Infection, Genetics and Evolution 39 (2016) 5154

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Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

Short communication

Interplay of autophagy and apoptosis during PRRSV infection of

Marc145 cell
Shuaifeng Li, Ao Zhou, Jiaxing Wang, Shujun Zhang
Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China

a r t i c l e i n f o a b s t r a c t

Article history: Autophagy and apoptosis play essential roles 'in virus infection. Our study was performed to investigate the in-
Received 21 August 2015 terplay between autophagy and apoptosis in PRRSV replication. In our present study, autophagy and apoptosis
Received in revised form 31 December 2015 were induced by PRRSV infection. Viral replication was dampened/attenuated by autophagy decient and poten-
Accepted 11 January 2016
tiated by apoptosis inhibition. Furthermore, PRRSV replication was restored by apoptosis inhibition in autophagy
Available online 13 January 2016
decient cells. Taken together, our ndings unveil the functional relationship between autophagy and apoptosis
Keywords:
during PRRSV replication.
PRRSV 2016 Elsevier B.V. All rights reserved.
Autophagy
Apoptosis
Marc145 cell

1. Introduction 2. Materials and methods

The porcine reproductive and respiratory syndrome virus (PRRSV)
causes acute respiratory disease and persistent infection in piglets and
reproductive failure in pregnant sows, which have led to serious
economic losses to the swine industry worldwide (Meulenberg, 2000).
Highly pathogenic PRRSV (HP-PRRSV) emerged in China in 2006, is
characterized by high fever, high morbidity and high mortality in pigs
of all ages (Tian et al., 2007). Autophagy is a conserved catabolic process
that maintains cellular homeostasis by degrading and removing dam-
aged or excess cellular organelles and protein aggregates from the cyto-
plasm, thereby ensuring cell survival. However, autophagy is induced in
different infections, and certain viruses, for example, hepatitis C virus
(HCV) (Dreux et al., 2009), Japanese encephalitis virus (JEV) (Ho et al.,
2013) and dengue virus (DENV) (Heaton and Randall, 2010), have de-
veloped strategies to hijack autophagy to aid in their replication. Al-
though previous studies have revealed that both autophagy and
apoptosis were triggered after PRRSV infection, there is little de-
scription about the functional interaction between autophagy and
apoptosis during PRRSV infection (Wang et al., 2015). Therefore,
the purpose of the present study was to investigate the functional re-
lationship between autophagy and apoptosis during PRRSV
replication.
Corresponding author.
E-mail address: sjxiaozhang@mail.hzau.edu.cn (S. Zhang).
2.1. Cell and virus
Marc145 cells were cultured and maintained in Dulbecco's modied
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin/streptomycin at 37 C in a humidied 5% CO2 in-
cubator. PRRSV strain WUH3 was adapted to Marc145 cells at a MOI of
0.5. Virus stocks of PRRSV were prepared in Marc145 cells and infectious
virus titer was analyzed in Marc145 cells using the method of Reed and
Muench (1938). For virus infection, cells were initially incubated with
PRRSV for 1 h at 37 C. After 1 h of adsorption, cells were exchanged
to the medium containing 5% FBS and cultured for the indicated time.
2.2. Quantitative real-time PCR
Total cellular RNA were extracted from cells using Total RNAeasy kit
(Omega), and subsequently, complementary DNAs (cDNAs) were
synthesized using the First-Strand Synthesis System (Thermo Scientic)
according to the manufacturer's instructions. All PCRs were performed
under the following conditions: 95 C for 3 min followed by 40 cycles
of 95 C for 10 s and 60 C for 20 s. SYBR Premix ExTaqTM kit (Bio-Rad)
was used according to the manufacturer's instructions, and the real-
time PCR was performed on an iQ5 Real-Time PCR system (Bio-rad).
The real-time PCR results were analyzed and expressed as relative
expression of CT (threshold cycle) valuing the 2Ct method. The
primer pairs used for RT-PCR are as follows: Rab7 former: 5-AACATT
CCCTACTTTGAGACCA-3; reverse: 5-CACCTCCGTTTCCTGCTTA-3; Beta
actin former: 5-AGCAAGCAGGAGTATGACGAGT-3, reverse: 5-CAAG
AAAGGGTGTAACGCAACT-3.

http://dx.doi.org/10.1016/j.meegid.2016.01.011
1567-1348/ 2016 Elsevier B.V. All rights reserved.
52 S. Li et al. / Infection, Genetics and Evolution 39 (2016) 5154

Fig. 1. PRRSV infection induces both autophagy and apoptosis in Marc145 cells. Marc145 cells were mock- or PRRSV-infected at a MOI of 0.5. Cell samples were collected at 24 h, 36 h, and
48 h post-infection for further analysis. (A) Western blot analyzed the expression of LC3-I, LC3-II and Nsp2 with the progression of PRRSV infection. GAPDH was used as the protein loading
control. (B) Relative LC3-II expression was estimated by gray scan and normalization against GAPDH. (C) Apoptosis was analyzed by ow cytometry at 24 h and 36 h post-infection.
(D) Quantication of the apoptosis rate of PRRSV-infected Marc145 cells at different time points. Data are expressed as means with SEM of three independent experiments. ***, P b 0.001.

2.3. Western blot
The whole cell lysate was extracted with RIPA buffer containing 1%
protease inhibitor PMSF and 1% cocktail (Sigma). 1012% SDS-PAGE
gels were used to separate proteins which were then transferred to
polyvinylidene uoride (PVDF) membranes (Bio-Rad). The membranes
were blocked with 5% nonfat milk and incubated with primary antibody
overnight at 4 C. Membranes were washed and incubated with
HRP-conjugated secondary antibody. The proteins were detected by
the enhanced chemiluminescence (ECL) system (Bio-Rad) following
the manufacturer's instructions. The relative expression normalized
GAPDH was calculated in Image J software (NIH).
2014). The secondary antibodies were HRP-conjugated anti-mouse IgG
(BosterBio) and HRP-conjugated anti-rabbit IgG (Cell Signaling).
2.6. Transfection of siRNA
SiRNA was transfected into Marc145 cells at a concentration of
100 pM with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according
to the manufacturer's protocol. SiRNA is synthesized by Gene-Pharma
(Shanghai, China). The siRNA sequences are as follows: Atg5, GATTCA
TGGAATTGAGCCA (Zhao et al., 2012); Rab7, CACGTAGGCCTTCAACAC
AAT; NC (negative control), TTCTCCGAACGTGTCACGT.
2.7. Statistical analysis

2.4. Flow cytometry analysis
Marc145 cells were infected with PRRSV at a MOI of 0.5. At 24 h and
36 h postinfection, the cells were collected and washed for 3 times. The
cells were stained with Annexin V and PI for 5 min. The percentage of
apoptosis was analyzed by ow cytometry.
All experiments were performed independently three times, and
data were presented as the means with SEM. Data were analyzed by
one-way ANOVA followed by Tukey's honest signicant difference test
with SPSS version 16.0 (SPSS, Chicago, IL, USA). A p value b 0.05 was
considered as statistically signicant.
3. Results

2.5. Reagents and antibodies
The chemical reagent used in this study was z-VAD-fmk (Beyotime).
Primary antibodies used in this study include the following: rabbit mono-
clonal anti-LC3-II (Cell Signaling); rabbit anti-cleaved Caspase3 (Cell Sig-
naling); rabbit anti-cleaved PARP (Cell Signaling); rabbit anti-Atg5
(Proteintech); mouse monoclonal anti-GAPDH (Proteintech); and
mouse polyclonal anti-PRRSV Nsp2 (described previously by Jing et al.,
During autophagy, the microtubule-associated protein 1 light chain
kinase 3 (LC3-I), becomes lipidated LC3-II, which was inserted into the
autophagosomal membrane (Mizushima and Yoshimori, 2007). LC3-II
was signicantly induced in PRRSV-infected cells compared with
mock-infected cells (Fig. 1A and B). The results from ow cytometry
showed that apoptosis was robustly induced by PRRSV infection at
36 h post-infection (Fig. 1C and D). These results suggest that both
autophagy and apoptosis can be triggered by PRRSV infection. To
 S. Li et al. / Infection, Genetics and Evolution 39 (2016) 5154 53

Fig. 2. PRRSV replication is dampened by autophagy decient and potentiated by apoptosis inhibition. (A) Marc145 cells were transfected with specic siRNA for Atg5 and negative control
(NC). At 24 h post-transfection, cells were infected with PRRSV for another 24 h or 36 h, and the total expression of Nsp2 and LC3-II were veried. The silencing effect of Atg5 was veried
by Western blot. (B) Marc145 cells were transfected with specic siRNA for Rab7 and negative control (NC) and infected with PRRSV, and at 24 h and 36 h post-infection cells were lysed
for Western blotting analysis. (C) The silencing efciency of Rab7 was detected by quantitative real-time PCR. (D) Marc145 cells were pretreated with z-VAD-fmk for 1 h before PRRSV
infection. Cells were lysed at different time points post-infection and the expression of Nsp2 was analyzed. The Nsp2 protein gray was analyzed by Image J. **, P b 0.01; ***, P b 0.001.

investigate the role of autophagy and apoptosis in PRRSV replication, we
performed a RNA interference (RNAi) experiment to knockdown the
expression of Atg5 or Rab7. Knockdown of Atg5 or Rab7 largely
abrogated intracellular PRRSV yield at both 24 h and 36 h
post-infection, as conrmed by the level of Nsp2 (Fig. 2A and B). To
completely understand the role of apoptosis in PRRSV replication, a
caspase pan inhibitor, z-VAD-fmk, was used. Marc145 cells were treated
with z-VAD-fmk (100 M), which has been conrmed to sufciently
reduce apoptosis (Lee and Kleiboeker, 2007). We found that PRRSV
replication was signicantly up-regulated in z-VAD-fmk treated cells

Fig. 3. PRRSV replication was restored by apoptosis inhibition in autophagy decient cells. (A) Marc145 cells were transfected with siRNA targeted Atg5 for 24 h and then infected with
PRRSV for a further 24 h or 36 h. Total cellular protein was harvested and cleaved caspase3 and cleaved PARP were analyzed by Western blotting. (B) Expression of cleaved caspase3 and
cleaved PARP were monitored and quantied, respectively. (C) Atg5 silencing cells were treated with z-VAD-fmk for 1 h before PRRSV infection. Cells were harvested and lysed for Western
blot analysis. (D) Quantication of Nsp2 protein expression from (C) was veried. Data are expressed as means with SEM of three independent experiments. *, P b 0.05; **, P b 0.01; ***,
P b 0.001.
54 S. Li et al. / Infection, Genetics and Evolution 39 (2016) 5154
compared with untreated cells (Fig. 2D). Taken together, these results
emphasized that autophagy has a positive effect while apoptosis plays
the negative role on PRRSV replication.
To test whether there is a functional relationship between autopha-
gy and apoptosis during PRRSV infection, we examined the activation of
caspase3 and PARP in PRRSV-infected control- and siAtg5-Marc145
cells. It was striking that PRRSV-infected cells exhibited more caspase3
and poly (ADP-ribose) polymerase (PARP) cleavage when the cells suf-
fered from autophagy defection (Fig. 3A and B). To further investigate
whether PRRSV replication can be restored by apoptosis inhibition in
autophagy decient cells, Atg5-silencing Marc145 cells were treated
with z-VAD-fmk and virus replication was determined by Western
blot. Viral Nsp2 protein level was statistically signicantly potentiated
in z-VAD-fmk treated Atg5-silencing cells (Fig. 3C and D). Taken togeth-
er, the results in this paper consolidate the functional interplay between
autophagy and apoptosis during PRRSV replication.
4. Discussions
The recent study has shown that autophagy and apoptosis are close-
ly related (Mario et al., 2014; Yang and Yao, 2015). Previous studies
have suggested that autophagy counteracts caspase-dependent apopto-
sis after virus infection, including cytomegalovirus (Mo et al., 2014) and
chikungunya virus (Joubert et al., 2012). Previously, the work from our
group has shown that autophagy facilitates PRRSV replication by
delaying apoptosis through the interaction between Beclin1 and Bad
(Zhou et al., 2015). The results of the study reported herein support
the idea that autophagy attenuates apoptosis during virus infection. Re-
cently, a number of evidence have demonstrated that apoptosis is able
to induced by PRRSV replication (Lee and Kleiboeker, 2007; Wang
et al., 2015). But the role that apoptosis plays in PRRSV replication is de-
bated. In the present study, we found that PRRSV infection was able to
induce both autophagy and apoptosis in Marc145 cells. Silencing of
the autophagy-related gene led to a down-regulation of PRRSV yield
(Fig. 2A and B), while inhibition of apoptosis resulted in an obviously
potentiate in viral replication. Furthermore, PRRSV replication could
be restored by apoptosis inhibition in autophagy decient cells. The
study presented here provides a deeper understanding of the pathogen-
esis of PRRSV.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
We thank Professor Liurong Fang for providing us the Nsp2
antibody. This work was supported by grants from the Natural Science
Foundation of China (31272427 and 31572367), the Hubei Province In-
ternational Cooperation Project (36215001) and the Wuhan Huanghe
Yingcai Scholarship (YSF2015000414).
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