- Email: [email protected]
ERK signaling pathways
Coxsackievirus B3 Induces Autophagy in HeLa cells AMPK/MEK/ERK and Ras/Raf/MEK/ERK signaling pathways
via
the
Le Xin, Xiaolin Ma, Zonghui Xiao, Hailan Yao, Zhewei Liu PII: DOI: Reference:
S1567-1348(15)00349-4 doi: 10.1016/j.meegid.2015.08.026 MEEGID 2455
To appear in: Received date: Revised date: Accepted date:
9 May 2015 17 August 2015 20 August 2015
Please cite this article as: Xin, Le, Ma, Xiaolin, Xiao, Zonghui, Yao, Hailan, Liu, Zhewei, Coxsackievirus B3 Induces Autophagy in HeLa cells via the AMPK/MEK/ERK and Ras/Raf/MEK/ERK signaling pathways, (2015), doi: 10.1016/j.meegid.2015.08.026
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ACCEPTED MANUSCRIPT Coxsackievirus B3 Induces Autophagy in HeLa cells via the AMPK/MEK/ERK and
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Le Xin, Xiaolin Ma, Zonghui Xiao, Hailan Yao *, Zhewei Liu *
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Ras/Raf/MEK/ERK signaling pathways
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Department of Molecular Immunology, Capital Institute of Pediatrics
*Corresponding author:
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Name: Zhewei Liu; Hailan Yao
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Mail Address: Molecular Immunology, Capital Institute of Pediatrics Phone: +8601085695572
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Email: [email protected]; [email protected]
ACCEPTED MANUSCRIPT Abstract In a previous study, the number of autophagosomes increased after coxsackievirus (CVB3)
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infection. However, the exact mechanism by which CVB3 regulates the number of
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autophagosomes is unclear. Earlier studies have found that infection with CVB3 activates
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extracellular signal-regulated kinase (ERK). ERK is essential for CVB3 replication and can increase the number of autophagosomes. In the current study, extracellular signal-regulated kinase 1/2 was activated in HeLa cells after CVB3 infection. The ERK kinase inhibitor,
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U0126, was then used to inhibit the activity of ERK. Treatment with U0126 led to a
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significant reduction in the number of autophagosomes indicating that the CVB3-induced autophagosome accumulation occurred may via the ERK pathway. The relationship between
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CVB3 infection and ERK pathway activation was also investigated. The results showed that
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the RasGAP protein could be further cleaved, leading to activation of the Ras/Raf/MEK (mitogen/extracellular signal-regulated kinase)/ERK pathway and that CVB3 infection could
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result in an increase in the concentration of calcium in the cytoplasm, resulting in mitochondrial damage, a decrease in the concentration of ATP and activation of the AMPK
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(AMP-activated protein kinase)/MEK/ERK pathway. In summary, CVB3 might directly or indirectly induce autophagy via AMPK/MEK/ERK and Ras/Raf/MEK/ERK signaling pathways in the host cells, representing a pivotal mechanism for CVB3 pathogenesis.
Key words: Coxsackievirus; ERK; AMPK; autophagy; RasGAP
ACCEPTED MANUSCRIPT 1. Introduction
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Coxsackievirus B3 (CVB3) is a non-enveloped, single, positive-strand RNA virus,
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belonging to the picornavirus family and the enterovirus genus (Robinson et al., 2014). CVB3
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induces the formation of autophagosome-like double-membrane vesicles in many kinds of cells, including HeLa cells (Wong et al., 2008), pancreatic acinar cells (Alirezaei et al., 2012), HL-1 cells, as well as neural progenitor and stem cells (Tabor-Godwin et al., 2012). Thus, an
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increase in the number of autophagosomes during CVB3 infection is common. Investigating
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the relationship between CVB3 and autophagosomes can help to clarify the relationship between CVB3 and cellular processes.
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Autophagosomes are characteristic of macroautophagy (here referred to as autophagy).
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Autophagosomes can fuse with endosomes to form amphisomes or lysosomes to form autolysosomes. Fusion with lysosomes exposes the material within the autophagosomes to
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lysosomal hydrolases, resulting in autophagosome degradation; this process is referred to as autophagy (Delorme-Axford et al., 2014). Autophagy is a catabolic cellular process,
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conserved in all eukaryotes, which involves the degradation and turnover of cytoplasmic components. Autophagy is not only important in cellular homeostasis, but also plays a role in various physiological processes, such as responses to environmental stress, development, aging, and tumor suppression (Klionsky et al., 2005). Recent research has indicated that autophagy may act as a host defense mechanism against bacterial (Yuk et al., 2012) and viral infections (Sir and Ou, 2010). However, autophagy is beneficial for some viruses (Jheng et al., 2014), including herpes simplex virus type 1. These viruses encode autophagy-blocking viral proteins, which promotes survival of the viruses in host cells. In addition, it has been documented that certain positive-stranded RNA viruses, such as poliovirus (Suhy et al., 2000) and enterovirus 71 (Tallóczy et al., 2002), are able to subvert cellular autophagy in order to
ACCEPTED MANUSCRIPT facilitate their own replication. Furthermore, autophagy is involved in virion maturation (Richards and Jackson, 2012). The role of autophagy in the host seems to be dependent on the
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viral strain, but its overall relevance remains controversial.
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Autophagy is a “double-edged sword” for CVB3. On the one hand, autophagy can clear a
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small portion of CVB3 (Wong et al., 2008). On the other hand, the life cycle of CVB3 depends on autophagy (Alirezaei et al., 2012; Robinson et al., 2014). It is not yet known whether
CVB3
directly
regulates
autophagosome
levels
and,
if
so,
by
what
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mechanism(s).Autophagy is reported to be regulated by several signaling pathways, such as
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the mammalian target of rapamycin pathway (Esclatine et al., 2009) and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway (Huang et al., 2011). Recent studies have
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found that the extracellular signal-regulated kinase (ERK) pathway can induce
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microtubule-associated-Protein 1 light chain 3 (LC3)-I to convert to LC3-II and can promote the production of the Beclin-1 protein (Cheng et al., 2008; Choi et al., 2010; Sivaprasad and
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Basu, 2008; Zeng et al., 2012), both of which can induce an increase in autophagosome generation. In addition, sustained activation of ERK protein can activate cathepsin B (a
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protease present in lysosomes) and downregulate the level of Lysosomal-Associated Membrane Protein 1 and 2 (LAMP-1 and LAMP-2)(Fehrenbacher et al., 2008). This inhibits the fusion of autophagosomes with lysosomes, leading to autophagosome accumulation. Previously, it was thought that the Ras/Raf/MEK (mitogen/extracellular signal-regulated kinase)/ERK and AMPK (AMP-activated protein kinase)/MEK/ERK pathway could both activate ERK (Hardie, 2011; Wang et al., 2009). ERK activation has been observed after CVB3 infection and has been found to be essential for CVB3 replication (Huber et al., 1999; Luo et al., 2002), however, the relationship between ERK and autophagy, the pathway which activates ERK, as well as whether and how CVB3 regulates autophagy and ERK in CVB3-infected cells remain unclear.
ACCEPTED MANUSCRIPT In the present study, we further investigated the relationships of Raf/MEK/ERK,
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AMPK/MEK/ERK signaling pathway with autophagy induction by CVB3 infection.
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2. Methods
2.1. Cell culture and virus
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HeLa cells were grown and maintained in Modified Eagle’s medium (MEM) containing
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L-glutamine (Hyclone, Logan, UT, USA), supplemented with 10 % newborn bovine serum (Gibco, Carlsbad, CA, USA) plus penicillin (100 U/mL) and streptomycin (100 µg/mL) in a
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37C, 5% CO2 incubator. CVB3 was amplified in HeLa cells after infection. Viral titers were
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routinely determined prior to infection via a plaque assay on HeLa cell monolayers, as
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previously described (Xin et al., 2014).
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2.2. Antibodies and kinase inhibitors
Antibodies against Actin (Cat#4970), Phosph-c-Raf (Cat#9431), Phosph-AMPKa (Cat#2535), AMPKa (Cat#2535), Phosph-MEK1/2 (Cat#9154), MEK1/2 (Cat#9122), Phosph-p44/42MAPK-(ERK1/2) (Cat#4370), p44/42MAPK-(ERK1/2) (Cat#9102), Protein A Agarose Beads (Cat#9863) and Rabbit IgG (Cat#3900) were obtained from Cell Signaling Technology (Danvers, MA, USA). Raf-1 (Cat#ab32025 and ab24452), LC3 (Cat#62721), RasGAP (Cat#32625) and the Ras Activation Assay kit (Cat#173242) were purchased from Abcam (Cambridge, England). Fluo-4 (Cat#F-23917) was obtained from Life Technologies (Carlsbad, CA, USA). The mitochondrial membrane potential assay kit with JC-1 (Cat# C2006) and the ATP Assay Kit (Cat# S0026) were obtained from Beyotime Biotechnology
ACCEPTED MANUSCRIPT Company (Shanghai, China). The MEK Inhibitor (U0126) was obtained from Promega (Madison, Wisconsin, USA) (Cat#1121). The AMPK inhibitor (Compound C) (Cat#171260)
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and the Raf1 Kinase inhibitor I (GW5074) (Cat#553008) were purchased from Calbiochem
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Company (Darmstadt, Germany). LC3 (Cat#L7543) and Bafilomycin A1 (Cat#B1793) was
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purchased from Sigma –Aldrich (St. Louis, MO, USA). Enterovirus group specific monoclonal antibody (mAb) 5-D8/1(M6064) was purchased from Dako company (Trappes,
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technology company (New York, USA).
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France). Cyto-ID™ Autophagy detection kit (Cat#51031) was purchased from Enzo life
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2.3. Virus infection of HeLa cells
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HeLa cells were infected with CVB3 at a multiplicity of infection of 10; control cells were given serum-free MEM. All cells were then incubated for 1 hour. Host cells were
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washed twice with PBS, cultured in complete medium and harvested at various time points (see figure legends for specific experimental details). HeLa cells were treated or pre-treated
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with the kinase inhibitors U0126 (20 µM), compound C (3 µM), bafilomycin A1 (100 nM) or GW5074 (10 nM). The cells were then infected, as described above, and subsequently cultured in fresh medium containing the same concentrations of the drugs used for pretreatments; the corresponding concentration of dimethyl sulfoxide was used as a control.
2.4. Western blot
Western blots were performed according to previously described methods (Xin et al., 2014).
ACCEPTED MANUSCRIPT 2.5. CO-IP
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Immunoprecipitations were performed according to the manufacturer's instructions and
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previously described methods (Zhu et al., 2002). Cells were lysed for 20 min on ice with lysis
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buffer (CST, Cat#9803) in the presence of protease cocktail inhibitor (Roche, Mannheim, Germany). After centrifugation at 14,000 × g for 20 min, the remaining lysates were pretreated with protein A/G (CST) for 10 min at 4°C to eliminate nonspecific binding to the
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agarose gel. Antibodies against phospho-ERK1/2, phospho-AMPK or Raf (1 μg) were added
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to the lysates and the lysates were then rotated overnight at 4°C in order to pull down the protein complexes (500 μg). Protein A Agarose beads were washed twice with lysis buffer and centrifuged at 6,000 × g for 2 min (4°C) after each wash. The complexes were
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immobilized with 20 μl of protein A/G-coated beads, followed by incubation overnight, on a rocker, at 4°C. The Protein A Agarose beads were then washed three times with lysis buffer
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and centrifuged at 6,000 × g for 5 min (4°C) after each wash. The proteins were eluted in SDS loading buffer via heating at 100°C for 10 min. SDS-PAGE and immunoblotting were
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carried out, according to the methods described above. The blots were probed with antibodies against phospho-ERK1/2, phospho-AMPK and Raf1. The lysates of control HeLa cells were used as controls.
2.6. Ras activity assay
A Ras activity assay kit was used according to the manufacturer's instructions. Cells were lysed on ice for 20 min with lysis buffer, in the presence of protease cocktail inhibitor (Roche). Antibodies against RasGTP (1 μl) were added to the lysate and incubated for 3 h at 4°C. Protein A/G Agarose Beads were washed twice with lysis buffer and centrifuged at
ACCEPTED MANUSCRIPT 6,000 × g for 2 min after each wash. The complexes were then immobilized with 20 μl of protein A/G-coated beads, followed by incubation on a rocker for 1 h, at 4°C. Then Protein A
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Agarose beads were washed with lysis buffer three times, centrifuged at 6,000 × g for 5 min
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(4°C) after each wash and the proteins were eluted in 2 X SDS loading buffer via heating at
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100°C for 5 min. SDS-PAGE and immunoblotting were carried out, according to the methods described in 2.2 part. The blots were probed with antibodies against RasGTP and RasGDP.
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The lysates of control HeLa cells were used as controls.
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2.7. Measurement of mitochondrial membrane potential
The mitochondrial membrane potential was examined by staining the astrocytes with
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(5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine
iodide),
a
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JC-1
lipophilic, cationic dye that exhibits a fluorescence emission shift, upon aggregation, from
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530 nm (green monomer) to 590 nm (red “J-aggregates”). In healthy cells with a high mitochondrial membrane potential (Δψm), JC-1 enters the mitochondrial matrix in a
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potential-dependent manner and forms aggregates (Korenić et al., 2014).The examination of Δψm was performed according to the manufacturer's instructions. Infected HeLa cells were harvested at different time points, methods described above. The cells were washed twice with PBS, counted and 600,000 cells were resuspended in 0.5 ml MEM. Staining was performed at 37°C for 20 min using 0.5 ml JC-1 working buffer. After staining, cells were centrifuged at 6,000 × g for 4 min and then washed three times with JC-1 washing buffer. The fluorescence intensity was measured using a multifunctional microplate reader (Thermo Fisher Scientific Company, Waltham, MA USA). Normal mitochondrial integrity was set as excitation/emission=490/530
nm.
excitation/emission = 425/590 nm.
Loss
of
mitochondrial
integrity
was
set
as
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2.8. ATP assay
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The concentration of ATP was measured using a kit, according to the manufacturer's
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instructions. Infected HeLa cells were harvested at different time points, as previously described. The cells were washed twice with PBS, lysed for 20 min on ice with lysis buffer and then centrifuged at 14,000 × g for 20 min. A 3 μl aliquot of lysed protein was used to
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determine the protein concentration via BCA assay. A 100 μl volume of ATP working buffer
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was then added to each detecting well of a 96-well plate, followed by 10 μl of different concentrations of ATP standard and sample solutions. The OD value was then measured via a
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multifunctional microplate reader (Thermo Fisher Scientific Company). The standard curve
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was created and the ATP concentration in each sample was calculated from the standard curve. The concentration of ATP was converted into nmol/mg protein, according to the measured
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concentration of the protein in each sample. Each sample was analyzed in triplicate and each
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experiment was repeated three times.
2.9. Measurement of intracellular Ca2+
Labeled calcium indicators are molecules that exhibit an increase in fluorescence upon binding Ca2+, according to the company’s instructions. Fluo-4 has been used to image the spatial dynamics of Ca2+ signaling, which results in increased fluorescence excitation at 488 nm and consequently higher fluorescence signal levels. Cells may be loaded with the AM ester forms of these calcium indicators by adding the dissolved indicator directly to dishes containing cultured cells. These indicators are useful for fluorescence and confocal microscopy, flow cytometry and microplate screening applications. The Ca2+ concentration
ACCEPTED MANUSCRIPT was measured according to the methods described previously (Luo et al., 2012). In brief, infected HeLa cells were harvested at different time points, as described earlier. The HeLa
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cells were washed three times with PBS, Fluo-4 working buffer was added (final
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concentration: 10 µmol) and the cells were incubated at 37°C for 40 min. The Fluo-4 working
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buffer was then removed and discarded, the cells were washed another three times with PBS, resuspended with 500 µl PBS and incubated at 37°C for 30 min. The fluorescence intensity
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set as excitation/emission = 488/510 nm.
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was measured using a multifunctional microplate reader (Thermo Fisher Scientific Company)
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2.10. Cyto-ID® Green autophagy dye staining procedure for autophagy detection
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The Cyto-ID® Green autophagy dye was prepared following the protocol from the manufacturer. In brief, diluted the 10× assay buffer to 1×assay. The Cyto-ID® Green
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autophagy dye solution was prepared by mixing 8 µl of the dye and 4 ml of 1× assay buffer. The collected suspension cells should be adjusted to a final concentration of approximately 1
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× 106 cells/ml. The cell sample is centrifuged for 5 min at 1000 rpm, and 100 of the dye is pipetted. Then incubated sample 30 min at 37°C, followed by a wash and resuspension with 100 or 500 µl of 1× assay buffer before imaged-based. Then incubated cells with 10 µM of Hoechst 33342 for 5 min at 37°C before analysis. The stained HeLa cells were analyzed using a fluorescence microscope (OLYMPUS) with a 100× magnification. Cyto-ID® Green autophagy dye and Hoechst 33342 were examined using the FITC and DAPI, respectively.
2.11. Viral plaque assay
Viral plaque assay were performed according to previously described methods (Xin et
ACCEPTED MANUSCRIPT al., 2014).
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2.12. Statistical analysis
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Statistical analyses were performed using SPSS 11.0. One-way analysis of variance (ANOVA) was used to determine statistical significance among 3 or more groups of data and LSD or Games–Howell post-hoc tests were used for multiple comparisons within each group.
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The Student’s t-test was used to determine statistical significance between treated and
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non-treated groups. All data are presented as the mean standard error (SE). Values of P <
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0.05 were considered statistically significant.
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3. Results
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3.1. Autophagosome accumulation is related to ERK activation in CVB3-infected HeLa cells.
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It can be concluded that CVB3 infection induces increased autophagosome formation in our previous experiment (Xin et al., 2014). However, whether it is the viral infection that leads to the increase of autophagosomes formation or the reduced fusion of autophagosomes and lysosomes that inhibits the degradation of autophagosomes is unknown. Thus, firstly, bafilomycin A1 was used to inhibit the fusion of autophagosomes and lysosomes (Rubinsztein et al., 2009). We found that LC3-II/beta-actin ratio has further increased in bafilomycin A1-treated HeLa cells (Fig. 1A). While after further analyzing p62 (Fig. 1A), we found that p62 protein increased, while viral capsid protein (VP1) has also increased. This indicates that after CVB3 infection, autophagosomes and lysosomes still had the ability to combine. And the increase of
ACCEPTED MANUSCRIPT autophagosomes may be due to the increase of new autophagosome formation. Previous studies have shown that the ERK1/2 pathway plays a key role in regulating
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autophagy (Cagnol and Chambard, 2010) and that CVB3 infection activates ERK1/2
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signaling (Huber et al., 1999; Luo H et al., 2002). In the current study, we examined whether
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ERK1/2 is involved in autophagy induction in CVB3-infected HeLa cells. The total ERK protein level did not significantly change at various time points after infection, however there was a relative increase in the phosphorylated form of ERK (p-ERK) at 9 hours post-infection
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(h.p.i.) (Fig. 1B). Based on this result, 9 h.p.i. was chosen as the time point for subsequent
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experiments. Then we treated HeLa cells with U0126 to inhibit ERK1/2 phosphorylation and found that treatment with U0126 led to a significant reduction in the number of
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autophagosomes. Firstly, UO126 suppressed conversion of LC3-I to LC3-II in both
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CVB3-infected and non-infected HeLa cells (Fig. 1C). Fig. 1D showed that the immunofluorescence decreased in U0126-treated HeLa cells further improved that the level
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of autopahgosomes decreased. At the same time, as shown in Fig. 1C, treatment with U0126 resulted in reduction of VP1 expression. These results indicate that, the ERK1/2 signaling
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pathway may be involved in CVB3-mediated autophagosome accumulation in HeLa cells.
3.2. AMPK is the upstream regulator of ERK1/2 in CVB3-mediated autophagy
To determine whether ERK1/2 activation occurs downstream of the Ras/Raf1 or AMPK/MEK/ERK pathway, the activities of some key proteins in the AMPK/MEK/ERK pathway were examined. Fig. 2A shows that CVB3 infection significantly increased phosphorylation of AMPK- and MEK, whereas no change was detected in the levels of total AMPK and MEK. A co-immunoprecipitation-western blot was used to examine the interaction of p-AMPK and p-MEK to further demonstrate that MEK activation is related to
ACCEPTED MANUSCRIPT AMPK activation. Clearly, The p-AMPK and p-MEK binding complex was higher in CVB3-infected HeLa cells, compared to control cells at 9 h.p.i. (Fig. 2B). Moreover,
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compound C, which is known to inhibit AMPK (Meley et al., 2006), inhibited activation of
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ERK1/2 and MEK, the expression of VP1 as well as lipidation of AMPK and LC3 lipidation,
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in both CVB3-infected and uninfected HeLa cells (Fig. 2C), suggesting that AMPK might be an upstream regulator of ERK1/2 in CVB3-mediated autophagy. This indicates that ERK1/2 interacts with its upstream regulator, AMPK.
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The activation mechanism of AMPK was then investigated. AMPK is sensitive to energy
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states and plays an important role in the balance of energy in cells (Wang et al., 2009), thus the ATP content in CVB3-infected HeLa cells was initially examined. The ATP content
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decreased beginning 3 h.p.i. (Fig. 2D), indicating possible mitochondrial damage since the
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mitochondria are the main organelles in cells that produce energy. The mitochondrial membrane potential was then examined to determine whether there was mitochondrial
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damage during the process of CVB3 infection. Mitochondrial membrane potential refers to the process of mitochondrial oxidative respiration, which generates energy stored in the inner
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mitochondrial membrane as electrochemical potential energy; this electrochemical potential energy is referred to as the mitochondrial membrane potential (Singer, 2007). When the structure and function of the mitochondria are impaired, particularly when the permeability of the mitochondrial inner membrane increases and/or the function of mitochondrial respiratory chain complex decreases, the mitochondrial membrane potential decreases resulting in oxygen-use disorders in the body and ATP production deficiencies. The mitochondrial membrane potential reflects the integrity of the mitochondrial membrane and is a sensitive indicator for evaluating the comprehensive function of the mitochondria (Tychinsky et al., 2004). A JC-1 fluorescent probe was used to detect mitochondrial membrane potential in order to determine whether infection with CVB3 caused mitochondrial damage. The
ACCEPTED MANUSCRIPT fluorescence intensity of the JC-1 polymer represents normal mitochondrial membrane potential and the fluorescence intensity of a JC-1 monomer changes when the mitochondrial
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membrane potential changes. When the fluorescence intensity of a JC-1 monomer increases,
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the mitochondrial membrane potential decreases, indicating mitochondrial damage. The
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fluorescence intensity of the JC-1 polymer in HeLa cells gradually decreased beginning 3 h.p.i., while the fluorescence intensity of the JC-1 monomer began to increase at 6 h.p.i., suggesting that CVB3 infection induced mitochondrial damage (Fig. 2F). The concentration
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of free calcium within the cytoplasm was then examined to further elucidate the mechanism(s)
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of mitochondrial damage. The fluorescence intensity indirectly represents the concentration of free calcium in the cytoplasm. Fig. 2E showed that the fluorescence intensity of Fluo-4
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increased beginning 3 h.p.i., indicating that CVB3 infection can cause an increase in the
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concentration of intracellular calcium, which is induced by CVB3 Protein 2B (van Kuppeveld et al., 1997). In summary, it appears that the CVB3 viral protein can cause an increase in the
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concentration of free calcium in the cytoplasm. This leads to mitochondrial damage, along with a reduction in the generation of ATP as well as activation of AMPK, MEK and ERK,
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which increases the number of autophagosomes.
3.3. ERK can also be activated by Ras/Raf/MEK/ERK pathway
To determine whether ERK1/2 activation also occurs downstream of the Ras/Raf1 pathway, activation of Ras and Raf1, by infection with CVB3, was also analyzed. RasGTP (the activated state of Ras) appeared beginning 9 h.p.i., though the Ras total protein level did not change during the infection process (Fig. 3A). And CVB3 infection significantly increased phosphorylation of Raf, whereas no change was detected in the levels of total Raf (Fig. 3A). Fig. 3B shows the p-MEK and Raf binding complex increased in CVB3-infected
ACCEPTED MANUSCRIPT HeLa cells, compared to the control group at 9 h.p.i., as indicated by the Co-IP method. HeLa cells were then treated with GW5074. Total protein expressions of ERK and MEK were
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unaffected, however Fig. 3C shows the levels of VP1, LC3-II, p-ERK and p-MEK were
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significantly reduced in both HeLa cells and in CVB3-infected HeLa cells treated with
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GW5074, an agent known to inhibit Raf (Li et al., 2014), indicating that ERK proteins and autophagosomes can also be activated through the Ras/Raf/MEK/ERK pathway. Published data (Huber et al., 1999) indicates that activation of the Ras pathway is
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induced by RasGAP cleavage during CVB3 infection of host cells. Therefore, the expression
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of the RasGAP protein was further examined in the current study. Fig. 3A shows that the cleavage fragments of RasGAP began to appear at 9 h.p.i.. In summary, it appears that CVB3
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directly or indirectly cleaves the RasGAP protein (Huber et al., 1999) and inducing activation
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4. Discussion
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of the entire Ras/Raf/MEK/ERK pathway.
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Autophagy is often viewed as the second defense of the body against invading pathogens. For instance, Epstein-Barr virus nuclear antigen 1 (EBNA1) escapes capture by proteasomes, but can still be recognized in autophagosomes (Paludan et al., 2005). However some viruses, such as EV71, use autophagosomes for replication (Xi et al., 2013), while some specific pathogens can inhibit the fusion of autophagosomes and lysosomes (Kyei et al., 2009) and/or can inhibit the lysosomal degradation function (Liu et al., 2014), thereby providing conditions for the replication of pathogens. Furthermore, autophagy does not result in cell lysis and provides sufficient time for viral replication and packaging in host cells (Belov et al., 2007; Jackson et al., 2005). CVB3 has a complicated relationship with autophagy. First, inhibition of LAMP-2 protein expression (a lysosomal membrane protein critical for
ACCEPTED MANUSCRIPT autophagosome-lysosome fusion) enhances CVB3 replication indicating that some CVB3 virus can be removed via autophagy (Wong et al., 2008). Secondly, it has been proven that
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CVB3 replication relies on expression of core autophagy components and silencing or
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depleting these components in vitro (Wong et al., 2008) or in vivo (Alirezaei et al., 2012)
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suppresses CVB3 replication. Thirdly, the autophagy pathway has been reported to play a crucial role in CVB3 shedding and release (Robinson et al., 2014). Thus, we hypothesize that, cells can deal with CVB3 through the autophagy pathway, though the autophagy process is
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also beneficial for the life cycle of CVB3, including replication and release. However, it is
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unclear how the autophagosome level is regulated during CVB3 infection or if CVB3 plays a role in autophagosome regulation at all. It can be concluded that CVB3 infection induces
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increased autophagosome formation in our previous experiment (Xin et al., 2014). However,
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whether it is the viral infection that leads to the increase of autophagosomes formation or the reduced fusion of autophagosomes and lysosomes that inhibits the degradation of
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autophagosomes is unknown. In this experiment, we inhibited the fusion of autophagosomes and lysosomes and found that autophagosomes and lysosomes still had the ability to combine
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and autophagy pathway still has its function. This indicates that after CVB3 infection, the increase of autophagosomes may be due to the increase of new autophagosome formation. Then, in the current study, we further demonstrate that autophagy is related to ERK, which can be activated via the Ras/Raf/MEK/ERK and AMPK/MEK/ERK pathways during CVB3 infection. ERK1/2 is a serine/threonine protein kinase, which can be phosphorylated and also a proline-directed kinase that activates proline-adjacent serine/threonines. It belongs to the mitogen-activated protein kinase (MAPK) family. ERK1/2 activation is closely related to the accumulation of autophagosomes (Li et al., 2013; Roy et al., 2014). Studies have found that certain viruses, such as swine rounded virus (Zhu et al., 2002), can induce ERK1/2 activation
ACCEPTED MANUSCRIPT and induce autophagosome accumulation. In previous studies, CVB3 infection has been found to promote the activation of ERK1/2 at 10min and at 7 h.p.i. (Luo et al., 2002) and the
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activation of ERK1/2 is essential for CVB3 replication. One reason that CVB3 can’t replicate
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without ERK may be due to the fact that ERK1/2 activation plays a role in the regulation of
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autophagy and autophagy is important for the life cycle of CVB3. Another reason may be ERK that activation can inhibit the fusion of autophagosomes with lysosomes (Fehrenbacher et al., 2008), which helps to decrease the degradation of CVB3 via the autophagy pathway.
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The relationship between CVB3 and ERK1/2 activation was then examined in order to
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determine whether CVB3 can directly or indirectly regulate the autophagosome level via the ERK1/2 pathway. Previous studies suggest that, the classical, and only, signaling pathway for
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ERK1/2 activation is the Ras/Raf/MEK1/2/ERK1/2 pathway. The Ras protein is the product
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of the Ras oncogene. It is a small protein with a molecular weight of 20-25 kDa. Ras proteins can bind GTP and GDP. GDP-bound Ras is inactive and GTP-bound Ras is active
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(Wittinghofo and Nassar, 1996). A variety of stimuli can convert GDP into GTP bound with Ras, which then enable the Ras protein activation. Activated Ras can bind with Raf, which
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then activates Raf. The c terminal catalytic region of the activated Raf can then bind with and activate MEK1/2, leading to ERK1/2 activation (Morrion, 1990; Widmann et al., 1999). In the previous study (Huber et al., 1999), it was found that ERK was activated during CVB3 infection via the Ras/Raf/MEK/ERK pathway. However, information regarding this pathway is incomplete and the inference was based on the idea that the Ras/Raf/MEK/ERK pathway was the only pathway that could activate ERK. Yet AMPK has also been reported to be able to activate ERK and is not dependent on the Ras/Raf/MEK/ERK pathway (Zhu et al., 2002). In research on autophagy induced by cell starvation, it was found that ERK1/2 was not activated by the conventional Ras/Raf/MEK/ERK pathway, but by the AMPK/MEK/ERK pathway (Wang and Guan, 2009). Thus, the AMPK/MEK/ERK signaling pathway may play
ACCEPTED MANUSCRIPT an even more important role in the regulation of autophagy than the Ras/Raf/MEK/ ERK pathway. However, it was still unclear whether AMPK was activated during CVB3 infection,
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and if so, what mechanisms led to AMPK activation. In the current study, the two pathways
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that activate ERK were fully verified.
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First, it was found that ERK could be activated via the Ras/Raf/MEK pathway in CVB3-infected HeLa cells, along with cleavage of the RasGAP protein. Previous findings (Huber et al., 1999) have indicated that RasGAP cleavage is not due to caspase activation,
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although there is still no direct proof that CVB3 can directly cleave the RasGAP protein.
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However, a potential cleavage site (Gln938/Asn939) for the 3CDpro proteinases of CV and echoviruses was found in the C-terminal, catalytic region of RasGAP. Cleavage at this
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dipeptide would result in a 104 kDa protein, coinciding with the RasGAP fragment detected
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in enterovirus-infected cells. Scheffzek et al. (1996) found that 3CD pro-mediated proteolysis may lead to the impairment of RasGAP catalytic activity during enterovirus replication.
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Based on these findings, the possibility that RasGAP is cleaved by a viral proteinase during enterovirus infection is reinforced. However, this point needs to be further investigated.
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Secondly, the activation mechanism of AMPK was studied. The results suggest that AMPK was activated due to a decrease in ATP content resulting from mitochondrial damage during CVB3 infection. The mechanism for mitochondrial damage may be associated with an increase in free calcium ions in the cytoplasm (Lemasters et al., 1987; Murata et al., 1994). In a previous study (Singer, 2007), it was shown that CVB3 Protein 2B was sufficient to induce an influx of Ca2+ to pass through the cell membrane or to release Ca2+ from ER stores, inducing an increase in free calcium ions in the cytoplasm. Therefore, it appears that the Ras/Raf/MEK/ERK and the AMPK/MEK/ERK pathways can activate ERK in CVB3-infected cells. CVB3 might directly or indirectly induce autophagy via AMPK/MEK/ERK and Ras/Raf/MEK/ERK signaling pathways in the host
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Acknowledgements
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This work was supported by Beijing Natural Science Foundation (7144197).
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ACCEPTED MANUSCRIPT Abbreviation List Coxsackievirus B3
EV71
Enterovirus 71
mTOR
Mammalian target of rapamycin
ERK1/2
Extracellular signal-regulated kinase 1/2
LC3
Microtubule-associated-protein 1 light chain 3
LAMP
Lysosomal-associated membrane protein
MEM
Modified Eagle’s Medium
MOI
Multiplicity of infection
DMSO
Dimethyl sulfoxide
P
Phosphorylation
EBNA1
Epstein-Barr virus nuclear antigen 1
MAPK
Mitogen-activated protein kinase
JC-1
5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine
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CVB3
iodide
Mitogen/extracellular signal-regulated kinase
AMPK
AMP-activated protein kinase
GW5074
Raf1 kinase inhibitor I
Compound C
AMPK inhibitor MEK inhibitor
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U0126
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MEK
ACCEPTED MANUSCRIPT Figure legends Fig. 1. ERK activation and autophagosome accumulation in HeLa cells infected by CVB3. (A)
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Treated HeLa cells with 100nM bafilomycin A1 1 hour before infected with CVB3 at
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multiplicity of infection (MOI) = 10. Then harvest cells at 9 h.p.i.. The expression of LC3 and
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VP1 were detected by Western blot. (B) HeLa cells infected with CVB3 at MOI = 10. Cells were harvested at different time points. The expressions of ERK and p-ERK were detected by Western blot. (C) HeLa cells treated with 20 µm U0126 (ERK kinase inhibitor) for 30 min
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prior to infection with CVB3 at MOI = 10. Cells were harvested at 9 h.p.i.. The expressions
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of VP1, ERK, p-ERK and LC3 protein were detected by Western blot. The experiment was repeated at least three times. D. Formation of autophagosomes, shown as green punctae in
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CVB3/HeLa cells that were treated as described in panel B and analyzed by fluorescence
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microscopy (100*). * P <0.05. The experiment was repeated at least three times. Fig. 2. Activated signaling pathways and mitochondrial damage in HeLa cells after EVB3
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infection. (A, D, E, F) HeLa cells infected with CVB3 at multiplicity of infection (MOI) = 10 were harvested at different time points. (A) Western blot analysis of MEK, p-MEK, AMPK,
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and p-AMPK protein levels. (D) ATP concentration detected by chemiluminescence. E. Fluo-4 calcium within ion fluorescent probes was used to detect free calcium concentration in cytoplasm. F. Mitochondrial membrane potential detected by JC-1 fluorescent probe. (B) HeLa cells infected with CVB3 at MOI = 10 were harvested at 9 h.p.i.. The interaction between p-AMPK and p-MEK and the interaction between p-MEK and Raf were analyzed via co-immunoprecipitation-western assay. IgG: IP negative control. (C) Cells were treated with 3 μm compound C (AMPK kinase inhibitor) for 3 hours before infection with CVB3 at MOI = 10. Cells were collected at 9 h.p.i.. Western blot analysis of VP1, ERK, p-ERK, p-MEK and MEK protein levels are shown. * P <0.05. The experiment was repeated at least three times.
ACCEPTED MANUSCRIPT Fig. 3. CVB3 cleaves RasGAP to activate ERK in HeLa cells. (A) HeLa cells infected with CVB3 at multiplicity of infection (MOI) = 10 were harvested at different time points.
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Western blot detection of MEK, p-MEK, Raf and p-Raf are shown. The expressions of Ras
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and RasGTP were detected via immunoprecipitation-Western blot assay. (B) HeLa cells
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infected with CVB3 at MOI = 10 were harvested at 9 h.p.i.. The interactions between Raf and p-MEK were detected by immunoprecipitation -Western blot assay. IgG: IP negative control. (C) HeLa cells were treated with 10 nM GW5074 (Raf kinase inhibitor) 1 hour before
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infection with CVB3 at MOI = 10. Western blot was used to detect the levels of VP1, ERK,
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p-ERK, p-MEK and MEK. * P <0.05. The experiment was repeated at least three times. Fig. 4. A brief chart showing that Coxsackievirus B3 directly or indirectly induces accumulation
in
HeLa
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Figure 1
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ACCEPTED MANUSCRIPT Highlights 1. CoxsackievirusB3-induced autophagosomes accumulated via activated ERK
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protein.
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2.ERK activated through Ras/Raf/MEK/ERK pathways in CVB3-infected HeLa
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3. ERK activated through AMPK/MEK/ERK pathways in CVB3-infected HeLa
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Le Xin, Xiaolin Ma, Zonghui Xiao, Hailan Yao, Zhewei Liu PII: DOI: Reference:
S1567-1348(15)00349-4 doi: 10.1016/j.meegid.2015.08.026 MEEGID 2455
To appear in: Received date: Revised date: Accepted date:
9 May 2015 17 August 2015 20 August 2015
Please cite this article as: Xin, Le, Ma, Xiaolin, Xiao, Zonghui, Yao, Hailan, Liu, Zhewei, Coxsackievirus B3 Induces Autophagy in HeLa cells via the AMPK/MEK/ERK and Ras/Raf/MEK/ERK signaling pathways, (2015), doi: 10.1016/j.meegid.2015.08.026
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ACCEPTED MANUSCRIPT Coxsackievirus B3 Induces Autophagy in HeLa cells via the AMPK/MEK/ERK and
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Le Xin, Xiaolin Ma, Zonghui Xiao, Hailan Yao *, Zhewei Liu *
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Ras/Raf/MEK/ERK signaling pathways
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Department of Molecular Immunology, Capital Institute of Pediatrics
*Corresponding author:
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Name: Zhewei Liu; Hailan Yao
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Mail Address: Molecular Immunology, Capital Institute of Pediatrics Phone: +8601085695572
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Email: [email protected]; [email protected]
ACCEPTED MANUSCRIPT Abstract In a previous study, the number of autophagosomes increased after coxsackievirus (CVB3)
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infection. However, the exact mechanism by which CVB3 regulates the number of
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autophagosomes is unclear. Earlier studies have found that infection with CVB3 activates
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extracellular signal-regulated kinase (ERK). ERK is essential for CVB3 replication and can increase the number of autophagosomes. In the current study, extracellular signal-regulated kinase 1/2 was activated in HeLa cells after CVB3 infection. The ERK kinase inhibitor,
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U0126, was then used to inhibit the activity of ERK. Treatment with U0126 led to a
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significant reduction in the number of autophagosomes indicating that the CVB3-induced autophagosome accumulation occurred may via the ERK pathway. The relationship between
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CVB3 infection and ERK pathway activation was also investigated. The results showed that
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the RasGAP protein could be further cleaved, leading to activation of the Ras/Raf/MEK (mitogen/extracellular signal-regulated kinase)/ERK pathway and that CVB3 infection could
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result in an increase in the concentration of calcium in the cytoplasm, resulting in mitochondrial damage, a decrease in the concentration of ATP and activation of the AMPK
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(AMP-activated protein kinase)/MEK/ERK pathway. In summary, CVB3 might directly or indirectly induce autophagy via AMPK/MEK/ERK and Ras/Raf/MEK/ERK signaling pathways in the host cells, representing a pivotal mechanism for CVB3 pathogenesis.
Key words: Coxsackievirus; ERK; AMPK; autophagy; RasGAP
ACCEPTED MANUSCRIPT 1. Introduction
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Coxsackievirus B3 (CVB3) is a non-enveloped, single, positive-strand RNA virus,
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belonging to the picornavirus family and the enterovirus genus (Robinson et al., 2014). CVB3
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induces the formation of autophagosome-like double-membrane vesicles in many kinds of cells, including HeLa cells (Wong et al., 2008), pancreatic acinar cells (Alirezaei et al., 2012), HL-1 cells, as well as neural progenitor and stem cells (Tabor-Godwin et al., 2012). Thus, an
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increase in the number of autophagosomes during CVB3 infection is common. Investigating
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the relationship between CVB3 and autophagosomes can help to clarify the relationship between CVB3 and cellular processes.
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Autophagosomes are characteristic of macroautophagy (here referred to as autophagy).
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Autophagosomes can fuse with endosomes to form amphisomes or lysosomes to form autolysosomes. Fusion with lysosomes exposes the material within the autophagosomes to
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lysosomal hydrolases, resulting in autophagosome degradation; this process is referred to as autophagy (Delorme-Axford et al., 2014). Autophagy is a catabolic cellular process,
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conserved in all eukaryotes, which involves the degradation and turnover of cytoplasmic components. Autophagy is not only important in cellular homeostasis, but also plays a role in various physiological processes, such as responses to environmental stress, development, aging, and tumor suppression (Klionsky et al., 2005). Recent research has indicated that autophagy may act as a host defense mechanism against bacterial (Yuk et al., 2012) and viral infections (Sir and Ou, 2010). However, autophagy is beneficial for some viruses (Jheng et al., 2014), including herpes simplex virus type 1. These viruses encode autophagy-blocking viral proteins, which promotes survival of the viruses in host cells. In addition, it has been documented that certain positive-stranded RNA viruses, such as poliovirus (Suhy et al., 2000) and enterovirus 71 (Tallóczy et al., 2002), are able to subvert cellular autophagy in order to
ACCEPTED MANUSCRIPT facilitate their own replication. Furthermore, autophagy is involved in virion maturation (Richards and Jackson, 2012). The role of autophagy in the host seems to be dependent on the
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viral strain, but its overall relevance remains controversial.
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Autophagy is a “double-edged sword” for CVB3. On the one hand, autophagy can clear a
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small portion of CVB3 (Wong et al., 2008). On the other hand, the life cycle of CVB3 depends on autophagy (Alirezaei et al., 2012; Robinson et al., 2014). It is not yet known whether
CVB3
directly
regulates
autophagosome
levels
and,
if
so,
by
what
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mechanism(s).Autophagy is reported to be regulated by several signaling pathways, such as
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the mammalian target of rapamycin pathway (Esclatine et al., 2009) and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway (Huang et al., 2011). Recent studies have
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found that the extracellular signal-regulated kinase (ERK) pathway can induce
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microtubule-associated-Protein 1 light chain 3 (LC3)-I to convert to LC3-II and can promote the production of the Beclin-1 protein (Cheng et al., 2008; Choi et al., 2010; Sivaprasad and
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Basu, 2008; Zeng et al., 2012), both of which can induce an increase in autophagosome generation. In addition, sustained activation of ERK protein can activate cathepsin B (a
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protease present in lysosomes) and downregulate the level of Lysosomal-Associated Membrane Protein 1 and 2 (LAMP-1 and LAMP-2)(Fehrenbacher et al., 2008). This inhibits the fusion of autophagosomes with lysosomes, leading to autophagosome accumulation. Previously, it was thought that the Ras/Raf/MEK (mitogen/extracellular signal-regulated kinase)/ERK and AMPK (AMP-activated protein kinase)/MEK/ERK pathway could both activate ERK (Hardie, 2011; Wang et al., 2009). ERK activation has been observed after CVB3 infection and has been found to be essential for CVB3 replication (Huber et al., 1999; Luo et al., 2002), however, the relationship between ERK and autophagy, the pathway which activates ERK, as well as whether and how CVB3 regulates autophagy and ERK in CVB3-infected cells remain unclear.
ACCEPTED MANUSCRIPT In the present study, we further investigated the relationships of Raf/MEK/ERK,
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AMPK/MEK/ERK signaling pathway with autophagy induction by CVB3 infection.
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2. Methods
2.1. Cell culture and virus
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HeLa cells were grown and maintained in Modified Eagle’s medium (MEM) containing
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L-glutamine (Hyclone, Logan, UT, USA), supplemented with 10 % newborn bovine serum (Gibco, Carlsbad, CA, USA) plus penicillin (100 U/mL) and streptomycin (100 µg/mL) in a
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37C, 5% CO2 incubator. CVB3 was amplified in HeLa cells after infection. Viral titers were
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routinely determined prior to infection via a plaque assay on HeLa cell monolayers, as
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previously described (Xin et al., 2014).
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2.2. Antibodies and kinase inhibitors
Antibodies against Actin (Cat#4970), Phosph-c-Raf (Cat#9431), Phosph-AMPKa (Cat#2535), AMPKa (Cat#2535), Phosph-MEK1/2 (Cat#9154), MEK1/2 (Cat#9122), Phosph-p44/42MAPK-(ERK1/2) (Cat#4370), p44/42MAPK-(ERK1/2) (Cat#9102), Protein A Agarose Beads (Cat#9863) and Rabbit IgG (Cat#3900) were obtained from Cell Signaling Technology (Danvers, MA, USA). Raf-1 (Cat#ab32025 and ab24452), LC3 (Cat#62721), RasGAP (Cat#32625) and the Ras Activation Assay kit (Cat#173242) were purchased from Abcam (Cambridge, England). Fluo-4 (Cat#F-23917) was obtained from Life Technologies (Carlsbad, CA, USA). The mitochondrial membrane potential assay kit with JC-1 (Cat# C2006) and the ATP Assay Kit (Cat# S0026) were obtained from Beyotime Biotechnology
ACCEPTED MANUSCRIPT Company (Shanghai, China). The MEK Inhibitor (U0126) was obtained from Promega (Madison, Wisconsin, USA) (Cat#1121). The AMPK inhibitor (Compound C) (Cat#171260)
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and the Raf1 Kinase inhibitor I (GW5074) (Cat#553008) were purchased from Calbiochem
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Company (Darmstadt, Germany). LC3 (Cat#L7543) and Bafilomycin A1 (Cat#B1793) was
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purchased from Sigma –Aldrich (St. Louis, MO, USA). Enterovirus group specific monoclonal antibody (mAb) 5-D8/1(M6064) was purchased from Dako company (Trappes,
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technology company (New York, USA).
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France). Cyto-ID™ Autophagy detection kit (Cat#51031) was purchased from Enzo life
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2.3. Virus infection of HeLa cells
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HeLa cells were infected with CVB3 at a multiplicity of infection of 10; control cells were given serum-free MEM. All cells were then incubated for 1 hour. Host cells were
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washed twice with PBS, cultured in complete medium and harvested at various time points (see figure legends for specific experimental details). HeLa cells were treated or pre-treated
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with the kinase inhibitors U0126 (20 µM), compound C (3 µM), bafilomycin A1 (100 nM) or GW5074 (10 nM). The cells were then infected, as described above, and subsequently cultured in fresh medium containing the same concentrations of the drugs used for pretreatments; the corresponding concentration of dimethyl sulfoxide was used as a control.
2.4. Western blot
Western blots were performed according to previously described methods (Xin et al., 2014).
ACCEPTED MANUSCRIPT 2.5. CO-IP
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Immunoprecipitations were performed according to the manufacturer's instructions and
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previously described methods (Zhu et al., 2002). Cells were lysed for 20 min on ice with lysis
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buffer (CST, Cat#9803) in the presence of protease cocktail inhibitor (Roche, Mannheim, Germany). After centrifugation at 14,000 × g for 20 min, the remaining lysates were pretreated with protein A/G (CST) for 10 min at 4°C to eliminate nonspecific binding to the
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agarose gel. Antibodies against phospho-ERK1/2, phospho-AMPK or Raf (1 μg) were added
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to the lysates and the lysates were then rotated overnight at 4°C in order to pull down the protein complexes (500 μg). Protein A Agarose beads were washed twice with lysis buffer and centrifuged at 6,000 × g for 2 min (4°C) after each wash. The complexes were
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immobilized with 20 μl of protein A/G-coated beads, followed by incubation overnight, on a rocker, at 4°C. The Protein A Agarose beads were then washed three times with lysis buffer
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and centrifuged at 6,000 × g for 5 min (4°C) after each wash. The proteins were eluted in SDS loading buffer via heating at 100°C for 10 min. SDS-PAGE and immunoblotting were
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carried out, according to the methods described above. The blots were probed with antibodies against phospho-ERK1/2, phospho-AMPK and Raf1. The lysates of control HeLa cells were used as controls.
2.6. Ras activity assay
A Ras activity assay kit was used according to the manufacturer's instructions. Cells were lysed on ice for 20 min with lysis buffer, in the presence of protease cocktail inhibitor (Roche). Antibodies against RasGTP (1 μl) were added to the lysate and incubated for 3 h at 4°C. Protein A/G Agarose Beads were washed twice with lysis buffer and centrifuged at
ACCEPTED MANUSCRIPT 6,000 × g for 2 min after each wash. The complexes were then immobilized with 20 μl of protein A/G-coated beads, followed by incubation on a rocker for 1 h, at 4°C. Then Protein A
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Agarose beads were washed with lysis buffer three times, centrifuged at 6,000 × g for 5 min
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(4°C) after each wash and the proteins were eluted in 2 X SDS loading buffer via heating at
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100°C for 5 min. SDS-PAGE and immunoblotting were carried out, according to the methods described in 2.2 part. The blots were probed with antibodies against RasGTP and RasGDP.
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The lysates of control HeLa cells were used as controls.
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2.7. Measurement of mitochondrial membrane potential
The mitochondrial membrane potential was examined by staining the astrocytes with
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(5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine
iodide),
a
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JC-1
lipophilic, cationic dye that exhibits a fluorescence emission shift, upon aggregation, from
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530 nm (green monomer) to 590 nm (red “J-aggregates”). In healthy cells with a high mitochondrial membrane potential (Δψm), JC-1 enters the mitochondrial matrix in a
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potential-dependent manner and forms aggregates (Korenić et al., 2014).The examination of Δψm was performed according to the manufacturer's instructions. Infected HeLa cells were harvested at different time points, methods described above. The cells were washed twice with PBS, counted and 600,000 cells were resuspended in 0.5 ml MEM. Staining was performed at 37°C for 20 min using 0.5 ml JC-1 working buffer. After staining, cells were centrifuged at 6,000 × g for 4 min and then washed three times with JC-1 washing buffer. The fluorescence intensity was measured using a multifunctional microplate reader (Thermo Fisher Scientific Company, Waltham, MA USA). Normal mitochondrial integrity was set as excitation/emission=490/530
nm.
excitation/emission = 425/590 nm.
Loss
of
mitochondrial
integrity
was
set
as
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2.8. ATP assay
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The concentration of ATP was measured using a kit, according to the manufacturer's
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instructions. Infected HeLa cells were harvested at different time points, as previously described. The cells were washed twice with PBS, lysed for 20 min on ice with lysis buffer and then centrifuged at 14,000 × g for 20 min. A 3 μl aliquot of lysed protein was used to
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determine the protein concentration via BCA assay. A 100 μl volume of ATP working buffer
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was then added to each detecting well of a 96-well plate, followed by 10 μl of different concentrations of ATP standard and sample solutions. The OD value was then measured via a
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multifunctional microplate reader (Thermo Fisher Scientific Company). The standard curve
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was created and the ATP concentration in each sample was calculated from the standard curve. The concentration of ATP was converted into nmol/mg protein, according to the measured
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concentration of the protein in each sample. Each sample was analyzed in triplicate and each
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experiment was repeated three times.
2.9. Measurement of intracellular Ca2+
Labeled calcium indicators are molecules that exhibit an increase in fluorescence upon binding Ca2+, according to the company’s instructions. Fluo-4 has been used to image the spatial dynamics of Ca2+ signaling, which results in increased fluorescence excitation at 488 nm and consequently higher fluorescence signal levels. Cells may be loaded with the AM ester forms of these calcium indicators by adding the dissolved indicator directly to dishes containing cultured cells. These indicators are useful for fluorescence and confocal microscopy, flow cytometry and microplate screening applications. The Ca2+ concentration
ACCEPTED MANUSCRIPT was measured according to the methods described previously (Luo et al., 2012). In brief, infected HeLa cells were harvested at different time points, as described earlier. The HeLa
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cells were washed three times with PBS, Fluo-4 working buffer was added (final
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concentration: 10 µmol) and the cells were incubated at 37°C for 40 min. The Fluo-4 working
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buffer was then removed and discarded, the cells were washed another three times with PBS, resuspended with 500 µl PBS and incubated at 37°C for 30 min. The fluorescence intensity
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set as excitation/emission = 488/510 nm.
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was measured using a multifunctional microplate reader (Thermo Fisher Scientific Company)
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2.10. Cyto-ID® Green autophagy dye staining procedure for autophagy detection
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The Cyto-ID® Green autophagy dye was prepared following the protocol from the manufacturer. In brief, diluted the 10× assay buffer to 1×assay. The Cyto-ID® Green
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autophagy dye solution was prepared by mixing 8 µl of the dye and 4 ml of 1× assay buffer. The collected suspension cells should be adjusted to a final concentration of approximately 1
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× 106 cells/ml. The cell sample is centrifuged for 5 min at 1000 rpm, and 100 of the dye is pipetted. Then incubated sample 30 min at 37°C, followed by a wash and resuspension with 100 or 500 µl of 1× assay buffer before imaged-based. Then incubated cells with 10 µM of Hoechst 33342 for 5 min at 37°C before analysis. The stained HeLa cells were analyzed using a fluorescence microscope (OLYMPUS) with a 100× magnification. Cyto-ID® Green autophagy dye and Hoechst 33342 were examined using the FITC and DAPI, respectively.
2.11. Viral plaque assay
Viral plaque assay were performed according to previously described methods (Xin et
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2.12. Statistical analysis
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Statistical analyses were performed using SPSS 11.0. One-way analysis of variance (ANOVA) was used to determine statistical significance among 3 or more groups of data and LSD or Games–Howell post-hoc tests were used for multiple comparisons within each group.
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The Student’s t-test was used to determine statistical significance between treated and
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non-treated groups. All data are presented as the mean standard error (SE). Values of P <
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0.05 were considered statistically significant.
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3. Results
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3.1. Autophagosome accumulation is related to ERK activation in CVB3-infected HeLa cells.
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It can be concluded that CVB3 infection induces increased autophagosome formation in our previous experiment (Xin et al., 2014). However, whether it is the viral infection that leads to the increase of autophagosomes formation or the reduced fusion of autophagosomes and lysosomes that inhibits the degradation of autophagosomes is unknown. Thus, firstly, bafilomycin A1 was used to inhibit the fusion of autophagosomes and lysosomes (Rubinsztein et al., 2009). We found that LC3-II/beta-actin ratio has further increased in bafilomycin A1-treated HeLa cells (Fig. 1A). While after further analyzing p62 (Fig. 1A), we found that p62 protein increased, while viral capsid protein (VP1) has also increased. This indicates that after CVB3 infection, autophagosomes and lysosomes still had the ability to combine. And the increase of
ACCEPTED MANUSCRIPT autophagosomes may be due to the increase of new autophagosome formation. Previous studies have shown that the ERK1/2 pathway plays a key role in regulating
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autophagy (Cagnol and Chambard, 2010) and that CVB3 infection activates ERK1/2
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signaling (Huber et al., 1999; Luo H et al., 2002). In the current study, we examined whether
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ERK1/2 is involved in autophagy induction in CVB3-infected HeLa cells. The total ERK protein level did not significantly change at various time points after infection, however there was a relative increase in the phosphorylated form of ERK (p-ERK) at 9 hours post-infection
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(h.p.i.) (Fig. 1B). Based on this result, 9 h.p.i. was chosen as the time point for subsequent
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experiments. Then we treated HeLa cells with U0126 to inhibit ERK1/2 phosphorylation and found that treatment with U0126 led to a significant reduction in the number of
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autophagosomes. Firstly, UO126 suppressed conversion of LC3-I to LC3-II in both
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CVB3-infected and non-infected HeLa cells (Fig. 1C). Fig. 1D showed that the immunofluorescence decreased in U0126-treated HeLa cells further improved that the level
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of autopahgosomes decreased. At the same time, as shown in Fig. 1C, treatment with U0126 resulted in reduction of VP1 expression. These results indicate that, the ERK1/2 signaling
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pathway may be involved in CVB3-mediated autophagosome accumulation in HeLa cells.
3.2. AMPK is the upstream regulator of ERK1/2 in CVB3-mediated autophagy
To determine whether ERK1/2 activation occurs downstream of the Ras/Raf1 or AMPK/MEK/ERK pathway, the activities of some key proteins in the AMPK/MEK/ERK pathway were examined. Fig. 2A shows that CVB3 infection significantly increased phosphorylation of AMPK- and MEK, whereas no change was detected in the levels of total AMPK and MEK. A co-immunoprecipitation-western blot was used to examine the interaction of p-AMPK and p-MEK to further demonstrate that MEK activation is related to
ACCEPTED MANUSCRIPT AMPK activation. Clearly, The p-AMPK and p-MEK binding complex was higher in CVB3-infected HeLa cells, compared to control cells at 9 h.p.i. (Fig. 2B). Moreover,
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compound C, which is known to inhibit AMPK (Meley et al., 2006), inhibited activation of
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ERK1/2 and MEK, the expression of VP1 as well as lipidation of AMPK and LC3 lipidation,
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in both CVB3-infected and uninfected HeLa cells (Fig. 2C), suggesting that AMPK might be an upstream regulator of ERK1/2 in CVB3-mediated autophagy. This indicates that ERK1/2 interacts with its upstream regulator, AMPK.
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The activation mechanism of AMPK was then investigated. AMPK is sensitive to energy
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states and plays an important role in the balance of energy in cells (Wang et al., 2009), thus the ATP content in CVB3-infected HeLa cells was initially examined. The ATP content
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decreased beginning 3 h.p.i. (Fig. 2D), indicating possible mitochondrial damage since the
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mitochondria are the main organelles in cells that produce energy. The mitochondrial membrane potential was then examined to determine whether there was mitochondrial
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damage during the process of CVB3 infection. Mitochondrial membrane potential refers to the process of mitochondrial oxidative respiration, which generates energy stored in the inner
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mitochondrial membrane as electrochemical potential energy; this electrochemical potential energy is referred to as the mitochondrial membrane potential (Singer, 2007). When the structure and function of the mitochondria are impaired, particularly when the permeability of the mitochondrial inner membrane increases and/or the function of mitochondrial respiratory chain complex decreases, the mitochondrial membrane potential decreases resulting in oxygen-use disorders in the body and ATP production deficiencies. The mitochondrial membrane potential reflects the integrity of the mitochondrial membrane and is a sensitive indicator for evaluating the comprehensive function of the mitochondria (Tychinsky et al., 2004). A JC-1 fluorescent probe was used to detect mitochondrial membrane potential in order to determine whether infection with CVB3 caused mitochondrial damage. The
ACCEPTED MANUSCRIPT fluorescence intensity of the JC-1 polymer represents normal mitochondrial membrane potential and the fluorescence intensity of a JC-1 monomer changes when the mitochondrial
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membrane potential changes. When the fluorescence intensity of a JC-1 monomer increases,
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the mitochondrial membrane potential decreases, indicating mitochondrial damage. The
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fluorescence intensity of the JC-1 polymer in HeLa cells gradually decreased beginning 3 h.p.i., while the fluorescence intensity of the JC-1 monomer began to increase at 6 h.p.i., suggesting that CVB3 infection induced mitochondrial damage (Fig. 2F). The concentration
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of free calcium within the cytoplasm was then examined to further elucidate the mechanism(s)
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of mitochondrial damage. The fluorescence intensity indirectly represents the concentration of free calcium in the cytoplasm. Fig. 2E showed that the fluorescence intensity of Fluo-4
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increased beginning 3 h.p.i., indicating that CVB3 infection can cause an increase in the
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concentration of intracellular calcium, which is induced by CVB3 Protein 2B (van Kuppeveld et al., 1997). In summary, it appears that the CVB3 viral protein can cause an increase in the
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concentration of free calcium in the cytoplasm. This leads to mitochondrial damage, along with a reduction in the generation of ATP as well as activation of AMPK, MEK and ERK,
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which increases the number of autophagosomes.
3.3. ERK can also be activated by Ras/Raf/MEK/ERK pathway
To determine whether ERK1/2 activation also occurs downstream of the Ras/Raf1 pathway, activation of Ras and Raf1, by infection with CVB3, was also analyzed. RasGTP (the activated state of Ras) appeared beginning 9 h.p.i., though the Ras total protein level did not change during the infection process (Fig. 3A). And CVB3 infection significantly increased phosphorylation of Raf, whereas no change was detected in the levels of total Raf (Fig. 3A). Fig. 3B shows the p-MEK and Raf binding complex increased in CVB3-infected
ACCEPTED MANUSCRIPT HeLa cells, compared to the control group at 9 h.p.i., as indicated by the Co-IP method. HeLa cells were then treated with GW5074. Total protein expressions of ERK and MEK were
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unaffected, however Fig. 3C shows the levels of VP1, LC3-II, p-ERK and p-MEK were
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significantly reduced in both HeLa cells and in CVB3-infected HeLa cells treated with
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GW5074, an agent known to inhibit Raf (Li et al., 2014), indicating that ERK proteins and autophagosomes can also be activated through the Ras/Raf/MEK/ERK pathway. Published data (Huber et al., 1999) indicates that activation of the Ras pathway is
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induced by RasGAP cleavage during CVB3 infection of host cells. Therefore, the expression
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of the RasGAP protein was further examined in the current study. Fig. 3A shows that the cleavage fragments of RasGAP began to appear at 9 h.p.i.. In summary, it appears that CVB3
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directly or indirectly cleaves the RasGAP protein (Huber et al., 1999) and inducing activation
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4. Discussion
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of the entire Ras/Raf/MEK/ERK pathway.
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Autophagy is often viewed as the second defense of the body against invading pathogens. For instance, Epstein-Barr virus nuclear antigen 1 (EBNA1) escapes capture by proteasomes, but can still be recognized in autophagosomes (Paludan et al., 2005). However some viruses, such as EV71, use autophagosomes for replication (Xi et al., 2013), while some specific pathogens can inhibit the fusion of autophagosomes and lysosomes (Kyei et al., 2009) and/or can inhibit the lysosomal degradation function (Liu et al., 2014), thereby providing conditions for the replication of pathogens. Furthermore, autophagy does not result in cell lysis and provides sufficient time for viral replication and packaging in host cells (Belov et al., 2007; Jackson et al., 2005). CVB3 has a complicated relationship with autophagy. First, inhibition of LAMP-2 protein expression (a lysosomal membrane protein critical for
ACCEPTED MANUSCRIPT autophagosome-lysosome fusion) enhances CVB3 replication indicating that some CVB3 virus can be removed via autophagy (Wong et al., 2008). Secondly, it has been proven that
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CVB3 replication relies on expression of core autophagy components and silencing or
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depleting these components in vitro (Wong et al., 2008) or in vivo (Alirezaei et al., 2012)
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suppresses CVB3 replication. Thirdly, the autophagy pathway has been reported to play a crucial role in CVB3 shedding and release (Robinson et al., 2014). Thus, we hypothesize that, cells can deal with CVB3 through the autophagy pathway, though the autophagy process is
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also beneficial for the life cycle of CVB3, including replication and release. However, it is
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unclear how the autophagosome level is regulated during CVB3 infection or if CVB3 plays a role in autophagosome regulation at all. It can be concluded that CVB3 infection induces
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increased autophagosome formation in our previous experiment (Xin et al., 2014). However,
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whether it is the viral infection that leads to the increase of autophagosomes formation or the reduced fusion of autophagosomes and lysosomes that inhibits the degradation of
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autophagosomes is unknown. In this experiment, we inhibited the fusion of autophagosomes and lysosomes and found that autophagosomes and lysosomes still had the ability to combine
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and autophagy pathway still has its function. This indicates that after CVB3 infection, the increase of autophagosomes may be due to the increase of new autophagosome formation. Then, in the current study, we further demonstrate that autophagy is related to ERK, which can be activated via the Ras/Raf/MEK/ERK and AMPK/MEK/ERK pathways during CVB3 infection. ERK1/2 is a serine/threonine protein kinase, which can be phosphorylated and also a proline-directed kinase that activates proline-adjacent serine/threonines. It belongs to the mitogen-activated protein kinase (MAPK) family. ERK1/2 activation is closely related to the accumulation of autophagosomes (Li et al., 2013; Roy et al., 2014). Studies have found that certain viruses, such as swine rounded virus (Zhu et al., 2002), can induce ERK1/2 activation
ACCEPTED MANUSCRIPT and induce autophagosome accumulation. In previous studies, CVB3 infection has been found to promote the activation of ERK1/2 at 10min and at 7 h.p.i. (Luo et al., 2002) and the
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activation of ERK1/2 is essential for CVB3 replication. One reason that CVB3 can’t replicate
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without ERK may be due to the fact that ERK1/2 activation plays a role in the regulation of
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autophagy and autophagy is important for the life cycle of CVB3. Another reason may be ERK that activation can inhibit the fusion of autophagosomes with lysosomes (Fehrenbacher et al., 2008), which helps to decrease the degradation of CVB3 via the autophagy pathway.
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The relationship between CVB3 and ERK1/2 activation was then examined in order to
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determine whether CVB3 can directly or indirectly regulate the autophagosome level via the ERK1/2 pathway. Previous studies suggest that, the classical, and only, signaling pathway for
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ERK1/2 activation is the Ras/Raf/MEK1/2/ERK1/2 pathway. The Ras protein is the product
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of the Ras oncogene. It is a small protein with a molecular weight of 20-25 kDa. Ras proteins can bind GTP and GDP. GDP-bound Ras is inactive and GTP-bound Ras is active
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(Wittinghofo and Nassar, 1996). A variety of stimuli can convert GDP into GTP bound with Ras, which then enable the Ras protein activation. Activated Ras can bind with Raf, which
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then activates Raf. The c terminal catalytic region of the activated Raf can then bind with and activate MEK1/2, leading to ERK1/2 activation (Morrion, 1990; Widmann et al., 1999). In the previous study (Huber et al., 1999), it was found that ERK was activated during CVB3 infection via the Ras/Raf/MEK/ERK pathway. However, information regarding this pathway is incomplete and the inference was based on the idea that the Ras/Raf/MEK/ERK pathway was the only pathway that could activate ERK. Yet AMPK has also been reported to be able to activate ERK and is not dependent on the Ras/Raf/MEK/ERK pathway (Zhu et al., 2002). In research on autophagy induced by cell starvation, it was found that ERK1/2 was not activated by the conventional Ras/Raf/MEK/ERK pathway, but by the AMPK/MEK/ERK pathway (Wang and Guan, 2009). Thus, the AMPK/MEK/ERK signaling pathway may play
ACCEPTED MANUSCRIPT an even more important role in the regulation of autophagy than the Ras/Raf/MEK/ ERK pathway. However, it was still unclear whether AMPK was activated during CVB3 infection,
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and if so, what mechanisms led to AMPK activation. In the current study, the two pathways
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that activate ERK were fully verified.
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First, it was found that ERK could be activated via the Ras/Raf/MEK pathway in CVB3-infected HeLa cells, along with cleavage of the RasGAP protein. Previous findings (Huber et al., 1999) have indicated that RasGAP cleavage is not due to caspase activation,
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although there is still no direct proof that CVB3 can directly cleave the RasGAP protein.
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However, a potential cleavage site (Gln938/Asn939) for the 3CDpro proteinases of CV and echoviruses was found in the C-terminal, catalytic region of RasGAP. Cleavage at this
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dipeptide would result in a 104 kDa protein, coinciding with the RasGAP fragment detected
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in enterovirus-infected cells. Scheffzek et al. (1996) found that 3CD pro-mediated proteolysis may lead to the impairment of RasGAP catalytic activity during enterovirus replication.
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Based on these findings, the possibility that RasGAP is cleaved by a viral proteinase during enterovirus infection is reinforced. However, this point needs to be further investigated.
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Secondly, the activation mechanism of AMPK was studied. The results suggest that AMPK was activated due to a decrease in ATP content resulting from mitochondrial damage during CVB3 infection. The mechanism for mitochondrial damage may be associated with an increase in free calcium ions in the cytoplasm (Lemasters et al., 1987; Murata et al., 1994). In a previous study (Singer, 2007), it was shown that CVB3 Protein 2B was sufficient to induce an influx of Ca2+ to pass through the cell membrane or to release Ca2+ from ER stores, inducing an increase in free calcium ions in the cytoplasm. Therefore, it appears that the Ras/Raf/MEK/ERK and the AMPK/MEK/ERK pathways can activate ERK in CVB3-infected cells. CVB3 might directly or indirectly induce autophagy via AMPK/MEK/ERK and Ras/Raf/MEK/ERK signaling pathways in the host
ACCEPTED MANUSCRIPT cells (see Fig. 4), representing a pivotal mechanism for CVB3 pathogenesis.
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Acknowledgements
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This work was supported by Beijing Natural Science Foundation (7144197).
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ACCEPTED MANUSCRIPT Abbreviation List Coxsackievirus B3
EV71
Enterovirus 71
mTOR
Mammalian target of rapamycin
ERK1/2
Extracellular signal-regulated kinase 1/2
LC3
Microtubule-associated-protein 1 light chain 3
LAMP
Lysosomal-associated membrane protein
MEM
Modified Eagle’s Medium
MOI
Multiplicity of infection
DMSO
Dimethyl sulfoxide
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Phosphorylation
EBNA1
Epstein-Barr virus nuclear antigen 1
MAPK
Mitogen-activated protein kinase
JC-1
5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine
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Mitogen/extracellular signal-regulated kinase
AMPK
AMP-activated protein kinase
GW5074
Raf1 kinase inhibitor I
Compound C
AMPK inhibitor MEK inhibitor
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multiplicity of infection (MOI) = 10. Then harvest cells at 9 h.p.i.. The expression of LC3 and
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VP1 were detected by Western blot. (B) HeLa cells infected with CVB3 at MOI = 10. Cells were harvested at different time points. The expressions of ERK and p-ERK were detected by Western blot. (C) HeLa cells treated with 20 µm U0126 (ERK kinase inhibitor) for 30 min
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prior to infection with CVB3 at MOI = 10. Cells were harvested at 9 h.p.i.. The expressions
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of VP1, ERK, p-ERK and LC3 protein were detected by Western blot. The experiment was repeated at least three times. D. Formation of autophagosomes, shown as green punctae in
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CVB3/HeLa cells that were treated as described in panel B and analyzed by fluorescence
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microscopy (100*). * P <0.05. The experiment was repeated at least three times. Fig. 2. Activated signaling pathways and mitochondrial damage in HeLa cells after EVB3
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infection. (A, D, E, F) HeLa cells infected with CVB3 at multiplicity of infection (MOI) = 10 were harvested at different time points. (A) Western blot analysis of MEK, p-MEK, AMPK,
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and p-AMPK protein levels. (D) ATP concentration detected by chemiluminescence. E. Fluo-4 calcium within ion fluorescent probes was used to detect free calcium concentration in cytoplasm. F. Mitochondrial membrane potential detected by JC-1 fluorescent probe. (B) HeLa cells infected with CVB3 at MOI = 10 were harvested at 9 h.p.i.. The interaction between p-AMPK and p-MEK and the interaction between p-MEK and Raf were analyzed via co-immunoprecipitation-western assay. IgG: IP negative control. (C) Cells were treated with 3 μm compound C (AMPK kinase inhibitor) for 3 hours before infection with CVB3 at MOI = 10. Cells were collected at 9 h.p.i.. Western blot analysis of VP1, ERK, p-ERK, p-MEK and MEK protein levels are shown. * P <0.05. The experiment was repeated at least three times.
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infected with CVB3 at MOI = 10 were harvested at 9 h.p.i.. The interactions between Raf and p-MEK were detected by immunoprecipitation -Western blot assay. IgG: IP negative control. (C) HeLa cells were treated with 10 nM GW5074 (Raf kinase inhibitor) 1 hour before
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infection with CVB3 at MOI = 10. Western blot was used to detect the levels of VP1, ERK,
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p-ERK, p-MEK and MEK. * P <0.05. The experiment was repeated at least three times. Fig. 4. A brief chart showing that Coxsackievirus B3 directly or indirectly induces accumulation
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3. ERK activated through AMPK/MEK/ERK pathways in CVB3-infected HeLa
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