ORIGINAL RESEARCH

published: 29 May 2018

doi: 10.3389/fmicb.2018.01091

Autophagy Benefits the Replication

of Egg Drop Syndrome Virus in Duck
Embryo Fibroblasts
Xueping Wang † , Xuefeng Qi † , Bo Yang, Shuying Chen and Jingyu Wang*
College of Veterinary Medicine, Northwest A&F University, Yangling, China

Egg drop syndrome virus (EDSV) is an economically important pathogen with a broad
host range, and it causes disease that leads to markedly decreased egg production.
Although EDSV is known to induce apoptosis in duck embryo fibroblasts (DEFs), the
interaction between EDSV and its host needs to be further researched. Here, we
provide the first evidence that EDSV infection triggers autophagy in DEFs through
increases in autophagosome-like double-membrane vesicles, the conversion of LC3-I
to LC3-II, and LC3 colocalization with viral hexon proteins. Conversely, P62/SQSTM1
Edited by:
degradation, LC3-II turnover, and colocalization of LAMP and LC3 confirmed that EDSV
Akio Adachi,
Kansai Medical University, Japan infection triggers complete autophagy. Furthermore, we demonstrated that inhibition
Reviewed by: of autophagy by chloroquine (CQ) and 3-methyladenine (3MA) or RNA interference
Deepak Shukla, targeting ATG-7 decreased the yield of EDSV progeny. In contrast, induction of
University of Illinois at Chicago,
United States
autophagy by rapamycin increased the EDSV progeny yield. In addition, we preliminarily
Anchun Cheng, demonstrated that the class I phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway
Sichuan Agricultural University, China
contributes to autophagic induction following EDSV infection. Altogether, these finding
Dwayne G. Stupack,
University of California, San Diego, lead us to conclude that EDSV infection induces autophagy, which benefits its
United States own replication in host cells. These findings provide novel insights into EDSV–host
*Correspondence: interactions.
Jingyu Wang
[email protected] Keywords: egg drop syndrome disease virus, autophagy, virus replication, autophagic flux, DEF cells
† These authors have contributed
equally to this work.
INTRODUCTION
Specialty section:
This article was submitted to Egg drop syndrome (EDS) is one of the most economically important diseases in the poultry
Virology, industry, and it has wide host range, such as turkey breeder flocks, healthy laying birds, quail, and
a section of the journal geese (Brugh et al., 1984; Bidin et al., 2007; Hafez, 2011). EDS is characterized by healthy laying hens
Frontiers in Microbiology
apparently changing to produce eggs of poor quality, with clinical signs including soft, thin-shelled
Received: 27 December 2017 eggs, and shell-less eggs (Huang J. et al., 2015). EDS was first reported in Holland in 1976, and
Accepted: 07 May 2018 the infection then spread throughout the world and more recently appeared in China (Baxendale,
Published: 29 May 2018
1978; McFerran and Smyth, 2000; Ezeibe et al., 2008; Fu et al., 2013). The causative agent, egg drop
Citation: syndrome virus (EDSV) belong to the genus Atadenovirus within the family Adenoviridae, which
Wang X, Qi X, Yang B, Chen S and
are non-enveloped viruses with an approximately 30–35 kb linear double-stranded DNA genome
Wang J (2018) Autophagy Benefits
the Replication of Egg Drop
(Fu et al., 2013; Larson et al., 2015). The adenovirus size range is 75–80 nm, and the replicative
Syndrome Virus in Duck Embryo cycle is divided into two phases: early protein expression (E1–E4) and late protein expression
Fibroblasts. Front. Microbiol. 9:1091. (L1–L5) (Larson et al., 2015). Accumulating evidence has demonstrated that adenoviral proteins
doi: 10.3389/fmicb.2018.01091 E1A and E1B play an essential role in establishing a productive virus infection by regulating cell

Frontiers in Microbiology | www.frontiersin.org 1 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

proliferation, apoptosis, and autophagy (Kirn, 2000; Dobbelstein, from the Green Square Biological Engineering Company (Shanxi,
2004; Rodriguez-Rocha et al., 2011). In addition, hexon is the China) and was propagated in the allantoic fluid of 10-day-
main building block of the adenovirus capsid. In recent years, old specific-pathogen-free embryonated duck eggs at 38.5◦ C for
numerous studies have revealed that hexon has a role in receptor 120 h. Virus titers for DEFs were determined on the basis of
binding, entry into the cell and apoptosis (Solanki et al., 2016; the 50% tissue culture infective dose (TCID50 )/mL using the
Yan et al., 2016; Qi et al., 2017). Despite the elucidation of some Reed–Muench method. The EDSV127 strain was at a titer of
aspects of EDSV pathogenesis, the underlying mechanism of 106.1 /0.1 mL. To obtain a replication-incompetent EDSV strain,
EDSV replication is still largely unknown. virus suspensions (2 mL) were irradiated with 254 nm UV light
Autophagy is an evolutionarily conserved process in for 1 h. The lack of replication of UV-inactivation of EDSV was
eukaryotes that maintain cellular homeostasis, including confirmed in DEFs. pcDNA3.1-hexon plasmids, or pCDNA3.1-
metabolic balance (Klionsky, 2007; Kroemer et al., 2010). control plasmids were previously established by our team (Qi
Autophagic activity is known to be controlled by a series of et al., 2017) and kept in our laboratory. All experiments related
autophagy-related genes (Atg). Up to now, over 30 specific to EDSV were done in a P2 biosafety laboratory and strictly
genes that take part in autophagy pathways have been identified carried out according to the Laboratory Biosafety Manual in our
in yeast (Klionsky et al., 2003; Milan et al., 2015). Upon laboratory.
autophagy induction, oncolytic adenoviruses activate a series of
autophagy-related biomarker proteins, such as autophagy related Viral Infection and Cell Treatment
microtubule-associated protein 1 light chain 3 (ATG8/LC3), Duck embryo fibroblasts in 6-well plates were infected with
Atg5 and Atg12, which form a complex that accumulates at EDSV at a multiplicity of infection (MOI) of 1 at 37◦ C
the isolation membrane, and the polyubiquitin binding protein (Supplementary Figure S1). Following a 2 h adsorption,
p62/SQSTM1 (p62), which assists in assessing autophagic flux unattached viruses were removed by aspiration, and the cells were
because of the functional link between LC3 and the ubiquitinated washed three times with sterile PBS (pH 7.4) and incubated at
substrates degraded by the autophagy pathway (Rodriguezrocha 37◦ C in DMEM supplemented with 2% FBS until samples were
et al., 2011). Although autophagy was originally recognized as a harvested at different times. For autophagy induction, the DEFs
protective function in the survival response of cells to a variety were pretreated with rapamycin (Rapa: 100 nM; R0395, Sigma)
of extracellular and intracellular stimuli, it also plays a role in dissolved in DMSO (D2650, Sigma) for 2 h, followed by a 2 h
pathogen infection (Shintani and Klionsky, 2004; Levine and absorption of EDSV. The cells were cultured in fresh medium
Kroemer, 2008; Deretic, 2010). Increasing evidence indicates in the presence of 100 nM rapamycin until being harvested. For
that the autophagosomal structures induced by many RNA or autophagy inhibition experiments, the DEFs were treated with
DNA viruses may serve as platforms for viral replication (Suhy chloroquine (CQ: 50 µM; C6628, Sigma) dissolved in PBS for
et al., 2000; Wong et al., 2008; Huang et al., 2009). In particular, 2 h prior to virus infection. In addition, cells were treated with 3-
recent research findings show that oncolytic adenovirus-induced methyladenine (3MA: 5 mM; M9281, Sigma) or 740Y-P (25 µM;
autophagy promotes virus replication (Rodriguezrocha et al., ApexBio Technology, Houston, TX, United States) dissolved in
2011). However, the extent to which autophagy is induced during DMSO for 2 h prior to virus infection. Then, the DEFs were
EDSV infection remains largely unknown. infected with EDSV at an MOI of 1. The inoculum was removed
The aim of the present study was to elucidate whether and cells were washed twice with PBS, and the cells were then
autophagy can be induced in permissive duck embryo fibroblasts incubated in fresh medium containing rapamycin (100 nM) or
(DEFs) during EDSV infection. By regulating autophagy 3MA (5 mM) until harvesting of the cells or the culture medium.
via pharmacological treatment and RNA interference, we Moreover, the same amount of dimethyl sulfoxide (DMSO) was
demonstrate that autophagy plays a positive and critical role in added to separate cells as the control group.
EDSV replication. In addition, we preliminarily revealed that
the P13K/Akt/mTOR signaling pathway contributes to triggering Cell Viability Detection
autophagy in EDSV-infected host cells. A better comprehension For cell viability experiments, the optimal concentration of the
of the interactions between EDSV and host autophagic responses corresponding drug used in this experiment was determined by
will provide new insights into viral pathogenesis and antiviral the Cell Counting Kit-8 (96992, Sigma) assay according to the
drug development. manufacturer’s instructions. In brief, 1 × 104 DEFs were seeded
in each well of 96-well culture plates, treated with each drug
at different concentrations and then cultured for 24 h. Then,
MATERIALS AND METHODS according to existing experimental procedures, the medium was
removed and replaced with fresh medium containing CQ (25,
Cells and Viruses 50, or 100 µM), rapamycin (50, 100, or 300 nM), 740Y-P (10,
Duck embryo fibroblasts were obtained from 11- to 12-day-old 25, or 50 µM), or 3MA (1, 5, or 10 mM) and incubated
duck embryos as previously described (Huang W.R. et al., 2015), for 24 h. Premixed WST-8 cell proliferation reagent (10 µl)
DEFs were maintained in high-glucose Dulbecco’s modified was then added to each well and incubated at 37◦ C for 4 h.
Eagle’s medium (DMEM, Gibco, United States) supplemented Cell viability was evaluated by the measuring the absorbance
with 10% fetal bovine serum (Gibco, United States) at 37◦ C in at 450 nm, which was recorded using a 96-well plate reader
5% CO2 . The EDSV127 strain (GenBank: Y09598) was obtained (Bio-Rad Laboratories, Inc.).

Frontiers in Microbiology | www.frontiersin.org 2 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

Transmission Electron Microscopy (TEM) were transiently transfected with ATG7 or scrambled siRNA
Duck embryo fibroblasts were mock infected or infected with with Lipofectamine 2000 (11668-27, Invitrogen) as previously
EDSV at an MOI of 1 for 2 h. Then, the cells were cultured described (Zhang et al., 2011). We used western blotting to test
in 2% FBS for 36 h and were then washed three times with the interference efficiency of ATG7; scrambled siRNA was used
PBS (pH 7.4) and scraped free. DEFs were then collected by as a negative control.
centrifugation at 1000 × g for 10 min. The cell pellets were
fixed with 2.5% glutaraldehyde/0.1 M phosphate buffer solution
overnight at 4◦ C, washed three times in phosphate buffer solution RESULTS
for 10 min, and post-fixed with 1% osmium tetroxide at 4◦ C for
1 h. Following dehydration in a graded series of ethanol solutions, EDSV Infection Upregulated the Levels
samples were embedded in a Spurr’s plastic resin. Finally, the of Autophagy in DEFs
autophagosome-like vesicles were examined under a Hitachi HT- To investigate whether autophagy is induced in DEFs by
7700 transmission electron microscopy (TEM) (Hitachi High EDSV infection, the cells were mock infected or infected with
Technologies, Co., Japan). the EDSV-127 strain or DEFs were treated with rapamycin
for 36 hpi, and they were then observed through TEM. As
Western Blot Analysis shown in Figure 1a, double-membrane vesicles were rarely
Duck embryo fibroblasts infected with EDSV or treated with observed in mock-infected cells, whereas a large number
a drug were harvested at the indicated time, and cell samples of single-membrane vesicles and double-or single-membrane
were lysed with RIPA lysis buffer (P0013, Beyotime, Beijing, vesicles were observed in the cytoplasm of EDSV-infected DEFs
China). The lysates were briefly sonicated and cleared by (Figure 1b). These double-membrane structures were similar
centrifugation at 12,000 × g for 5 min at 4◦ C. Protein samples to the autophagosomes in DEFs induced by rapamycin, a well-
were diluted in 5× SDS-PAGE loading buffer and were heated known autophagy inducer, suggesting that the autophagosomes
at 100◦ C for 5 min; after centrifugation at 12,000 × g for were induced in DEFs by EDSV infection (Figure 1c). Higher-
5 min, the samples were then separated on 12% SDS-PAGE magnification images of autophagosome-like vesicles in EDSV-
gels and transferred to nitrocellulose membranes (Millipore, infected cells are show in Figures 1d,e,f. Quantitative analyses
ISEQ00010). After incubation in blocking buffer (5% non- showed that the number of autophagosome-like vesicles in the
fat dry milk solution containing 0.05% Tween-20) at room cytoplasm of EDSV-infected DEFs was significantly higher than
temperature for 2 h, membranes were incubated overnight the number when DEFs were treated with rapamycin and the
with the primary antibodies rabbit anti-LC3B (L7543, Sigma), number in mock-infected DEFs (Figure 1g).
rabbit anti-p62/SQSTM1 (P0067, Sigma), rabbit anti-ATG-7 Next, to further examine the autophagy induced by EDSV
(A2856, Sigma), and antibodies for PI3-k (#4257), phospho- infection, we performed western blot assays to analyze the
PI3kinase p85 (Tyr458) (#4228), Akt (#4691),phospho-Akt conversion of LC3-I to LC3-II. As shown in Figure 2A,
(Ser473) (#9271), mTOR (#2983), phospho-mTOR (ser2448) we observed that LC3-II levels in EDSV-infected cells began
(#2971) were purchased from cell signaling Technology, Inc. to increase at 24 hpi, and LC3-l-to-LC3-II conversion was
(Danvers, MA, United States). After thorough washing with significantly greater in EDSV-infected DEFs than in mock-
Tris-buffered saline with Tween (TBST), the membranes infected cells at the indicated time. The difference in the
were reacted with corresponding HRP-conjugated secondary ratio of LC3-II/LC3-I between the EDSV-infected cells and the
antibodies (LI-COR Biosciences) for 2 h at room temperature. mock-infected cells was significant (P < 0.001) at 48 and 60
The target protein bands were visualized by an enhanced hpi (Figure 2B). Meanwhile, hexon proteins that specifically
chemiluminescence detection kit (Thermo Fisher Scientific) recognize EDSV were used to track the progression of infection.
with the ECL Plus western blot detection system (Amersham As shown in Figure 2A, increased viral levels were detected in
Biosciences, Piscataway, NJ, United States). EDSV-infected cells from 24 to 48 hpi.
In addition, EDSV infection led to a significant enhancement
Cell Transfection of punctate staining signals for LC3 distributed throughout the
Duck embryo fibroblasts were seeded in 12-well plated grown entire cytoplasm (Figure 2C), whereas the mock-infected DEFs
to 80% confluence were transfected with pCDNA3.1-hexon exhibited no LC3 punctate accumulation. More importantly,
or an empty vector (pCDNA3.1-control). Four milligrams signals of hexon protein from EDSV in EDSV-infected cells
of expression plasmids combined with 10 µl Lipofectamine were colocalized with the punctate green fluorescent staining
2000 reagent (Thermo, Lithuania) was added to the cells of LC3 (Figure 2C). Based on these experiment, we concluded
according to the manufacturer’s instructions. Twenty-four hours that autophagy was significantly enhanced in DEFs upon EDSV
post-transfection, the cells were seeded in 6-well plates for infection.
immunoblotting or cell viability assay.
Increased Level of Autophagic Flux in
RNA Interference EDSV-Infected DEFs
Duck embryo fibroblasts were seeded in six-well cell culture P62 also known as sequestosome-1 (SQSTM1), is multifunctional
plates. When cells had grown to 60 to 70% confluence, they protein that has been recognized as an indicator for assessing

Frontiers in Microbiology | www.frontiersin.org 3 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

FIGURE 1 | Egg drop syndrome virus (EDSV) infection triggers autophagosome-like vesicles formation in duck embryo fibroblasts (DEFs). (a) TEM observation. DEFs
were mock-treated for 36 hpi, (b) infected with EDSV at an MOI of 1 for 36 hpi or (c) treated with rapamycin for 36 hpi and then processed for observation. Scale
bar: 2 µm. (d,e) High magnification images of the typical autophagosome-like vesicles in EDSV-infected and (f) rapamycin-treated cells are shows. Scale bar:
0.5 µm. (g) The number of autophagosome-like vesicles per cell profile in mock- and EDSV-infected DEFs and rapamycin-treated DEFs were quantified. The results
are shown as the mean ± SEM (n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

autophagic flux (Lippai and Low, 2014). As shown in Figure 3A, demonstrated in Figure 4C, the colocalization of LC3 and
immunoblotting revealed significant progressive degradation of LAMP1 was detected in EDSV-infected DEFs, while mock-
p62 in EDSV-infected cells compared with the unaltered p62 infected cells showed no overlap (Figure 4D). These results
levels in mock-infected cells, suggesting that autophagosomes indicated that DEFs go through a complete autophagic process
were able to fuse with lysosomes to degrade the cargos. The ratios following EDSV infection.
of p62/β-actin shown in Figure 3B indicate that p62 reduction
was notable at 48 hpi and peaked at 60 hpi. Virus Replication Is Required for EDSV
Then, we used western blot-based measurements of LC3-ll Induction of Autophagy
turnover in the presence or absence of the lysosomal protease To reveal whether EDSV replication is necessary for the
inhibitor chloroquine (CQ), which can inhibit the fusion induction of autophagy, ultraviolet (UV)-irradiated EDSV was
of autophagosomes with lysosomes and leads to a marked used to infect DEFs. As shown in Figures 5A,B, LC3 proteins in
accumulation of autophagic vacuoles, inducing intense LC3- UV-inactivated EDSV-infected DEFs did not undergo conversion
II accumulation (Boya et al., 2005; Geng et al., 2010). As from LC3-l to LC3-ll, and the expression of hexon proteins
demonstrated in Figures 4A,B the accumulation of LC3-II and was not detected in this group. In contrast, the levels of both
p62 was observed with CQ treatment, representing enhanced of LC3-I and LC3-II in DEFs indicated apparent conversion in
autophagic flux at 24 and 48 hpi post-infection in EDSV-infected DEFs inoculated with live EDSV, which was accompanied with
DEFs compared with the mock-treated cells. an increase in hexon protein levels. Furthermore, the results of
Furthermore, we measured LC3 protein colocalization confocal microscopy further verified that UV-inactivated EDSV
with lysosome-associated membrane protein 1 (LAMP1). As was not able to induce the LC3 colocalized with hexon proteins in

Frontiers in Microbiology | www.frontiersin.org 4 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

FIGURE 2 | Modification of LC3 proteins during EDSV infection. (A) The significant conversion of LC3-l to LC3-ll was detected in mock-or EDSV-infected DEFs using
an anti-LC3B antibody, and the expression of hexon from EDSV was also detected using anti-hexon antibodies. (B) The optical densities of each protein band were
measured by densitometric scanning, and the optical density ratio of LC3-ll/LC3-l was calculated. The results are shown as the mean ± SEM (n = 3). ∗ P < 0.05;
∗∗ P < 0.01; ∗∗∗ P < 0.001. (C) Confocal immunofluorescence microscopy images of mock-infected control and EDSV-infected DEFs. Mock- and EDSV-infected

cells were fixed at 36 hpi and stained with antibodies against LC3 and EDSV hexon and then merged. Scale bar: 10 µm. Cell nuclei were counterstained with
Hoechst. Fluorescence images were examined under a confocal laser scanning microscope. Scale bar = 10 µm.

FIGURE 3 | Egg drop syndrome virus infection enhances p62 degradation in DEFs. (A) A representative image of a western blot analysis of relative levels of p62 in
mock- or EDSV-infected DEFs at indicated time points. (B) The optical densities of each protein band were measured by densitometric scanning, and the optical
density ratio of p62/β-actin was calculated. The results are shown as the mean ± SEM (n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; # Means no significant
difference, P > 0.05.

EDSV-infected DEFs (Figure 5C). In brief, the results indicated (Cina et al., 2012). Cells were harvested at 24 and 48 hpi
that virus replication was necessary for induction of autophagy and then subjected to western blot analysis. Meanwhile, virus
by EDSV infection. progeny were collected at the corresponding time points for
titration.
The Replication of EDSV in DEFs Is Compared with the mock-infected DEFs (Figure 6A), LC3
Enhanced by the Induction of Autophagy punctate staining increased in the DEFs treated with rapamycin
To investigate whether the replication of EDSV in DEFs (Figure 6A). Moreover, we further observed that LC3-ll exhibited
is enhanced by autophagy induction, DEFs, prior to virus an increasing trend in rapamycin-treated cells compared to the
inoculation, were treated with rapamycin, which can specifically cells treated with DMSO (Figures 6B,C). These results show
induce the autophagy pathway by directly inhibiting mTOR that rapamycin treatment enhanced autophagy in DEFs. Along

Frontiers in Microbiology | www.frontiersin.org 5 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

FIGURE 4 | Egg drop syndrome virus infection enhances autophagic flux. (A) Western blot analyses of the effect of CQ treatment on the expression of both LC3-ll
and p62 proteins in DEFs. Cell lysates were subjected to western blot analyses using antibodies against LC3, p62, and β-actin. Representative profiles are shown,
and similar results were obtained in three independent experiments. (B,C) Analysis of changes in the relative amounts of both LC3-ll and p62 in rapamycin-
pretreated and EDSV-infected DEFs in the absence and presence of CQ. (D) Representative images of colocalization of LC3 with the endosomal/lysosomal marker
LAMP1 in mock- and EDSV-infected DEFs. Cell nuclei were counterstained with Hoechst. Fluorescence images were examined under a confocal laser scanning
microscope. The results are shown as the mean ± SEM (n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. Scale bar = 10 µm.

with the enhancement of autophagy in EDSV-infected DEFs, inhibition will have on virus replication. 3MA is widely used
the expression of EDSV hexon proteins was increased following to inhibit autophagy at the early stage by targeting class III
treatment with rapamycin (Figures 6B,C). Additionally, we phosphatidylinositol-3-kinase (P13k) (Seglen and Gordon, 1982).
observed that the titers of EDSV in the infected DEFs treated with 3MA treatment not only reduced LC3-l/LC3-ll conversion but
rapamycin were higher than those in the infected DEFs treated also reduction expression of hexon proteins and the yields of virus
with DMSO (Figure 6D). These results suggest that autophagy titers at 24 and 48 hpi (Figures 7A,B).
enhanced the replication of EDSV in DEFs. To further confirm the relationship between autophagy
and EDSV replication. We depleted the essential endogenous
The Replication of EDSV Is Reduced by ATG7, which encodes an E1-like enzyme that is essential
the Inhibition of Autophagy to the formation of autophagic vacuoles (Kim et al., 1999),
Since the replication of EDSV in DEFs is enhanced by the and knockdown of the ATG7 gene can seriously impair
induction of autophagy, we may ask what effect autophagy the autophagic pathway (Komatsu et al., 2005). As expected,

Frontiers in Microbiology | www.frontiersin.org 6 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

FIGURE 5 | Western blot analysis of the ability of UV-inactivated EDSV to induce the conversion of LC3-l to LC3-ll in DEFs. (A) Cells infected with replication
-competent EDSV or with UV-inactivated EDSV (UV-EDSV) at an MOI of 1. (B) Densitometric analysis of relative levels of LC3-II and LC3-I in EDSV- or UV-EDSV
-infected DEFs. The results are shown as the mean ± SEM (n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. (C) Confocal microscopy analysis of the ability of
UV-EDSV to induce autophagy in DEFs. Scale bar = 15 µm.

transfection with ATG7-targeting siRNA resulted in an obvious DEFs with PCDNA-hexon. As shown in Figure 9A, the hexon
decrease of endogenous Atg7 proteins at 48 hpi. Meanwhile, protein expression levels increased in parallel with an increase
the expression of LC3-ll was slightly decreased (Figure 7C). in LC3-l-to-LC3-ll conversion, and P62 gradually decreased in
In addition, we found that the expression of EDSV hexon hexon-transfected DEFs. These results show that EDSV hexon
proteins (Figure 7C) as well as the yield of EDSV progeny expression can induce autophagy in DEFs. To examined whether
were significantly reduced (Figure 7D). These data support the pI3K/Akt/mTOR signaling pathway was associated with
our hypothesis that autophagy induction could facilitate EDSV hexon-induced autophagy in hexon-transfected DEFs. As show in
replication. Figures 9A,B. The p-P13k, p-Akt, and p-mTOR phosphorylation
protein expression levels was decreased in hexon-transfected
EDSV Triggered Autophagy in DEFs via DEFs at 48 hpi. To further verify the above findings, hexon-
transfected DEFs were treated with 740Y-P or without 740Y-P.
PI3K/Akt/mTOR Signaling Pathway The results showed that the downregulation of p-P13k, p-mTOR
The role of the pI3K/Akt/mTOR pathway in induction of
and p-Akt was significantly recovered in the presence of 740Y-
autophagy was examined in EDSV-infected DEFs. As shown
P (Figures 9A,B). Altogether, these results indicated that hexon
in Figure 8. EDSV infection resulted in significant reductions
not only induced autophagy, but reduced the phosphorylation of
in phosphorylation of p-pI3k, p-Akt, and p-mTOR in DEFs
p-P13k, p-mTOR, and p-Akt in DEFs at 48 hpi.
at indicated time points post-infection (Figures 8A,B).
However, the expression of LC3-II was increased after
Modulation of Autophagy Activity With
EDSV infection (Figures 8A,B). To further verify the
above findings, EDSV-infected DEFs were treated with or Autophagy Regulators Does Not Affect
without 740Y-P, which is a PI3K activator that can lead to Cell Viability
the upregulation of p-mTOR and p-Akt (Zhang et al., 2015). Moreover, we tested whether high concentrations of drugs used
The results showed that the downregulation of p-mTOR to pharmacologically alter autophagy, including CQ treatment,
and p-Akt was significantly recovered in the presence of 3MA treatment, rapamycin treatment and 740Y-P treatment,
740Y-P (Figure 8C). These results indicate that EDSV reduces affected the capability of EDSV to replicate. Statistical analyses
p-P13k, p-mTOR, and p-Akt protein phosphorylation in infected revealed no significant effects by Cell Counting Kit-8 assay
DEFs. (Figure 10).

Hexon Triggered Autophagy in DEFs via DISCUSSION

PI3K/Akt/mTOR Signaling Pathway
In order to study whether hexon expression also induces As an economically important disease of the poultry industry,
autophagy in DEFs as similar as EDSV infection, we transfected EDSV infection reduces egg production in chickens and induces

Frontiers in Microbiology | www.frontiersin.org 7 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

FIGURE 6 | Autophagy induction enhanced the replication of EDSV. (A) A representative image from confocal immunofluorescence microscopy analysis of LC3
expression in EDSV-infected DEFs or DEFs treated with 100 nM rapamycin prior to EDSV infection. Scale bar = 10 µm. (B) Western blot analyses the cells infected
with DMSO (mock), cells treated with rapamycin for 4 h and then infected with EDSV at an MOI of 1 (Rapa + v), cells treated with DMSO for 4 h and then infected
with EDSV at an MOI of 1 (DMSO + v), which were harvested at 24 and 48 hpi. (C) Densitometric analysis of both LC3-ll and β-actin proteins in mock, Rapa + v, and
DMSO + v groups. (D) Quantification of the yields of virus progeny in EDSV-infected DEFs pretreated with DMSO (DMSO+V) and in EDSV-infected cells pretreated
with rapamycin (Rapa+v) were determined according to the TCID50/mL. The results are shown as the mean ± SEM (n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

an acute respiratory disease in goslings, leading to substantial evidence of egg drop syndrome (EDS) has been reported in a
economic losses. It is believed that ducks are a natural host variety of wild waterfowl species (Cha et al., 2013a,b; Huang J.
of EDSV since EDSV and EDSV antibodies have been found et al., 2015). DEFs are the natural target cells of EDSV, and they
repeatedly in various domesticated ducks, and global serologic provide a valuable in vitro model reference to study host–virus

Frontiers in Microbiology | www.frontiersin.org 8 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

FIGURE 7 | Inhibition of autophagy reduces EDSV replication in EDSV cells. (A) Western blot analysis of the effect of 3MA (5 mM) on the expression of both LC3 and
hexon proteins in DEFs. Cells infected with DMSO (mock), cells infected with EDSV at an MOI of 1 (DMSO+V) and cells treated with 3MA for 4 h and then infected
with EDSV an MOI of 1 (3MA+V) were harvested at 24 and 48 hpi. Cell samples were processed for western blot analysis, using antibodies against LC3, hexon, and
β-actin, at 24 and 48 hpi. (C) Western blot analysis of the effect of ATG7 silencing on the expression of both LC3 and hexon proteins in DEFs. DEFs were transfected
with either ATG7 siRNA (ATG7) or non-targeting siRNA (Scramble). Cell samples were processed for western blot analysis using antibodies against ATG7, LC3,
hexon, and β-actin at 48 hpi. (B,D) Quantification of the yields of progeny virus in EDSV-infected DEFs were determined according to the TCID50/mL. The results are
shown as the mean ± SEM (n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

interaction (Wang et al., 2015; Qi et al., 2017; Yin et al., 2017). et al., 2010). Notably, the autophagic response to different
Many researchers have mainly focused on adenovirus infections viruses is not always the same. The autophagic flux induced by
in human cells, and little is known about adenovirus infections adenoviruses and duck enteritis virus is a complete autophagic
in avian-derived cells. Huang J. et al. (2015) found that DEFs response (Jiang et al., 2011; Yin et al., 2017). However, influenza
are suitable hosts for EDSV replication. Qi et al. (2017) study A virus and Rotavirus, by blocking autophagosome degradation,
demonstrated that in vitro infection of DEFs with EDSV caused induce an incomplete autophagic response (Gannage et al.,
apoptosis, which may have been associated with virus replication. 2009; Crawford et al., 2012). This implies that autophagic
Recently, many researchers have documented that apoptosis has flux is virus-specific. To clarify whether infection with EDSV
a complex interaction with autophagy or can even be induced induces complete autophagy, we used several means to measuring
simultaneously by pathogen infection (Gougeon and Piacentini, autophagic flux, including monitoring p62 degradation after
2009; Xin et al., 2014). Thus, it is essential to resolve the question EDSV infection. The lysosome inhibitor CQ increased the
of whether EDSV infection in host cells triggers autophagy and levels of LC3-II and p62 in virus-infected cells. Furthermore,
what its role is in this process. fluorescent LAMP1 puncta colocalized with LC3-positive puncta
In this study, we demonstrated that EDSV triggers autophagy in EDSV-infected cells. These findings demonstrated that
in DEFs, as shown by TEM, including the presence double- complete autophagic flux was triggered upon EDSV infection.
and single-membrane vesicles, indicating that autophagosomes Egg drop syndrome virus infection can trigger complete
were formed. Meanwhile, we monitored the conversion of LC3- autophagy in DEFs; however the role of the EDSV replication
l/LC3-ll in EDSV-infected DEFs by western blot analysis. In process in regulating autophagy is unclear. Autophagy is a
addition, we found that hexon proteins of EDSV were colocalized balancing mechanism that maintains the homeostasis of cells.
with LC3 proteins in the EDSV-infected DEFs, as shown by Autophagy commonly serves as a defense mechanism to clear
confocal microscopy analysis. Our findings provide evidence viruses and reduce viral growth (Yakoub and Shukla, 2015).
that EDSV infection is able to induce autophagy in DEFs. We Nevertheless, growing evidence suggests that some DNA viruses
also assessed whether EDSV infection of DEFs triggers complete such as hepatitis B virus, duck enteritis virus, and adenoviruses
autophagy. Complete autophagy involves the dynamic process of have evolved strategies to use autophagic vesicles for replication
autophagosome synthesis, including autophagosome formation (Li et al., 2011; Rodriguezrocha et al., 2011; Yin et al., 2017). In
and maturation, and degradation of autophagosomes within further determining what role autophagy plays in the replication
lysosomes; thus, autophagic flux is a more accurate indicator of of EDSV, we observed that EDSV infection resulted in LC3-
autophagy activity than autophagosome formation (Mizushima l/LC3-ll conversion, which continued to increase with infection

Frontiers in Microbiology | www.frontiersin.org 9 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

FIGURE 8 | Egg drop syndrome virus induced autophagy in DEFs via the P13k/Akt/mTOR pathway. (A) DEFs infected with EDSV was harvested at the indicated
time points post-infection and analyzed for the expression of p-P13k, P13k, p-mTOR, mTOR p-Akt, and Akt by western blot assay. (C) The levels of Akt, p-Akt,
p-mTOR, and mTOR in DEFs treated with 0.1% DMSO (control group), EDSV, EDSV + 740Y-P (25 µM), 740Y-P (25 µM) were measured by western blot assay.
(B,D) The optical densities of each protein band were measured by densitometric scanning, and the optical density ratios of p-P13k/P13k, p-mTOR/mTOR, and
p-Akt/Akt were calculated. The results are shown as the mean ± SEM (n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001, “#” means no significant difference, P > 0.05.

FIGURE 9 | Hexon triggered autophagy in DEFs via the P13k/Akt/mTOR pathway. (A) Western blot analysis of the effect of 740Y-P (25 mM) on the expression of
p-PI3k, PI3k, p-Akt, Akt, p-mTOR, mTOR, LC3, P62, and hexon proteins in DEFs. Cell lysates were subjected to western blot analyses using antibodies against
p-PI3k, PI3k, LC3, p62, hexon, and β-actin. Representative profiles are shown, and similar results were obtained in three independent experiments. (B) The optical
densities of each protein band were measured by densitometric scanning, and the optical density ratios of LC3II/I, P62/β-actin, hexon/β-actin, p-P13k/P13k,
p-mTOR/mTOR, and p-Akt/Akt were calculated. The results are shown as the mean ± SEM (n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001, “#” means no significant
difference, P > 0.05.

Frontiers in Microbiology | www.frontiersin.org 10 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

regulating autophagy (Jung et al., 2010; Kim et al., 2011;

Cao et al., 2017). However, adenoviral and adenoviral-related
protein regulation of autophagy through the PI3K/Akt/mTOR
signaling pathway has rarely been reported. In our study, we
observed that at the middle to late stages of EDSV infection,
there was a reduction in p-P13K, p-Akt, and p-mTOR protein
phosphorylation in infected DEFs, indicating a critical role of the
PI3K/Akt/mTOR pathway in EDSV-triggered autophagy.
Increasing evidence indicates that PI3K activation is affected
by many factors, such as different sources of virus strains, cell
lines and different viral proteins, that may cause significant
differences in the regulation of P13K signaling pathways (Shin
et al., 2007; Ma et al., 2011; Huang W.R. et al., 2015; Xie et al.,
2016). Some studies reported that adenovirus penton proteins
interact with αv integrins to promote adenovirus internalization
and that this specifically involves mediation of PI3K activation
(Li et al., 1998). However, previous studies of EDSV penton
proteins are limited to serological surveys and the complete viral
FIGURE 10 | Pharmacological alterations had no effects on cell viability. DEF
viability was determined by WST-8 cell proliferation assay after treatments with
genome analysis and what role dose the penton proteins play
rapamycin (Rapa), chloroquine (CQ), 3-methyladenine (3MA) or 740Y-P or during EDSV infection and replication are unknown (Rohn et al.,
transfection with siATG7 for 48 h. Data are presented as the mean ± SEM of 1997; Xiao et al., 2009). Therefore, some questions naturally arise,
three independent experiments. “#” means no significant difference, P > 0.05. such as whether the penton protein promotes the internalization
of EDSV and whether the process requires the downregulation
or activation of PI3K. Moreover, the expression of E4 induced
time. These data demonstrate that the autophagy induced by by E1A via cellular transcription factor E4F, participates in the
EDSV infection may be closely correlated with viral replication. activation of the PI3K/mTOR pathway and play an important
Notably, UV-inactivated EDSV does not trigger LC3-l/LC3- role in adenovirus-infection (Raychaudhuri et al., 1987; O’Shea
ll conversion in DEFs. This result was further confirmed by et al., 2005). Whereas, according to published researches, E1A
confocal microscopy, which showed that UV-inactivated EDSV cannot be found in EDSV (Qi et al., 2017). In terms of this,
did not result in LC3 colocalizing with hexon proteins in further study is needed to clarify how the lack of E1A proteins
EDSV-infected DEFs. Therefore, it may be implied that EDSV affects the function of other proteins or which proteins can
replication is necessary during autophagosome formation. Then, replace E1A proteins and perform its function in EDSV-infected
we analyzed the specific effect of autophagy on EDSV infection host cells. Hexon is the major protein of the virus capsid and
by administering pharmacological regulators. Our data showed is important for the uptake of adenovirus into cells. Given the
that rapamycin treatment not only upregulated the expression findings that there is a temporal correlation between changes
of viral hexon proteins but also increased the yield of EDSV in autophagy and hexon expression levels observed in EDSV-
progeny. We also demonstrated that inhibition of autophagy infected DEFs, we asked whether hexon can induce autophagy
with 3MA or shRNA-based depletion of the essential autophagy during EDSV infection. In this study, we found that hexon
protein ATG-7 downregulated hexon expression and decreased protein expression levels gradually increased in parallel with LC3-
the yield of EDSV progeny. Meanwhile, the data from the CCK- l-to-LC3-ll conversion rates, and P62 levels gradually decreased
8 assay indicated that pharmacological alterations of autophagy in hexon transfected DEFs, as shown by western blot analysis.
had no significant effects on cell viability following treatment with These results clearly support the hypothesis that EDSV hexon
rapamycin, CQ or 3MA. Taken together, these findings reveal that expression can induce autophagy in DEFs. In addition, we found
induction of autophagy during EDSV infection promotes virus preliminary verification that EDSV hexon transfection reduces
replication. p-P13k, p-mTOR, and p-Akt protein phosphorylation in infected
Although EDSV induces autophagy to help its replication, the DEFs.
exact mechanism underlying this process is unclear. Previous To summarize, our findings suggest that autophagy is
studies demonstrated that adenovirus proteins E1A and E1B play triggered in host cells upon EDSV infection or hexon transfection
an essential role in the induction of pro-autophagic signaling and that both are involved in the replication of EDSV, which
pathways (Polager et al., 2008; Piya et al., 2011). Moreover, it has implies that EDSV can utilize the autophagic pathway to sustain
been shown that suppression of the mTOR signaling pathway by replication in host cells. In addition, from our current study, it
rapalogs such as rapamycin and everolimus enhances autophagic was found that both EDSV and EDSV hexon protein can reduce
cell death in oncolytic adenoviral therapy (Yokoyama et al., 2008; the phosphorylation of p-P13K, p-Akt, and p-mTOR protein in
Rajecki et al., 2009). It is well-known that the serine/threonine infected DEF cells. Clearly, our understanding of the molecular
kinase mTOR in mammalian cells is a central regulator of mechanisms driving the interplay between EDSV and autophagy
autophagy. mTOR mainly functions downstream from the PI3K- is still insufficient in several aspects; for example, how hexon
Akt signaling pathway and plays important roles in negatively proteins regulate the mechanism of EDSV-induced autophagy via

Frontiers in Microbiology | www.frontiersin.org 11 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

PI3K/Akt/mTOR pathway remains unclear. Besides, it not FUNDING

yet clear whether other proteins or virulence factors in
EDSV affect the process that hexon proteins regulate the This work was financially supported by Shaanxi Science
autophagy through different mechanisms in infected DEFs. and Technology Overall Innovation Project Plan (Grant No.
However, this basic data presented here are helpful for further 2015KTCL02-16).
exploring the pathogenesis related to the interaction between
EDSV and host cells. This knowledge will provide novel
insights into autophagy abrogation and may be used to help SUPPLEMENTARY MATERIAL
develop potential antiviral strategies or drugs against EDSV
infection. The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2018.01091/full#supplementary-material
AUTHOR CONTRIBUTIONS
FIGURE S1 | (A) The DEFs cells were infected by EDSV at different MOI for 48 h.
XW, XQ, and JW conceived and designed the experiments. XW The cells were lyzed and western bolt were performed. (B) The optical densities of
each protein band were measured by densitometric scanning, and the optical
performed the experiments, analyzed the data, and wrote the density ratios of LC3II/I were calculated. Data are presented as the mean ± SEM
paper. JW contributed reagents, materials and analysis tools. XW, of three independent experiments. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
XQ, SC, and BY prepared the figures and tables. “#” means no significant difference, P > 0.05.

REFERENCES Gannage, M., Dormann, D., Albrecht, R., Dengjel, J., Torossi, T., Ramer, P. C., et al.
(2009). Matrix protein 2 of influenza A virus blocks autophagosome fusion with
Baxendale, W. (1978). Egg drop syndrome 76. Vet. Rec. 102, 285–286. doi: 10.1136/ lysosomes. Cell Host Microbe 6, 367–380. doi: 10.1016/j.chom.2009.09.005
vr.102.13.285 Geng, Y., Kohli, L., Klocke, B. J., and Roth, K. A. (2010). Chloroquine-induced
Bidin, Z., Lojkic, I., Mikec, M., and Pokric, B. (2007). Naturally occurring egg autophagic vacuole accumulation and cell death in glioma cells is p53
drop syndrome infection in turkeys. Acta Vet. Brno 76, 415–421. doi: 10.2754/ independent. Neuro Oncol. 12, 473–481. doi: 10.1093/neuonc/nop048
avb200776030415 Gougeon, M. L., and Piacentini, M. (2009). New insights on the role of apoptosis
Boya, P., Gonzalez-Polo, R. A., Casares, N., Perfettini, J. L., Dessen, P., and autophagy in HIV pathogenesis. Apoptosis 14, 501–508. doi: 10.1007/
Larochette, N., et al. (2005). Inhibition of macroautophagy triggers apoptosis. s10495-009-0314-1
Mol. Cell. Biol. 25, 1025–1040. doi: 10.1128/mcb.25.3.1025-1040.2005 Hafez, H. M. (2011). Avian adenoviruses infections with special attention to
Brugh, M., Beard, C. W., and Villegas, P. (1984). Experimental infection of laying inclusion body hepatitis/hydropericardium syndrome and egg drop syndrome.
chickens with adenovirus 127 and with a related virus isolated from ducks. Pak. Vet. J. 31, 85–92.
Avian Dis. 28, 168–178. doi: 10.2307/1590139 Huang, J., Dan, T., Yang, W., Liu, C., Xu, J., and Wang, J. (2015). Egg drop
Cao, Z. X., Yang, Y. T., Yu, S., Li, Y. Z., Wang, W. W., Huang, J., et al. (2017). syndrome virus enters duck embryonic fibroblast cells via clathrin-mediated
Pogostone induces autophagy and apoptosis involving PI3K/Akt/mTOR axis endocytosis. Virus Res. 210, 69–76. doi: 10.1016/j.virusres.2015.07.014
in human colorectal carcinoma HCT116 cells. J. Ethnopharmacol. 202, 20–27. Huang, S. C., Chang, C. L., Wang, P. S., Yu, T., and Liu, H. S. (2009). Enterovirus
doi: 10.1016/j.jep.2016.07.028 71-induced autophagy detected in vitro and in vivo promotes viral replication.
Cha, S. Y., Kang, M., Moon, O. K., Park, C. K., and Jang, H. K. (2013a). Respiratory J. Med. Virol. 81, 1241–1252. doi: 10.1002/jmv.21502
disease due to current egg drop syndrome virus in Pekin ducks. Vet. Microbiol. Huang, W. R., Chiu, H. C., Liao, T. L., Chuang, K. P., Shih, W. L., and Liu,
165, 305–311. doi: 10.1016/j.vetmic.2013.04.004 H. J. (2015). Avian reovirus protein p17 functions as a nucleoporin Tpr
Cha, S. Y., Kang, M., Park, C. K., Choi, K. S., and Jang, H. K. (2013b). Epidemiology suppressor leading to activation of p53, p21 and PTEN and Inactivation
of egg drop syndrome virus in ducks from South Korea. Poult. Sci. 92, of PI3K/AKT/mTOR and ERK signaling pathways. PLoS One 10:e0133699.
1783–1789. doi: 10.3382/ps.2013-03067 doi: 10.1371/journal.pone.0133699
Cina, D. P., Onay, T., Paltoo, A., Li, C., Maezawa, Y., De Arteaga, J., et al. (2012). Jiang, H., White, E. J., Rios-Vicil, C. I., Xu, J., Gomez-Manzano, C., and Fueyo, J.
MTOR regulates autophagic flux in the glomerulus. Autophagy 8, 696–698. (2011). Human adenovirus type 5 induces cell lysis through autophagy and
doi: 10.4161/auto.19386 autophagy-triggered caspase activity. J. Virol. 85, 4720–4729. doi: 10.1128/jvi.
Crawford, S. E., Hyser, J. M., Utama, B., and Estes, M. K. (2012). Autophagy 02032-10
hijacked through viroporin-activated calcium/calmodulin-dependent kinase Jung, C. H., Ro, S. H., Cao, J., Otto, N. M., and Kim, D. H. (2010). mTOR
kinase-beta signaling is required for rotavirus replication. Proc. Natl. Acad. Sci. regulation of autophagy. FEBS Lett. 584, 1287–1295. doi: 10.1016/j.febslet.2010.
U.S.A. 109, E3405–E3413. doi: 10.1073/pnas.1216539109 01.017
Deretic, V. (2010). Autophagy in infection. Curr. Opin. Cell Biol. 22, 252–262. Kim, J., Dalton, V. M., Eggerton, K. P., Scott, S. V., and Klionsky, D. J.
doi: 10.1016/j.ceb.2009.12.009 (1999). Apg7p/Cvt2p is required for the cytoplasm-to-vacuole targeting,
Dobbelstein, M. (2004). Replicating adenoviruses in cancer therapy. Curr. Top. macroautophagy, and peroxisome degradation pathways. Mol. Biol. Cell 10,
Microbiol. Immunol. 273, 291–334. doi: 10.1007/978-3-662-05599-1_9 1337–1351. doi: 10.1091/mbc.10.5.1337
Ezeibe, M. C., Okoroafor, O. N., Eze, J. I., and Eze, I. C. (2008). Seroprevalence Kim, J., Kundu, M., Viollet, B., and Guan, K. L. (2011). AMPK and mTOR regulate
of egg drop syndrome–76 virus as cause of poor egg productivity of poultry autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, 132–141.
in Nsukka. South East Nigeria. Trop. Anim. Health Prod. 40, 137–140. doi: 10.1038/ncb2152
doi: 10.1007/s11250-007-9072-7 Kirn, D. (2000). Replication-selective oncolytic adenoviruses: virotherapy aimed at
Fu, G., Chen, H., Huang, Y., Cheng, L., Fu, Q., Shi, S., et al. (2013). Full genetic targets in cancer. Oncogene 19, 6660–6669. doi: 10.1038/sj.onc.1204094
genome sequence of egg drop syndrome virus strain FJ12025 isolated from Klionsky, D. J. (2007). Autophagy: from phenomenology to molecular
muscovy duckling. Genome Announc. 1:e00623-13. doi: 10.1128/genomeA.00 understanding in less than a decade. Nat. Rev. Mol. Cell Biol. 8, 931–937.
623-13 doi: 10.1038/nrm2245

Frontiers in Microbiology | www.frontiersin.org 12 May 2018 | Volume 9 | Article 1091

Wang et al. The Mechanisms of EDSV Triggering Autophagy

Klionsky, D. J., Cregg, J. M., Dunn, W. A. Jr., Emr, S. D., Sakai, Y., Sibirny, A., et al. Shin, Y. K., Liu, Q., Tikoo, S. K., Babiuk, L. A., and Zhou, Y. (2007). Influenza
(2003). A unified nomenclature for yeast autophagy-related genes. Dev. Cell 5, A virus NS1 protein activates the phosphatidylinositol 3-kinase (PI3K)/Akt
539–545. doi: 10.1016/S1534-5807(03)00296-X pathway by direct interaction with the p85 subunit of PI3K. J. Gen. Virol. 88,
Komatsu, M., Waguri, S., Ueno, T., Iwata, J., Murata, S., Tanida, I., et al. (2005). 13–18. doi: 10.1099/vir.0.82419-0
Impairment of starvation-induced and constitutive autophagy in Atg7-deficient Shintani, T., and Klionsky, D. J. (2004). Autophagy in health and disease:
mice. J. Cell Biol. 169, 425–434. doi: 10.1083/jcb.200412022 a double-edged sword. Science 306, 990–995. doi: 10.1126/science.
Kroemer, G., Mariño, G., and Levine, B. (2010). Autophagy and the integrated 1099993
stress response. Mol. Cell. 40, 280–293. doi: 10.1016/j.molcel.2010.09.023 Solanki, M., Zhang, W., Jing, L., and Ehrhardt, A. (2016). Adenovirus
Larson, C., Oronsky, B., Scicinski, J., Fanger, G. R., Stirn, M., Oronsky, A., et al. hexon modifications influence in vitro properties of pseudotyped human
(2015). Going viral: a review of replication-selective oncolytic adenoviruses. adenovirus type 5 vectors. J. Gen. Virol. 97, 160–168. doi: 10.1099/jgv.0.0
Oncotarget 6, 19976–19989. doi: 10.18632/oncotarget.5116 00328
Levine, B., and Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell Suhy, D. A., Giddings, T. H., and Kirkegaard, K. (2000). Remodeling the
132, 27–42. doi: 10.1016/j.cell.2007.12.018 endoplasmic reticulum by poliovirus infection and by individual viral proteins:
Li, E., Stupack, D., Klemke, R., Cheresh, D. A., and Nemerow, G. R. (1998). an autophagy-like origin for virus-induced vesicles. J. Virol. 74, 8953–8965.
Adenovirus Endocytosis via Î ± v Integrins requires phosphoinositide-3-OH doi: 10.1128/JVI.74.19.8953-8965.2000
Kinase. J. Virol. 72, 2055–2061. Wang, Q. X., Wu, Y. J., Cai, Y. L., Zhuang, Y. B., Xu, L. H., Wu, B. C., et al. (2015).
Li, J., Liu, Y., Wang, Z., Liu, K., Wang, Y., Liu, J., et al. (2011). Subversion of cellular Spleen transcriptome profile of muscovy ducklings in response to infection
autophagy machinery by hepatitis B virus for viral envelopment. J. Virol. 85, with muscovy duck reovirus. Avian Dis. 59, 282–290. doi: 10.1637/10992-1125
6319–6333. doi: 10.1128/jvi.02627-10 14-Reg
Lippai, M., and Low, P. (2014). The role of the selective adaptor p62 and ubiquitin- Wong, J., Zhang, J., Si, X., Gao, G., Mao, I., Mcmanus, B. M., et al. (2008).
like proteins in autophagy. Biomed. Res. Int. 2014:832704. doi: 10.1155/2014/ Autophagosome supports coxsackievirus B3 replication in host cells. J. Virol.
832704 82, 9143–9153. doi: 10.1128/JVI.00641-08
Ma, J., Sun, Q., Mi, R., and Zhang, H. (2011). Avian influenza A virus H5N1 Xiao, M., Zhidan, Y. U., Bo, M. A., and Wang, J. (2009). Cloning and expression
causes autophagy-mediated cell death through suppression of mTOR signaling. of vecombinent of protein Penton gene from egg drop syndrome virus and
J. Genet. Genomics 38, 533–537. doi: 10.1016/j.jgg.2011.10.002 identification of expressed protein antigenicity. J. Northeast Agric. Univ. 40,
McFerran, J. B., and Smyth, J. A. (2000). Avian adenoviruses. Rev. Sci. Tech. 19, 83–87.
589–601. doi: 10.20506/rst.19.2.1238 Xie, L., Xie, Z., Li, H., Fan, Q., Luo, S., Huang, J., et al. (2016). Avian reovirus
Milan, E., Perini, T., Resnati, M., Orfanelli, U., Oliva, L., Raimondi, A., et al. (2015). σA and σNS proteins activate the phosphatidylinositol 3-kinase-dependent
A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis Akt signalling pathway. Arch. Virol. 161, 2243–2248. doi: 10.1007/s00705-016-
and determines proteasome inhibitor susceptibility in multiple myeloma cells. 2908-6
Autophagy 11, 1161–1178. doi: 10.1080/15548627.2015.1052928 Xin, L., Xiao, Z., Ma, X., He, F., Yao, H., and Liu, Z. (2014). Coxsackievirus
Mizushima, N., Yoshimori, T., and Levine, B. (2010). Methods in mammalian B3 induces crosstalk between autophagy and apoptosis to benefit its release
autophagy research. Cell 140, 313–326. doi: 10.1016/j.cell.2010.01.028 after replicating in autophagosomes through a mechanism involving caspase
O’Shea, C., Klupsch, K., Choi, S., Bagus, B., Soria, C., Shen, J., et al. (2005). cleavage of autophagy-related proteins. Infect. Genet. Evol. 26, 95–102.
Adenoviral proteins mimic nutrient/growth signals to activate the mTOR doi: 10.1016/j.meegid.2014.05.005
pathway for viral replication. EMBO J. 24, 1211–1221. doi: 10.1038/sj.emboj. Yakoub, A. M., and Shukla, D. (2015). Basal autophagy is required for
7600597 herpes simplex virus-2 infection. Sci. Rep. 5:12985. doi: 10.1038/srep1
Piya, S., White, E. J., Klein, S. R., Jiang, H., McDonnell, T. J., Gomez-Manzano, C., 2985
et al. (2011). The E1B19K oncoprotein complexes with Beclin 1 to regulate Yan, J., Dong, J., Wu, J., Zhu, R., Wang, Z., Wang, B., et al. (2016). Interaction
autophagy in adenovirus-infected cells. PLoS One 6:e29467. doi: 10.1371/ between hexon and L4-100K determines virus rescue and growth of hexon-
journal.pone.0029467 chimeric recombinant Ad5 vectors. Sci. Rep. 6:22464. doi: 10.1038/srep22464
Polager, S., Ofir, M., and Ginsberg, D. (2008). E2F1 regulates autophagy and the Yin, H. C., Zhao, L. L., Li, S. Q., Niu, Y. J., Jiang, X. J., Xu, L. J., et al.
transcription of autophagy genes. Oncogene 27, 4860–4864. doi: 10.1038/onc. (2017). Autophagy activated by duck enteritis virus infection positively
2008.117 affects its replication. J. Gen. Virol. 98, 486–495. doi: 10.1099/jgv.0.
Qi, X., Xu, J., Wang, Z., Wang, X., and Wang, J. (2017). The role of hexon in egg 000696
drop syndrome virus (EDSV) inducing apoptosis in duck embryo fibroblast Yokoyama, T., Iwado, E., Kondo, Y., Aoki, H., Hayashi, Y., Georgescu, M. M.,
cells. Res. Vet. Sci. 114, 395–400. doi: 10.1016/j.rvsc.2017.07.015 et al. (2008). Autophagy-inducing agents augment the antitumor effect of
Rajecki, M., af Hallstrom, T., Hakkarainen, T., Nokisalmi, P., Hautaniemi, S., telerase-selve oncolytic adenovirus OBP-405 on glioblastoma cells. Gene Ther.
Nieminen, A. I., et al. (2009). Mre11 inhibition by oncolytic adenovirus 15, 1233–1239. doi: 10.1038/gt.2008.98
associates with autophagy and underlies synergy with ionizing radiation. Int. Zhang, C., Yuan, X. R., Li, H. Y., Zhao, Z. J., Liao, Y. W., Wang, X. Y., et al.
J. Cancer 125, 2441–2449. doi: 10.1002/ijc.24608 (2015). Anti-cancer effect of metabotropic glutamate receptor 1 inhibition
Raychaudhuri, P., Rooney, R., and Nevins, J. R. (1987). Identification of an E1A- in human glioma U87 cells: involvement of PI3K/Akt/mTOR pathway. Cell
inducible cellular factor that interacts with regulatory sequences within the Physiol. Biochem. 35, 419–432. doi: 10.1159/000369707
adenovirus E4 promoter. EMBO J. 6, 4073–4081. Zhang, Y., Li, Z., Ge, X., Guo, X., and Yang, H. (2011). Autophagy promotes the
Rodriguez-Rocha, H., Gomez-Gutierrez, J. G., Garcia-Garcia, A., Rao, X. M., replication of encephalomyocarditis virus in host cells. Autophagy 7, 613–628.
Chen, L., McMasters, K. M., et al. (2011). Adenoviruses induce autophagy to doi: 10.4161/auto.7.6.15267
promote virus replication and oncolysis. Virology 416, 9–15. doi: 10.1016/j.virol.
2011.04.017 Conflict of Interest Statement: The authors declare that the research was
Rodriguezrocha, H., Gomezgutierrez, J. G., Garciagarcia, A., Rao, X. M., conducted in the absence of any commercial or financial relationships that could
Chen, L., Mcmasters, K. M., et al. (2011). Adenoviruses induce autophagy be construed as a potential conflict of interest.
to promote virus replication and oncolysis. Virology 416, 9–15. doi:
10.1016/j.virol.2011.04.017 Copyright © 2018 Wang, Qi, Yang, Chen and Wang. This is an open-access article
Rohn, K., Prusas, C., Monreal, G., and Hess, M. (1997). Identification and distributed under the terms of the Creative Commons Attribution License (CC BY).
characterization of penton base and pVIII protein of egg drop syndrome virus. The use, distribution or reproduction in other forums is permitted, provided the
Virus Res. 47, 59–65. doi: 10.1016/S0168-1702(96)01407-4 original author(s) and the copyright owner are credited and that the original
Seglen, P. O., and Gordon, P. B. (1982). 3-Methyladenine: specific inhibitor of publication in this journal is cited, in accordance with accepted academic practice.
autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc. No use, distribution or reproduction is permitted which does not comply with these
Natl. Acad. Sci. U.S.A. 79, 1889–1892. doi: 10.1073/pnas.79.6.1889 terms.

Frontiers in Microbiology | www.frontiersin.org 13 May 2018 | Volume 9 | Article 1091