoding an ER localization signal), were expressed in HeLa cells,suggesting that IPS-1 localizes to mitochondria, but not to the ER(Fig. 3D). When YFP-Atg5 was coexpressed, the FRET signal was
also observed from IPS-1 WT, but not from the CARD-null mutant
IPS-1 T54A, suggesting that the Atg5–Atg12 conjugate physicallyassociates with IPS-1 within a cell by targeting the CARD (Fig. 3D).
The Atg5–Atg12 Conjugate Directly Associates with IPS-1 and RIG-I
Through the CARDs. Next, we set out to identify domains in IPS-1

that are responsible for interaction with the Atg5–Atg12 conjugate
by employing IPS-1 mutants (Fig. 4A). The deletion of the transJounai et al.

membrane domain (TMD) abrogated the interaction (Fig. 4B).
IPS-1 CARD alone was sufficient for the association with the
Atg5–Atg12 conjugate. Site-directed mutagenesis in the third ␣-helical region of the CARD by T54A substitution resulted in loss ofinteraction with the Atg5–Atg12 conjugate. Such an interaction was
also abrogated in the presence of proline rich domain (PRD)
immediately juxtaposed to the CARD (IPS-1 CARD–PRD), suggesting that the PRD blocks this interaction. Collectively, theseresults suggest that the IPS-1 CARD is the target of the Atg5–Atg12
conjugate and IPS-1 TMD supports this interaction by interferingwith the PRD blockade of the CARD (Fig. 4B).
To confirm the direct interaction between the Atg5–Atg12
conjugate and IPS-1, a pull-down assay was performed using
recombinant proteins. Because recombinant Atg5 and Atg12 are
not conjugated in vitro system, we tested free Atg5 for interaction

with IPS-1. Atg5 alone was sufficient for direct interaction with
IPS-1 and RIG-I through the CARD, suggesting that the Atg5–
Atg12 conjugate directly interacts with the CARDs of both IPS-1
and RIG-I within the cells (SI Fig. 12).
Because the CARD–CARD homotypic interactions between
IPS-1 and RIG-I are essential for subsequent signaling, we examined whether the Atg5–Atg12 conjugate interferes with this interaction or not. As shown in Fig. 4C, the interaction between IPS-1
and RIG-I was enhanced by increasing the amount of the Atg5–
Atg12 conjugate but did not change with control molecule expression (ARPC-2). Such evidence suggests that RIG-I CARDs may
gain access to both IPS-1 CARD and the Atg5–Atg12 conjugate
upon isRNA stimulation, although the Atg5–Atg12 conjugate does
not dissociate RIG-I CARDs from IPS-1, but intercalates between
RIG-I and IPS-I by binding directly to the CARDs of thesemolecules.

Fig. 4.
The Atg5–Atg12 conjugate associates with IPS-1 and
RIG-I through the CARDs. (A) A
schematic diagram of IPS-1 andtruncated or site-directed mutants. (B) HEK293 cells were transiently transfected with expression plasmids for HA hAtg5 and
HA hAtg12 plus FLAG-tagged
hIPS-1 in either full-length ortruncated form. (C) HEK293 cells
were transfected with expression
plasmids for FLAG hRIG-I ⌬C (0.75
␮g) and HA hIPS-1 (0.75 ␮g) in thepresence of 0, 1, or 4 ␮g of that for
HA hAtg5 and HA hAtg12 or biotinylated actin related protein
complex p34 (ARPC-2). Fortyeight hours after transfection, celllysates were prepared, immunoprecipitated with anti-FLAG antibody, and then subjected to immunoblotting analysis using
anti-FLAG antibody, anti-HA antibody, or streptavidin-HRP. Three to five independent experiments gave similar results.

Jounai et al.

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Fig. 3. The Atg5–Atg12 conjugate associates with IPS-1, RIG-I, and MDA5. (A)HEK293 cells were transiently transfectedwith expression plasmids for HA hAtg5 and
HA hAtg12 plus FLAG-tagged hRIG-I,
hMDA5, hIPS-1, or GFP in either full-length
or truncated form. Forty-eight hours after
transfection, cell lysates were prepared, immunoprecipitated with anti-FLAG antibody, and then subjected to immunoblotting analysis using anti-FLAG or anti-HA
antibody. (B) HEK293 cells were incubatedwith or without VSV at moi ⫽ 1.0. The celllysates were immunoprecipitated with anti-Atg5 or control antibody and were thensubjected to immunoblotting analysis using anti-Atg5 or MAVS (IPS-1) antibody. (C)HeLa cells were treated with Mitotracker
reagent, fixed, and incubated with goatanti-APG5 (Atg5) and rabbit anti-MAVS(IPS-1) antibodies. The cells were thenwashed, treated with Alexa 488-conjugated anti-goat IgG and Alexa 405-conjugated anti-rabbit IgG antibodies, and thenanalyzed under a confocal microscope. Fluorescent signals were depicted as black and white in ‘‘Atg5,’’ ‘‘IPS-1,’’ and ‘‘Mitotracker’’ views. In the ‘‘Merge’’ view,Atg5, IPS-1, or Mitotracker was represented in blue, green, or red, respectively. (D) FRET signal was examined after CFP-IPS-1 plus YFP-Mit, CFP-IPS-1 plus
YFP-KDEL, CFP-IPS-1 plus YFP-Atg5, or CFP-IPS-1 T54A plus YFP-Atg5 were expressed in HeLa cells. (Left and Center) Cells were imaged for CFP (donor) (Left) andYFP (acceptor) (Center). The acceptor bleaching was completed within 20 sec. (Right) FRET efficiency based on the YFP bleaching. The ratio of the increase in
CFP fluorescence signal to its signal after bleaching is represented as reported in ref. 41. Three to five independent experiments gave similar results.

Discussion
Autophagy is one of the most primitive degradation processes,
eliminating both intracellular microbes and existing cellular proteins that are no longer required. It serves fundamental functions
in lower organisms such as yeast, metazoans, or Caenorhabditis
elegans (30). Host’s antivirus protection mechanism is also conserved in plants and primitive animals. In mammals, the IPS-1dependent cellular signaling plays a pivotal role in innate antiviral
immune responses. Data from the present study suggest that the
Atg5–Atg12 conjugate directly interacts with both IPS-1 and RIG-I
and inhibits transmission of CARD-mediated signaling. Such aninteraction results in a suppression of isRNA-mediated type I IFN
production and subsequent innate antiviral immune responses.
Therefore, the present study provides a link between the autophagicmachinery and innate immune signaling against viral infection.
Because the autophagic process and the signaling pathway leading
to type I IFN production direct opposite outcomes of RNA virus
infection, i.e., the former helps viral replication, whereas the latteris central to antiviral responses, the Atg5–Atg12 conjugate interaction with CARDs may be a turning point toward the pathway
leading to viral proliferation or the host’s elimination of infecting
viruses. Moreover, our results regarding the interaction between
the Atg5–Atg12 conjugate and CARD-mediated signaling molecules provide important evidence with respect to the developmental
process in the nascent immune system and suggest that the interaction of these two systems plays a key role in the primary defense
against various pathogenic agents. In addition, because the interaction between the Atg5–Atg12 conjugate and IPS-1 was observed
without VSV infection, the Atg5–Atg12 conjugate may repress
signaling under physiological conditions and play an important rolein maintaining cellular homeostasis. Previous reports and ourcurrent data demonstrated that VSV induces autophagy and may
use autophagosomes as a replication scaffold like other RNA
viruses, such as mouse hepatitis virus (MHV) (9, 10). Thus, it ispossible that this association is regulated by the autophagic process.
It is also plausible that RIG-I function is not permissive for
autophagy, that perhaps RIG-I signaling can serve to block autophagy, and that is what this pathway is targeted by the Atg5–Atg12
conjugate. However, because autophagy occurs even under physiological conditions in vitro and because there is no specific reagent
that inhibits the autophagic process and not other signaling pathways, additional efforts are required to clarify the regulatory
mechanism of this interaction through the autophagy induced byvarious biological situations.
Our current data suggest that some RNA viruses have evolved touse the Atg5–Atg12 conjugate to interfere with antivirus immune
signaling. Human hepatitis C virus is known to encode the NS3/4A
protease to directly cleave IPS-1 to shut off its capacity to transmit
subsequent signaling (18, 31). Because IPS-1 is ubiquitously expressed in a wide-variety of cells and is essential for isRNAmediated type I IFN signaling, it is possible for some pathogenic
RNA viruses to engage the Atg5–Atg12 conjugate for evasion of
IPS-1-mediated host innate immune responses.
Other than its canonical role as a regulator of the autophagic
process, Atg5 has been shown to interact with exogenous molecules,
such as VirG of Shigella. According to the amino acid sequence andsecondary structure prediction analysis, Atg5 contains a large
number of acidic residues, a potential glycosylation site, and potential casein kinase II, protein kinase C, and tyrosine kinase sites.
However, we could not identify specific sequences predicted toform CARD or CARD-like domains in Atg5. In addition, previous
reports have shown the possibility of Atg5 interaction with Fasassociated protein with death domain or Bcl-XL (28, 32). Further
analysis using site-directed mutagenesis or crystal structure isneeded to identify precise domains and the amino acids of Atg5
required for such interactions.
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Fig. 5. A scheme of the molecular association among RIG-I, IPS-1, and the
Atg5–Atg12 conjugate during RNA virus infection.

In conclusion, our present work demonstrated functional andmolecular associations among Atg family members and innate
immune signaling as summarized in Fig. 5. The model predicts that(i) the Atg5–Atg12 conjugate is interacting with IPS-1 but weakly
with RIG-I under physiological conditions; (ii) infection of some
RNA viruses may cause a conformational change of RIG-I andallows NH2-terminal RIG-I CARDs to interact with the CARD of
IPS-1; and (iii) the Atg5–Atg12 conjugate intercalates between the
CARDs of RIG-I and IPS-1 and inhibits signal transmission,
resulting in suppression of type I IFN production and innate
antiviral immune responses. Further investigation is needed toclarify the precise mechanisms bridging autophagy and the host’s
antiviral responses through the association among the Atg5–Atg12
conjugate and CARD-containing signaling molecules. Our work
provides a framework for the design and development of antiviraltherapeutic applications by targeting such mechanisms.
Materials and Methods
Cells, Viruses, and Reagents. HEK293, HeLa, and Vero cells werepurchased from American Type Culture Collection (Manassas,
VA). Atg5 KO MEFs were kindly provided by N. Mizushima(Tokyo Medical and Dental University, Tokyo, Japan) and havebeen described in refs. 27 and 33–35. These cells were maintainedin DMEM supplemented with 10% FCS and 50 ␮g/ml penicillin/
streptomycin. VSV was provided by National Institute of Animal
Health (Tokyo, Japan). Human HSV type I (KOS strain) was kindly
provided by T. Suzutani (Fukushima Medical University, Fukushima, Japan). Endotoxin free poly(I:C) was obtained from Invivogen (San Diego, CA). Anti-LC3 and Atg5 antibodies were
provided by N. Mizushima (25, 35, 36). Anti-APG5 (Atg5) andanti-Sp1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MAVS (IPS-1) or anti-phospho-IRF-3(Ser-396) antibody was purchased from Bethyl Laboratories(Montgomery, TX) or Cell Signaling Technology (Danvers, MA),
respectively.
Expression Plasmids. The LC3-GFP expression plasmid was provided by N. Mizushima (35). The YFP-Mit and YFP-KDEL
expression plasmids were described in refs. 25 and 37. The expression plasmid for biotinylated actin-related protein complex p34(pcDNA3.2/capTEV-NT/V5-GW/ARPC2) was purchased from
Invitrogen (Carlsbad, CA). The IPS-1 expression plasmid was
described in ref. 17. The truncated form of IPS-1 cDNA wasgenerated by ligating the amino acid 1-100 and the amino acid170-540 of hIPS-1 ORF amplified by PCR (IPS-1⌬PRD); amplifying the amino acid 1-514 by PCR (IPS-1⌬TMD); amplifying theamino acid 1-170 by PCR (IPS-1 PRD); amplifying the amino acid
1-100 by PCR (hIPS-1 CARD); ligating the amino acid 1-100 ofhIPS-1 ORF and EGFP cDNA fragment amplified by PCR (IPS1CARD-GFP); ligating the CARD and TMD (amino acid 514-540)
cDNA amplified by PCR (IPS-1 CARD-TMD); or ligating the
CARD, TMD, and EGFP cDNA amplified by PCR (IPS-1CARDTMD-GFP). Atg5 and Atg12 cDNAs were kind gift from N.
Mizushima. RIG-I, MDA5, RIG-I ⌬C (amino acid 1-604), andMDA5 ⌬C (amino acid 1-575) cDNAs were amplified by PCR using
a human spleen cDNA library (TAKARA, Tokyo, Japan). The
Jounai et al.

Fluorescence and Confocal Microscopy Analysis. Two million WT or
Atg5 KO MEFs were transfected with the LC3-GFP expression
plasmid by using MEF Nucleofector Kit 1 (Amaxa, Gaithersburg,MD) according to the manufacture’s protocol and 4 ⫻ 105 cells
were plated on a slide glass. Twenty-four hours after transfection,
the cells were infected with or without VSV at multiplicity of
infection (moi) ⫽ 1.0. Eight hours after infection, the cells were
fixed and analyzed by a fluorescent deconvolution microscope,
BIOZERO (Keyence, Tokyo, Japan). HeLa cells were treated with
Mitotracker (Invitrogen) 45 min before fixation with 3% paraformaldehyde and incubated with goat anti-APG5 (Atg5) and rabbit
anti-MAVS (IPS-1) antibodies followed by treatment with Alexa
488-conjugated anti-goat IgG and Alexa 405-conjugated anti-rabbit
IgG antibodies (Invitrogen). Confocal microscopy analysis was
performed using FV500 (Olympus, Tokyo Japan).
Virus Titration. WT or Atg5 KO MEFs (5 ⫻ 105 cells per ml) were

infected with VSV at moi ⫽ 0.1 or 1.0 in the presence or absenceof 20 ␮g/ml rabbit normal IgG (Cedarlane, Ontario, Canada) or a
mixture of 1,000 units/ml of anti-mouse IFN-␣ and IFN-␤ antibodies (PBL Biomedical Laboratories, Piscataway, NJ). Twenty-fourhours after infection, the cells and culture supernatants wererecovered and kept at ⫺80°C. Vero or HeLa cells (5 ⫻ 105 cells perml) were used in the plaque assay for measuring virus titer. Afterthe addition of the supernatants, the cells were overlaid with MEM
containing 1% agarose and 5% FCS and were incubated for 24 h.
After staining with neutral red, the number of plaques wasenumerated.

Luciferase Assay. Luciferase assay was conducted as described inref. 40.
Immunoprecipitation and Immunoblot Analysis. Immunoprecipita-tion and immunoblot analysis was performed as described in ref. 38
by using anti-FLAG M2 (Sigma), anti-HA (Covance, Berkeley,CA), anti-HA-Peroxidase (3F10) (Roche Diagnostics, Indianapolis, IN), anti-Atg5, anti-MAVS (IPS-1), or anti-phospho-IRF3(Ser-396) antibody.
FRET. HeLa cells were transfected with various combinations ofplasmid expressing CFP- or YFP-fusion proteins. Cells were imaged
on an inverted microscope (Olympus IX70) with a standard 75-W
xenon lamp and a 40⫻ objective lens (Uapo/340, N.A. 1.35).
Interference filters (excitation and emission filters) contained inwheels were automated using Lambda 10–2 hardware (Sutter
Instruments, Novato, CA). Interference filters used in this study
were as follows: donor channel excitation, 440DF20; donor channel
emission, 480DF30; acceptor channel excitation, 490DF20; acceptor channel emission, 535DF25; FRET channel excitation,
440DF20; FRET channel emission, 535DF25. Emitted light wascaptured by a cooled CCD camera (CoolSNAP fx; Roper Scientific,
Tucson, AZ). The whole system was controlled using the
MetaFluor 6.0 software (Universal Imaging, Media, PA). The
FRET signal is shown after following the calculation by using ImageJ software (National Institutes of Health, Bethesda, MD).
FRET ⫽ 1 ⫺ (donor emission before bleaching)/(donor emission after bleaching).
Statistical Analysis. Student’s t test was used for statistical analysis.

ref. 40.

We thank Drs. N. Mizushima, T. Yoshimori (Osaka University), and M.
Komatsu (Juntendo University, Tokyo, Japan) for providing experimental materials and suggestions; J. Huang (University of California, Los
Angeles, CA) for suggestions; and S. Saha and S. Yamazaki for technicalassistance. This work was supported by Ministry of Education, Culture,
Sports, Science, and Technology of Japan, Yokohama City University
Center of Excellence Program (N.J.); Ministry of Education, Culture,
Sports, Science, and Technology of Japan Grants 18590432 (to F.T.) and
18659126 (to K.O.); the National Institute of Biomedical Innovation(K.O.); Strategic Research Project of Yokohama City University Grant
K18022 (to F.T.); and Advancement of Medical Sciences from Yokohama Medical Foundation (F.T.).

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Real-Time PCR. Real-time PCR was performed as described in

Jounai et al.

PNAS 兩 August 28, 2007 兩 vol. 104 兩 no. 35 兩 14055

IMMUNOLOGYhIPS-1 CARD mutant (IPS-1 CARD-TMD T54A) and Atg5K130R expression plasmids were generated by site-directed mutagenesis. These fragments were introduced in-frame into pFLAGCMV4 (Sigma, St. Louis, MO), pCIneo-HA (38), pCAGGS-Flagm1SECFP (39), or pCAG-His Venus vector (39). Sequences of the
PCR products were confirmed using an ABI PRISM Genetic
Analyzer (Applied Biosystems, Foster City, CA)