CHIKV infection induces autophagosome formation in infected cells
During autophagy, Beclin-1–PI3K-III complex activation results in isolation membrane formation that surrounds its substrates to generate an autophagic vesicle characterized by a double membrane, which is called an autophagosome (Mehrpour et al., 2010
). Two ubiquitin-like systems have been shown to be essential for autophagosome formation. In the first, Atg12 (autophagy-related gene 12) is conjugated to Atg5, together forming a complex with Atg16L1, which decorates the outer membrane of the isolation membrane. Microtubule-associated protein 1 LC3 (light chain 3, also known as Atg8) constitutes the second ubiquitin-like system: LC3 conjugates phosphatidylethanolamine (PE) at the outer and inner autophagosomal membrane. Unlike the Atg12–Atg5–Atg16L1 complex that is recycled, the LC3-PE (referred to as LC3-II) remains associated with the inner membrane of autophagosome, making it a useful marker of autophagosomes (Kroemer et al., 2010
; Mehrpour et al., 2010
). To define the relationship between CHIKV infection and autophagy, we first examined autophagosome formation. Mouse embryonic fibroblasts (MEFs) were infected and LC3 puncta or LC3-II formation was measured by immunofluorescence or Western blot, respectively. Compared with uninfected MEFs, CHIKV-infected cells showed increased numbers of LC3 puncta () and stronger staining for LC3-II by Western blotting (). Autophagosome formation could be first observed after 5 h and peaked at 9 h after infection (), suggesting the requirement for CHIKV replication as the trigger for autophagy induction. Replication-defective CHIKV (achieved by UV-B irradiation) failed to induce autophagosome formation, thus supporting the requirement for viral replication (). Serum starvation served as a positive control for autophagy induction (; and not depicted).
Figure 1. Autophagosome induction within CHIKV-infected cells. (A and B) WT MEFs were incubated for 24 h in control media (uninfected), in nutrient-deprived media (starvation), or in the presence of live or UV-B–treated CHIKV (MOI = 1). (A) Immunofluorescence (more ...)
To evaluate if the induction of autophagosomes was occurring via a classical macroautophagy pathway, we evaluated CHIKV infection using MEFs deficient in the key autophagy gene Atg5
. As predicted, Atg5−/−
MEFs showed no evidence of LC3-II conversion upon CHIKV infection (). Similarly, immunofluorescence studies demonstrated that LC3 puncta observed during CHIKV infection were dependent on Atg5 expression (). Restoration of Atg5
expression, achieved by cDNA transfection, rescued the cells’ ability to induce autophagy (). We observed similar results using cells deficient for Atg7
, another key autophagy gene (data not depicted). Furthermore, we investigated the implication of Beclin-1 in CHIKV-induced autophagy using MEFs that express a mutant form of Bcl-2 (MEF-Bcl2AAA
). Notably, Bcl-2 directly regulates the activation of Beclin-1 and alanine substitution of the three phosphorylation sites (T69A/S70A/S87A) in Bcl-2 prevents dissociation of the Bcl-2–Beclin-1 complex, selectively inhibiting autophagy induction by Beclin-1 without affecting the antiapoptotic role of Bcl-2 (Wei et al., 2008
; He et al., 2012
). As expected, MEF-Bcl2AAA
cells were not able to induce autophagy under starvation conditions (). Similarly, CHIKV-induced autophagy was abrogated in Bcl-2AAA
MEFs, indicating that CHIKV-induced autophagy occurs via a Beclin-1–dependent mechanism. To confirm that CHIKV infection induced autophagosome formation in other cell types, we also investigated the appearance of autophagosomes in HeLa cells and human foreskin fibroblasts. Silencing of Atg5
genes using small interference RNA (siRNA) confirmed data shown using MEFs (unpublished data).
To analyze whether autophagosome formation was dependent on direct viral infection, we marked active replication using GFP-expressing recombinant CHIKV (Vanlandingham et al., 2005
) and analyzed LC3 puncta using ImageStreamX. In brief, multispectral cytometric analysis enables the capture of high-resolution images of cells in flow (up to 500 cells/s) and permits analysis of LC3 puncta (de la Calle et al., 2011
). 24 h after infection, GFP-expressing cells were gated (, R2), and LC3 bright detail intensity (BDI) was integrated for each cell as a measure of autophagosome formation. For comparison, GFP-negative cells were gated ( [R1] and I [blue line]), and histogram plots representing LC3 puncta indicate that CHIKV-infected cells (, red line) have higher LC3 BDI. Representative ImageStreamX images with median intensity levels of BDI are shown for CHIKV-infected and uninfected cell populations (), confirming that LC3 puncta (scored based on high BDI) correlated with the presence of both viral-encoded GFP and robust autophagosome accumulation. Using this method, we quantified the percentage of LC3-positive cells (BDIhi
) when bulk cultures are segregated for CHIKV infection (, R2 vs. R1, P < 0.05). Starved cells were used as positive control for autophagy induction (unpublished data). Based on these data, we conclude that autophagy induction occurs via a Beclin-1–dependent mechanism in a cell-intrinsic manner; in other words, viral replication within the cell, as opposed to secreted factors produced by neighboring infected cell, is the stimulus for autophagy induction.
Autophagosome/lysosome fusion remains intact during CHIKV infection
Upon maturation, autophagosomes fuse with late endosomes and lysosomes, which results in the formation of a degradative compartment referred to as autolysosomes (Deretic and Levine, 2009
). Some viruses encode inhibitors of this event (e.g., influenza virus), and as a result enhanced numbers of LC3 puncta could be a reflection of basal autophagy accumulation and not de novo autophagosome formation (Gannagé et al., 2009
). To discriminate between these two possibilities, we analyzed autophagy in the presence of lysosomal inhibitors leupeptin and E64D in transfected GFP-LC3 HeLa cells (). As expected, inhibition of autophagosome/lysosome fusion resulted in an increased number of LC3 puncta in control (basal autophagy flux) and starved (induced autophagy) HeLa cells. The presence of leupeptin and E64D also enhanced the number of autophagosomes in infected cells, allowing us to conclude that CHIKV infection induces de novo autophagosome formation (). To confirm this finding, we directly assessed colocalization of LC3 and the lysosomal-associated membrane protein LAMP-1 (). Optical sectioning of cells was performed and LC3+
, and double-labeled vesicles were enumerated on a per cell basis. Approximately 45% of the LC3+
puncta colocalized with LAMP-1+
lysosomes in CHIKV-infected cells (). Line scan analysis confirmed colocalization and indicated that the lysosomal structures were discrete vesicles within the cell ().
Figure 2. CHIKV induces autophagic flux. (A and B) GFP-LC3–expressing HeLa cells were starved (starvation) or infected with CHIKV (MOI = 1) alone or in the presence of leupeptin and E64D (L+E) for 24 h. The numbers of GFP+ vesicles per cell were enumerated. (more ...)
As a final measure of autophagic flux, we transfected MEF cells with a construct encoding an RFP-GFP-LC3 polyprotein. In this way, it is possible to distinguish autophagosomes (RFP+ GFP+ puncta) and autolysosomes (RFP+ GFP− vesicles), with the latter being GFP negative as a result of the quenching of signal by the acidic microenvironment of the lysosome. CHIKV infection increased the number of both autophagosomes and autolysosomes compared with uninfected cells (). Moreover, an equivalent ratio of autophagosomes and autolysosomes was measured in infected cells and starved cells (). Together, the data presented in and provide evidence for CHIKV infection inducing de novo autophagosome formation without inhibition of autophagosome maturation.
ER stress serves as a trigger for autophagy during CHIKV infection
The ER serves as an important sensor of cellular stress. It detects changes in cell homeostasis and responds by triggering pathways referred to as the unfolded protein response (UPR; Lee et al., 2003
; McGuckin et al., 2010
). Viral infection has been shown to activate UPR as a result of the accumulation of viral proteins. At least three different pathways may be activated during ER stress, which are regulated by the signaling molecules eIF2α, IRE1α, and ATF6, respectively (McGuckin et al., 2010
). We screened all three pathways (unpublished data) and identified a critical role for IRE1α. A role for IRE1α activation during CHIKV infection was first demonstrated by analyzing the phosphorylation of IRE1α (p-IRE-1α) at different time points after infection. Western blotting and immunofluorescence indicated that p-IRE1α was observed during the early phase of infection () and could be detected only in infected cells (as evaluated based on E3 colocalization; ). These data suggested that CHIKV leads to an intrinsic activation of ER stress. Importantly, p-IRE1α was no longer detected 3 d after infection. This shutdown seems to be the result of a decreased level of IRE1α protein, as indicated by Western blot analysis (). Remarkably, the kinetics of IRE1 phosphorylation correlated with the conversion of LC3-I to LC3-II ().
Figure 3. CHIKV-induced autophagy is regulated by ER and oxidative stress. (A–C) WT MEFs were infected with CHIKV at indicated time points and Western blotting was performed to detect phosphorylation of IRE1α (p-IRE1α) and JNK (p-JNK) as (more ...)
To define the molecular events triggered by p-IRE1α, we investigated the activation of XBP1 and c-Jun amino-terminal kinases (JNK; McGuckin et al., 2010
). Activation of XBP1 is regulated by a differential splice variant of XBP1
mRNA, which may be evaluated based on the expression of a protein of higher molecular weight and is referred to as XBP1s (for spliced XBP1; Yoshida et al., 2001
). This pathway has been shown to favor cell survival (McGuckin et al., 2010
). In contrast, IRE1α-induced phosphorylation of JNK is considered a link between cell stress and apoptosis (Urano et al., 2000
). During CHIKV infection, we observed an induced expression of XBP1s, but did not detect enhanced phosphorylation of JNK (p-JNK; ). To examine the functional link between IRE1α in CHIKV-induced autophagy, we silenced expression of IRE1α
using siRNA and analyzed CHIKV-induced LC3 puncta as well as LC3-II conversion (). Reduced IRE1α gene expression was confirmed by Western blot, and shown to result in fewer CHIKV-induced autophagosomes. These data define a role for CHIKV activation of ER stress, which induces autophagy via an IRE1α- and XBP1s-mediated signaling pathway.
CHIKV-induced oxidative stress favors autophagosome production through the inhibition of mTORC1
Oxidative stress, primarily caused by increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), is a feature of the host response to viral infections (Cataldi, 2010
and NO are considered to be the most important mediators among ROS and RNS, respectively. Free oxidative agents are known to induce autophagy and can also lead to cell death during strong and prolonged stimulation (Djavaheri-Mergny et al., 2007
; Filomeni et al., 2010
; Guo et al., 2010
). To assess the impact of ROS/RNS production in CHIKV-induced autophagy, we first investigated whether CHIKV infection induces ROS and/or RNS production. We infected WT MEF for 24 h and monitored the presence of oxygen species and free NO. As positive control, we used pycocyanin and l
-arginine, inducers of ROS and NO, respectively (). As expected, pycocyanin increased the percentage of cells that produced ROS, in the absence of NO production, whereas l
-arginine induced free NO but not ROS. Interestingly, we observed that CHIKV infection led to increased production of both ROS and NO (), which could be inhibited using the ROS inhibitor N
-cysteine or the RNS scavenger c-PTIO (). Of note, exposure to a RNS scavenger cross-inhibited ROS production, highlighting the interconnectivity between the ROS and RNS pathways. This phenomenon could be explained by NO reacting with O2−
to form the oxidant peroxynitrite (ONOO-; Djavaheri-Mergny et al., 2007
; Novo and Parola, 2008
To confirm the implication of ROS/RNS production in CHIKV-induced autophagy, we investigated LC3+ staining and the conversion of LC3-I to LC3-II in infected MEFs pretreated with N-acetyl-l-cysteine (). A significant decrease in LC3 puncta was observed, demonstrating the importance of ROS production in CHIKV-induced autophagy. We further evaluated potential overlap with the ER stress pathway by assessing autophagy in cells silenced for IRE1α mRNA and treated with N-acetyl-l-cysteine. Strikingly, we observed an additive inhibitory effect that reduced the number of autophagosomes per cell to near baseline levels (). This data suggests that ER stress and oxidative stress act via independent mechanisms to induce autophagy during CHIKV infection.
Although oxidative stress is known to induce autophagy upon microbial infection, the precise mechanism remains poorly documented. Based on recently established links between ROS and mTORC1 inhibition, which appears to be dependent on TSC2 (tuberous sclerosis complex 2), itself regulated by the AMP-activated protein kinase (AMPK; Alexander et al., 2010a
), we investigate the regulation of this complex during CHIKV infection. Importantly, phosphorylated mTOR can be integrated into two different complexes, called mTORC1 and mTORC2, depending on its interactions with Raptor and Rictor, respectively (Zoncu et al., 2011
). Although both complexes are implicated in protein synthesis, only mTORC1 is linked to autophagy (Thomson et al., 2009
). To discriminate the activation of mTORC1 from mTORC2, we analyzed both mTOR phosphorylation and the induction of p-S6K1, a specific substrate of mTORC1. As shown, we observed a diminished level of both p-mTOR and p-S6K1 24 h after infection (). This inhibition was transient, and 2–3 d after infection, a strong induction of the mTOR–S6K1 pathway could be detected. The kinetics of mTOR–S6K1 inhibition correlated with conversion of LC3-I to LC3-II, suggesting a role for mTORC1 as a mediator of CHIKV-induced autophagy. To define how CHIKV-induced ROS is capable of inhibiting mTORC1, we next evaluated the activation of AMPK. Strikingly, the active form of AMPK was detected 24 h after infection, coincident with the inhibition of mTORC1 (). Moreover, the implication of the AMPK pathway in ROS-mediated inhibition of mTORC1 could be confirmed using N
-cysteine (). Together, these data provide a mechanistic understanding of CHIKV-induced autophagy.
Autophagy is a prosurvival mechanism that limits CHIKV-induced cell death
In addition to autophagy, other forms of cell stress may be triggered as a result of viral infection, including activation of cell death pathways. Increasing evidence suggests that cell stress pathways intersect and in some instances cross-inhibit each other (Thorburn, 2008
). As CHIKV infection triggers a pronounced CPE (Sourisseau et al., 2007
), it was important to investigate the function of autophagy on CHIKV-induced cell death. Atg5−/−
MEFs and cells unable to engage the intrinsic apoptosis pathway (Bax−/− Bak−/−
MEFs) were infected with CHIKV and loss of membrane integrity was analyzed. Whereas CHIKV infection triggered cell death in WT cells in a time- and dose-dependant manner, Bax−/− Bak−/−
MEFs remained refractory, showing only minimal evidence of CPE at day 3 (). In contrast, Atg5−/−
MEFs showed a dramatic increase in cell death compared with its WT control (). Notably, the enhanced CPE was most prominent at low multiplicity of infection (MOI), suggesting a link between cell death and viral propagation throughout the cultured MEFs. Similar results were obtained by silencing expression of autophagy genes in WT MEFs (). To explore alternative cell death pathways, autophagy genes were silenced in the Bax−/− Bak−/−
MEFs. Importantly, the absence of both autophagy and apoptosis pathways did not further sensitize the cells to alternative forms of cell death (). Finally, we tested the importance of viral replication for cell death induction. WT MEFs were infected with live or UVB-inactivated CHIKV, and cell death was evaluated. As shown, replication defective CHIKV fails to induce cell death ().
Figure 4. Enhanced CHIKV-induced cell death in the absence of autophagy. (A–D) WT, Bax−/− Bak−/−, or Atg5−/− MEFs were infected with CHIKV at indicated doses and time points. Percentage of cell death was measured (more ...)
To define the form of programmed cell death responsible for the CPE in WT and Atg5−/− MEFs, we used pharmacological inhibitors of apoptosis (z-VAD, a broad spectrum caspase inhibitor) or necroptosis (necrostatin-1, an inhibitor of RIPK1). Infected cells treated with necrostatin-1 exhibited a similar level of cell death as compared with infected control cells, whereas z-VAD rescued both the WT and Atg5−/− MEFs from CHIKV-induced CPE. (). These data, as well as the absence of CPE in Bax−/− Bak−/− MEFs, demonstrate that the principle form of cell death induced by CHIKV is caspase-mediated apoptosis.
Autophagy delays both intrinsic and extrinsic apoptosis pathways
To define the interaction between autophagy and apoptosis at the single cell level, WT and Atg5−/−
or Bax−/− Bak−/−
cells were infected with CHIKV. As previously, LC3 BDI was used as a measure for autophagy induction, and apoptosis activity was characterized by labeling with antibodies specific for the active, cleaved form of caspase-3 (de la Calle et al., 2011
). Data are represented in with each dot indicating a single cell. Regions were established as detailed previously (de la Calle et al., 2011
), and the percentage of autophagic cells (LC3 BDIhi
, cleaved caspase-3lo
; defined by R1), apoptotic cells (LC3 BDIlo
, cleaved caspase-3hi
; defined by R2), or cells with evidence for both processes (LC3 BDIhi
, cleaved caspase-3hi
; defined by R3) were enumerated and represented graphically (). Supporting immunofluorescence results (), CHIKV infection triggered an increase in LC3 puncta in WT and Bax−/− Bak−/−
MEFs, but not in Atg5−/−
MEFs (). Strikingly, the number of cells exhibiting active caspase-3 was increased upon CHIKV infection as compared with uninfected cells, shown at 0 h (). The number of LC3+
cells was dramatically reduced in WT cells 3 d after infection. This observation correlated with high level of apoptosis and suggests that autophagy and apoptosis are mutually exclusive processes (, compare B with C). This observation was further supported by the absence of double-positive cells (region R3; ).
Figure 5. Autophagy limits CHIKV-induced apoptosis. (A–D) WT, Bax−/− Bak−/−, or Atg5−/− MEFs were infected with CHIKV (MOI = 1) for 24 h and were stained for LC3 and activated caspase-3. (A) Representative (more ...)
Two important observations emerged from the study of the knockout MEFs. The first intriguing finding concerned a marked increase in active caspase-3+ cells when Atg5 is absent (). These data suggested an important role for autophagy in the regulation of CHIKV-induced apoptosis. The second discovery concerned the possibility of the Bax−/− Bak−/− cells to activate caspase-3; the timing of apoptosis onset showed interexperimental variation with cells showing detectable levels of cleaved caspase-3 between 48 and 72 h (). Evidence for caspase-3 activation in Bax−/− Bak−/− cells indicated activation of the extrinsic apoptosis pathway, which may be induced independently of mitochondrial outer membrane permeabilization.
We then investigated whether autophagy is able to regulate apoptotic cell death in human fibroblasts cells (HFF), which are known cell targets of CHIKV infection (Sourisseau et al., 2007
). We down-regulated the expression of Atg5
genes in HFF by an siRNA strategy and analyzed the activation of caspase-3 after 24 h of infection with different viral inputs (). Inhibition of both Atg5
dramatically increased the percentage of cleaved caspase-3–positive cells according to viral doses, demonstrating that the antiapoptotic function of autophagy is also observed in human cells.
To distinguish the role of autophagy in limiting the distinct apoptosis pathways, we evaluated cleaved caspase-9 (a marker of the intrinsic pathway) and cleaved caspase-8 (indicative of activation of the extrinsic pathway) during CHIKV infection (). Early during the kinetics of viral infection (16 h), caspase-9 activation was evident in WT cells in the absence of detectable levels of cleaved caspase-8. In comparison, Atg5−/− MEFs displayed a twofold increase in the percentage of active caspase-9–positive cells, as well as early evidence for cleaved caspase-8. By 40 h after infection, both the intrinsic and extrinsic pathways were engaged in the WT cells and, again, Atg5−/− cells demonstrated higher levels of activation for both cell death pathways ().
Using GFP-expressing CHIKV, we next evaluated the relationship between infection and activation of caspase-3, -9, and -8 (). Whereas the intrinsic apoptotic pathway was engaged primarily in CHIKV+ cells, the extrinsic pathway was detectable in both infected and bystander uninfected cells. These data suggest that CHIKV infection induces apoptosis through both intra- and extracellular factors () and that autophagy, acting in a cell-intrinsic manner, preferentially protects infected cells from apoptosis.
CHIKV-induced apoptosis is independent of ER and oxidative stress
As introduced in the previous sections, both ER and oxidative stress may trigger proapoptotic pathways (McGuckin et al., 2010
). To determine the relative contribution of stress pathway induction on autophagy versus apoptosis, we again used Atg5−/−
MEFs. We first confirmed the antiapoptotic effect of autophagy by investigating the cleavage of pro–caspase-3 in WT and Atg5−/−
MEFs (). Whereas pro–capase-3 was cleaved only after 48 h of infection in WT cells, the active form of caspase-3 was detected 24 h after infection in Atg5−/−
MEFs. Moreover, the ratio of active caspase-3/GAPDH was increased in Atg5−/−
cell during all time points investigated ().
Figure 6. ER and oxidative stress don’t enhance in CHIKV-induced apoptosis. (A and B) WT or Atg5−/− MEFs were infected with CHIKV at the indicated time points. (A) Western blotting was performed using anti-LC3 and anti–caspase-3 (more ...)
We next investigated the activation of IRE1α and inhibition of mTOR in Atg5−/− MEFs. Interestingly, both pathways were similarly regulated by CHIKV infection as compared with WT MEFs ( compared with ). To ascertain the impact of these pathways on apoptosis, we evaluated by immunofluorescence the percentage of active caspase-3–positive cells in WT or Atg5−/− MEFs in which ER and oxidative stress had been inhibited (). IRE1α and/or ROS inhibition increased the percentage of WT cells with active caspase-3. Importantly, no change in active caspase-3 expression was observed in Atg5−/− MEFs. These data suggest that ER and oxidative stress occur before the role of Atg5 in facilitating autophagy and are not critical for the induction of CHIKV-induced apoptosis.
Together, the data presented in – demonstrate that during the early phase of CHIKV infection, autophagy is induced via the activation of ER stress and the inhibition of mTOR by ROS production. By triggering autophagy, CHIKV-induced cell stress indirectly limits apoptotic cell death. Nevertheless, after 48 h of infection, ER stress is blunted, in part secondary to IRE1α degradation, and mTOR becomes hyperphosphorylated. These events result in decreased autophagic tone and, via a still undefined mechanism, apoptosis pathways dominate, in turn resulting in pronounced CHIKV-induced CPE.
Regulation of apoptosis and in vitro CHIKV propagation
Both autophagy and apoptosis have been related to the regulation of viral replication and/or propagation. Although autophagy can result in the degradation of viral proteins (e.g., Sindbis) without significantly affecting viral infection, it has also been reported to enhance viral replication (e.g., HCV; Dreux and Chisari, 2009
). Similarly, apoptosis has been implicated in both pro- and antiviral responses (Li and Stollar, 2004
). As both pathways are engaged by CHIKV infection, it was important to evaluate the effect of autophagy and apoptosis on viral propagation. To achieve this, we measured viral load in the supernatant of WT, Atg5−/−
, or Bax−/− Bak−/−
MEFs. CHIKV titers seemed enhanced in supernatant of Atg5−/−
cells, suggesting that autophagy could restrict viral release (). However, modest viral titers were lower in Bax−/− Bak−/−
MEFs as compared with WT controls at day 1 after infection, thereby suggesting a role for apoptotic cell death in viral release during early phase infection (). Similar results were obtained in HeLa cells using siRNA specific for Atg5
Figure 7. Regulation of apoptotic cell death limits CHIKV propagation in vitro. (A and B) WT, Bax−/− Bak−/−, or Atg5−/− MEFs were infected with CHIKV at indicated MOI and extracellular viral titers were determined (more ...)
To further analyze the function of autophagy and apoptosis in viral infection, we assessed the percentage of infected cells after 24 h of infection by using GFP-expressing recombinant CHIKV as a marker of active infection (). At low viral dose (MOI = 0.1), Atg5−/− MEFs showed greater infection than WT controls. This difference is a result of enhanced infection and not differential viral entry, as suggested by a kinetic study of infected cells (data not depicted). Results for Bax−/− Bak−/− MEFs were even more striking as only a minority of cells were GFP+ cells, supporting that apoptotic cell death influences CHIKV propagation (). Cytometric assessment of the Bax−/− Bak−/− MEFs indicated that although fewer cells were infected, those cells that were infected showed higher expression of CHIKV E3 proteins as compared with its WT control (). This suggested that a delay in cell death allowed for greater per cell viral replication, but failure to undergo rapid cell death resulted in fewer infected cells at the population level. In contrast, autophagy-deficient cells expressed similar expression intensity to CHIKV E3 proteins as compared with its WT control, indicating that autophagy had minimal impact on virally encoded GFP expression within the cell (). These data were further confirmed in a secondary culture–based assay; cell supernatants from the respective cell lines were exposed to uninfected WT MEFs and GFP expression was scored after an additional 24-h incubation, indicating higher levels of infectious virus in the absence of autophagic flux and lower levels in apoptosis-deficient cells (unpublished data). Together, these data suggested that autophagy could regulate viral propagation by limiting the release of virus induced by apoptotic cell death.
To support this observation, we established an image analysis script using the ImageStreamX, which integrated cell area (unpublished data), thereby permitting quantification of cells in the final stages of apoptosis based on their being small and pyknotic. Importantly, analysis of the pyknotic cells indicated that ~80% expressed viral capsid protein as compared with larger, less dead cells, of which ~45% stained positive for E3 (), suggesting that ingestion of apoptotic bodies could contribute to infection of phagocytic neighboring cells (Krejbich-Trotot et al., 2011a
). Apoptotic body accumulation upon CHIKV infection was next determined in WT or Atg5−/−
MEFs (, R1). We also confirmed that accumulation of small area events was dependent on an apoptotic process, as use of z-VAD eliminated this population of cells (). Following from previous results, cells deficient for autophagy genes accumulated more apoptotic bodies as compared with WT MEFs (), whereas the Bax−/− Bak−/−
cells showed fewer numbers of events in the R1 gate ().
To establish the importance of apoptosis induction in viral propagation, WT and Atg5−/−
MEFs were treated with an apoptosis inhibitor and the percentage of infected cells was assessed (). Interestingly, apoptosis inhibition dramatically decreased the percentage of infected cells, demonstrating that apoptotic cell death, and consequently apoptotic body formation, is an important mechanism to enhance CHIKV propagation in cell culture. Based on these data we conclude that mechanistically, propagation of CHIKV can be controlled by autophagy, which acts via the limitation of infection-induced apoptosis. Although these data are in apparent contradiction with the recent study of Krejbich-Trotot et al. (2011b)
, they do not follow infectious particles, instead drawing their conclusions based on E1 expression and CHIKV mRNA transcription.
Although cell stress, cell death and viral propagation appear linked, one potential caveat is that autophagy induction enhances type I IFN production, thus providing an alternative explanation for decreased viral infection. Indeed, prior data suggests an intersection between Atg5 and RIG-I although, arguably, autophagy enhanced vesicular stomatitis virus (VSV) replication by limiting the innate immune response (Jounai et al., 2007
). To assess whether IFN-β expression is altered by autophagy induction, Atg5−/−
MEFs were infected by CHIKV and the culture supernatant was assayed at 24, 48, and 72 h after infection (unpublished data). Despite differences in viral propagation, there was no difference in IFN-β production in Atg5−/−
MEFs as compared with the WT control cells.
In vivo CHIKV infection of Atg16LHM mice results in increased apoptosis and greater lethality
Given the importance of autophagy in controlling CHIKV-induced apoptosis in vitro, we next investigated the role of autophagy in vivo using a neonatal model of infection. The disruption of autophagy genes results in embryonic lethality (Cadwell et al., 2008
). Therefore, to analyze the potential role of autophagy in CHIKV pathogenesis we used Atg16L hypomorphic mice (Atg16LHM
) in which Atg16L1
gene has been modified by gene trap mutagenesis. These mice display hypomorphic expression of Atg16L protein and reduced autophagy (not depicted; Cadwell et al., 2008
). 9-d-old WT and Atg16LHM
mice were infected with CHIKV and followed for lethality (). Although only 60% of WT mice succumbed to CHIKV, we observed a increase in lethality in Atg16LHM
mice. These data indicate that autophagy has a prosurvival function during CHIKV infection and limits disease pathogenesis. To understand how autophagy enhances survival, we first analyzed viral load in infected tissues in WT and Atg16LHM
mice (). Viral load in skin muscle and serum, important targets of CHIKV infection, were similar in WT and Atg16LHM
mice, suggesting that autophagy did not significantly affect in vivo viral infection. Similar results were obtained by analyzing the viral load in lung, liver, brain, and spleen (unpublished data). Interestingly, the one observed difference concerned a delayed clearance of CHIKV in the muscle at day 9 after infection. This is consistent with failure to thrive as a cause of death in neonatal animals (Couderc et al., 2008
). In the context of our in vitro data, and the fact that the Atg16LHM
have only a partial block in autophagy (Cadwell et al., 2008
), we suggest that the muscle tissue may be highly sensitive to the autophagy-mediated protective effects of CHIKV infection.
Figure 8. Autophagy limits apoptotic induction in CHIKV infected tissues and delays lethality of mice. (A–F) WT (n = 43) and Atg16LHM mice (n = 34) were infected at 9 d of age with 4 × 105 PFU CHIKV subcutaneously. (A) Mice were monitored for lethality (more ...)
To understand how autophagy could protect mice against CHIKV-induced CPE, we further investigated the impact of autophagy on apoptosis in infected tissues. We first analyzed whether apoptosis could be detected during CHIKV pathogenesis. 9-d-old WT mice were infected and, after 5 d, muscles, skin, liver, brain, BM, and spleen were collected and stained for the expression of E3 (, red) and active caspase-3 (a-CASP3; , green). Both E3 and a-casp3 could be readily detected in muscle and skin but not in other tissues. Of note, a-casp3 was detected in the BM; however, this could be attributed to higher basal apoptosis as uninfected animals had similar numbers of a-casp3–positive cells. Interestingly, apoptosis induction in infected tissues was observed in infected cells (determined by colocalization of E3 and a-casp3) as well as in bystander cells (no E3 staining), suggesting, as seen in our in vitro studies, that both intrinsic and extrinsic apoptosis could be induced. These studies were extended to Atg16LHM mice, and although similar levels of E3-expressing cells were found in the muscle and skin, we observed higher levels of a-casp3–positive cells (). Remarkably, the enhanced levels of apoptosis were observed only in infected cells and did not alter the level of bystander cell death, again supporting the relevance of our in vitro findings ( and ). Similar observations were shown in skin (unpublished data). These results demonstrate the relevance of our findings and support a role for autophagy as a regulator of apoptosis, thus characterizing a novel mechanism of host response during CHIKV infection.