Prototype foamy virus elicits complete autophagy involving the ER stress-related UPR pathway
- Peipei Yuan†1, 2,
- Lanlan Dong†1, 2, 3,
- Qingqing Cheng1,
- Shuang Wang1,
- Zhi Li4,
- Yan Sun4,
- Song Han1, 2,
- Jun Yin1, 2,
- Biwen Peng1, 2,
- Xiaohua He1, 2 and
- Wanhong Liu1, 2Email author
© The Author(s) 2017
Received: 24 November 2016
Accepted: 27 February 2017
Published: 7 March 2017
Prototype foamy virus (PFV) is a member of the Spumaretrovirinae subfamily of retroviruses, which maintains lifelong latent infection while being nonpathogenic to their natural hosts. Autophagy is a cell-programmed mechanism that plays a pivotal role in controlling homeostasis and defense against exotic pathogens. However, whether autophagy is the mechanism for host defense in PFV infection has not been investigated.
Our results revealed that PFV infection induced the accumulation of autophagosomes and triggered complete autophagic flux in BHK-21 cells. PFV infection also altered endoplasmic reticulum (ER) homeostasis. The PERK, IRE1 and ATF6 pathways, all of which are components of the ER stress-related unfolded protein response (UPR), were activated in PFV-infected cells. In addition, accelerating autophagy suppressed PFV replication, and inhibition of autophagy promoted viral replication.
Our data indicate that PFV infection can induce complete autophagy through activating the ER stress-related UPR pathway in BHK-21 cells. In turn, autophagy negatively regulates PFV replication.
KeywordsPFV Autophagy ER stress Viral replication
Prototype foamy virus (PFV) is a member of the foamy viruses (FVs; also known as spumaviruses) belonging to a subfamily of the Retroviridae family . In contrast to human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV), FVs appear to be nonpathogenic in either naturally or accidentally infected hosts and maintain a lifelong infection in the host [2, 3]. Several host factors, such as Trim5α, APOBEC3G and Nmi have been studied as restrictors during PFV replication [4–8]. Previously, we also reported that Pirh2 inhibits PFV replication by degrading the viral trans-activator Tas via ubiquitination . However, the mechanisms that PFV utilizes to maintain perpetual nonpathogenicity in host cells remain elusive.
Autophagy is a highly conserved catabolic pathway that maintains cellular homeostasis. As an intrinsic defense mechanism, host cells may utilize autophagy against invading viruses [10, 11]. Sagnier et al. reported that autophagy can be an antiviral process due to its degradation of the HIV-1 trans-activator Tat, which is a protein essential for viral replication . Inhibition of autophagy could lead to increased viral replication and virulence for herpes simplex virus type-1 (HSV-1) and Sindbis viruses [13, 14]. In addition, autophagy is primarily antiviral for Japanese encephalitis virus (JEV) and might have implications for the disease progression and pathogenesis of JEV .
However, the relationship between PFV infection and autophagy remains unexplored. In this paper, we reported that PFV infection could induce autophagy and investigated the mechanism underlying this phenomenon.
PFV infection triggers the accumulation of autophagosomes
Next, we analyzed whether PFV replication is required for the induction of autophagy. PFV was inactivated by ultraviolet (UV) radiation, and its ability to induce autophagy was examined. As shown in Fig. 1d, synthesis of the PFV structural protein Gag was dramatically decreased in BHK-21 cells infected with UV-inactivated PFV which was treated with UV for 1.5 and 2.0 h. Meanwhile, the levels of both LC3-I and LC3-II were detected in BHK-21 cells infected with UV-inactivated PFV. LC3-II conversion was not increased in UV-PFV-infected BHK-21 cells similar to mock-treated cells. Conversely, there was an apparent conversion from LC3-I to LC3-II in BHK-21 cells infected with normal PFV (Fig. 1e). Although PFV infectivity could be inactivated by UV radiation, the Gag expression were not been eliminated completely as the Fig. 1c shown. It could be speculated that the UV-radiated PFV might not been full deactivated and might remain very low infectious. UV-inactivated PFV might cause host cell response, while there were no significant statistical differences in LC3-II conservation and LC3 accumulation in mock-infected and UV-PFV-infected groups (Additional file 1: Figure S1a, S1b, S1c). These results suggested that the replication of PFV was required for the induction of autophagy.
PFV-induced autophagy is a complete autophagic process
ER stress contributes to PFV-induced autophagy
PFV induces autophagy by activating ER stress-related UPR signaling
Induction of autophagic vesicles formation in infected cells can be driven by recognition of viral RNA by innate immune sensors, virus binding to receptors, expression of viral proteins that usually leads to the induction of UPR due to ER stress and/or production of Reactive Oxygen Species (ROS) . We have confirmed that PFV infection also triggered ER-stress related UPR. It was likely that ER-stress UPR signaling partly contributed to PFV induced complete autophagy. It has been reported that NDV (Newcastle disease virus) can induce autophagy, which is associated with activating the ER-stress-related UPR by viral proteins to benefit its replication . In addition, Datan et al. found that dengue virus could up-regulate ER stress and ataxia telangiectasia mutated (ATM) signaling followed by the production of ROS to enhance autophagy, and the increased autophagy enabled dengue to reproduce . However, our results showed that promoting PFV-induced autophagy though ER stress suppressed the viral replication (Fig. 3d). This implies a novel mechanism for regulating PFV replication caused by PFV-induced autophagy via ER-stress-related UPR signaling.
PFV-induced autophagy negatively regulates viral replication
Recently, autophagy as a cellular adaptive response has been found to be involved in various viral infections . Inhibition of PFV-induced autophagy could reinforce PFV replication and enhancing this process could attenuate viral replication, suggesting that autophagy negative regulated PFV replication and might play an antiviral role in PFV infection. PFV-induced complete autophagy was a process that the autophagosome fuses with a lysosome to form an autolysosome where the captured cytosol component and the inner membrane were degraded .
In this paper, we report for the first time that PFV infection induces the complete autophagic process to promote autophagosome accumulation. Activation of the ER stress-related UPR contributes to PFV-induced autophagy, and PFV-induced autophagy may act as an antiviral mechanism by negatively regulating PFV replication.
Cells, viruses and plasmids
BHK-21 cells were stored by our laboratory. BHK-21 cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) (SV30087; HyClone). The cells were cultured at 37 °C in a humidified incubator with 5% CO2.
The PFV virions were acquired by transient transfection 293T cells with the pHSRV13 proviral plasmid using the PEI transfection reagent [38, 39]. Mock-infected cells were similarly produced by transient transfection of pAT153 blank vector using the PEI transfection reagent to generate negative controls . The cells and the culture medium were freeze-thawed for three cycles to release viruses at 48 h after transfection. To prepare cell-free virus stocks or mock-infected supernatant, the culture supernatant was centrifuged at 4000×g for 10 min and filtered through a 0.22 μm-pore-size filter membrane, and stored at −80 °C. To assess the viral titer, the BHK-21 cells were seeded into 96-well plates, and the medium was removed after the cells were incubated with virus for 1.5 h in an incubator. Then, the supernatant was replaced with growth medium and maintained for 48 h. Virus titers were calculated as 50% tissue culture infectious doses (TCID50) using the Reed-Münch method .
The following plasmids were constructed by our laboratory: cDNA encoding LC3 was cloned into pEGFP-N1 (Clontech, #6085-1); fragments encoding PFV-Bet and Tas were cloned into pCMV-HA (Clontech, #631604); cDNAs encoding PFV-Gag were cloned into pCMV-His (Clontech); pLKO.1-TRC (Addgene, #10878). The primers are shown in Additional file 2: Table S1. The tandem fluorescent monomeric red fluorescent protein mRFP-GFP-LC3 (ptfLC3, Addgene, #21074), and GFP-LAMP1 were purchased from Addgene. The infectious pHSRV13 provirus DNA and the empty plasmid pAT153 were a gift from Professor Rolf M. Flügel (German Cancer Research Center) . GRP78-specific siRNA (5′-GGAGCGCAUUGAUACUAGATT-3′) and a non-silencing siRNA (NC) (5′-UUCUCCGGACGUGUCACGUTT-3′, used as a negative control) were purchased from (GenePharma Shanghai, China) . Plasmids and siRNA transfections were performed by using lipofectamine 2000 reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions.
Reagents and antibodies
Chloroquine (CQ), Rapamycin (Rapa) and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich. Dimethyl sulfoxide (DMSO), dl-dithiothreitol (DTT) and 4-phenyl butyric acid (4-PBA) were purchased from Biosharp. Rabbit anti-LC3, anti-Atg5, anti-p62/SQSTM1, and anti-HA polyclonal antibodies (Abs) were purchased from Cell Signaling Technology. Anti-phospho-JNK/JNK, anti-phospho-ERK1/2pY204, anti-phospho-IRE1, anti-phospho-ATF6 and anti-β-actin polyclonal Abs were purchased from Abcam. Antibody against PFV Gag was kindly provided by Professor Li Zhi, and anti-Tas was produced by immunizing mouse with prokaryotic expressed Tas and purified according to standard procedures . HRP-conjugated goat anti-mouse or HRP-conjugated goat anti-rabbit secondary antibodies were from PM BIOPRIMACY.
Viral infection and drug treatment
BHK-21 cells were seeded into 6-well or 12-well plates and cultured until 80% confluency was reached. Then, the cells were infected with PFV at a MOI of 0.5. After 1.5 h infection, the cells were washed three times with phosphate-buffered saline (PBS) to remove unattached viruses and were incubated in maintenance medium (2% FBS) at 37 °C for the indicated time. BHK-21 cells were pretreated with optimal concentrations of CQ (50 μM), Rapa (400 nM), DTT (1 mM), or 4-PBA (1 mM) for 4 h, followed by infection with PFV. The cells were further cultured in maintenance medium in the absence or presence of drugs. For 3-MA (10 mM) treatment, cells were pretreated for 2 h with 3-MA, followed by infection with PFV. The cells were further cultured in maintenance medium in the absence or presence of 3-MA until the samples were harvested.
PFV supernatant was prepared as methods above. PFV supernatant was radiated with UV (254 nm) for 1.5 h. BHK-21 cells were incubated with UV-inactivated PFV supernatant for 1.5 h in an incubator. Then, the supernatant was replaced with growth medium and maintained for indicated time. The inactivation efficiency of cells which were infected with UV-inactivated PFV was measured by western blotting with specific viral protein Gag antibody.
Transmission electron microscopy (TEM)
BHK-21 cells were collected at 24 hpi with PFV at an MOI of 0.5. Cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) overnight and subjected to preparation for TEM observation . Autophagosomes were defined as double- or single-membrane vesicles measuring 0.3–2.0 μm in diameter.
Confocal fluorescence microscopy
BHK-21 cells were seeded in 12- or 24-well plates that contained coverslips and were grown to 70% confluence. Then, BHK-21 cells were transfected with GFP-LC3, ptfLC3 or GFP-LAMP1 using Turbofect (Thermo Fisher #R0531) according to the manufacturer’s guideline. The cells were infected with PFV or treated with drugs as described above at 24 hpi. Treated cells were washed twice with PBS and fixed in 4% paraformaldehyde in PBS for 15 min. Coverslips were inverted onto slides containing 50% glycerol, and fluorescence signals were visualized with a confocal fluorescence microscope (Leica-LCS-SP8-STED) or fluorescence microscope.
Following the indicated treatment or transfection, BHK-21 cells were washed twice with ice-cold 1× phate-buffered saline (PBS) and lysed on ice with RIPA buffer (Beyotime Biotechnology #P0013B) containing a 1× protease inhibitor cocktail. Thereafter, the cell lysates were centrifuged at 13,000 rpm for 15 min at 4 °C. The samples were boiled at 100 °C for 10 min in sample loading buffer (5% SDS, 10% glycerol, 60 mM Tris pH 6.8, 5% β-mercaptoethanol, and 0.01% bromophenol blue) before electrophoretic separation. The protein samples were resolved by 12.5 or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Roche). The membranes were blocked in 5% nonfat milk-TBST for 3 h at room temperature. Next, the membranes were incubated with primary antibodies overnight at 4 °C followed by washing with 1× TBST for 10 min × 3 times. Then the membranes were hybridized with horseradish peroxidase (HRP)-conjugated secondary antibody (Tianjin Sungene Biotech Co., Ltd) for 1.5 h at room temperature. Antibody–antigen complexes were observed using enhanced chemiluminescence (ECL) system (Advansta, Menlo Park, CA, USA) with a Kodak imager (Carestream Health). The quantitative analysis of the relative intensities of proteins (normalized to β-actin) was performed with Quantity One Software (Bio-Rad, Hercules, CA, USA) and GraphPad Prism 5. All data are representative of three independent experiments with triplicate samples. Statistical significance was analyzed with a two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. All experiments in this study are repeated at least for three times.
Quantitative PCR (qPCR) was used to determine the relative quantities of RNA (cDNA) and DNA. Total RNA was extracted from harvested cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was obtained by reverse transcription with the Revert Aid™ First Strand cDNA Synthesis Kit (Thermo Scientific, Rockford, USA) according to the manufacturer’s protocol. QPCR was performed with a SYBR green Real-Time PCR master mix kit (Toyobo) according to the manufacturer’s protocol. All primers are listed in Additional file 2. The program set on the CFX96 sequence detection system (BIO-RAD) was 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s, 58 °C for 20 s, and 72 °C for 15 s. Values for the relative quantification were calculated by the ∆∆Ct method. Melting curve analysis was performed to verify the specificity of the products, and each sample was tested in triplicate.
Quantitative measurement of spliced XBP1 mRNA
1 × 106 BHK-21 cells were seeded in 6-well. 48 h after PFV infection, cells were washed three times with phosphate-buffered saline (PBS), and total RNAs were extracted by Trizol reagment (Invitrogen, Carlsbad, CA, USA). Then, 1 μg of total RNA samples were reverse transcribed using the Revert Aid™ First Strand cDNA Synthesis Kit (Thermo Scientific, Rockford, USA) according to the manufacturer’s protocol. For measurement of spliced XBP1 mRNA, XBP1 double-stranded cDNA was synthesized under the following thermal cycling conditions: 94 °C 5 min, 95 °C 30 s–55 °C 30 s–72 °C 30 s 30 cycles. Then, 7.5 U of restriction enzyme Pst I (TaKaRa Bio, Shiga, Japan) was added to the reaction mixture for 1 h to digest the double-stranded DNA of unspliced XBP1. The spliced XBP1 DNA wouldn’t be digested. β-actin mRNA expression was used as an internal control. PCR products were analyzed by electrophoresis through 1.5% agarose gel, and their identity was checked by DNA sequencing.
Data were expressed as the means ± standard deviations. Statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA) to evaluate the differences between experimental groups. Statistical significance was analyzed with a two-tailed Student’s t test. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. All data are representative of three independent experiments with triplicate samples. All experiments in this study are repeated at least for three times.
cyclic AMP-dependent transcription factor 4
activating transcription factor 6
baby hamster kidney cell line
CCAAT/enhancer binding protein homologous protein
fetal bovine serum
4-phenyl butyric acid
growth arrest and DNA damage-inducible protein 34
green fluorescent protein
glucose-regulated protein 78
human immunodeficiency virus
human T cell leukemia virus
Jun N-terminal kinase
inositol-requiring enzyme 1
microtubule-associated protein 1 light chain 3
lysosomal-associated membrane protein 1
minimum essential medium
multiplicity of infection
protein kinase R-like endoplasmic reticulum kinase
prototype foamy virus
red fluorescent protein
reactive oxygen species
unfolded protein response
X box-binding protein 1
WL, PY and LD designed the experiments. PY, LD and WL analyzed data and wrote the paper. PY and LD performed the majority of the experiments. QC and SW performed part of the experiments. ZL and YS offered some experimental materials. SH, JY, BP, XH and WL supervised this study and reviewed and edited the paper. All authors read and approved the final manuscript.
This work was supported by the National Natural Sciences Foundation of China (Nos. 81371790, 81641093, 81371422, 81571481, 31670163 and 31170154), Major AIDS and Viral Hepatitis and Other Major Infectious Disease Prevention and Control project of China (2014ZX10001003), the Fundamental Research Funds for the Central Universities of China and the Translational Medical Research Fund of Wuhan University School of Medicine.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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