Avian metapneumovirus (aMPV) is the causative agent of respiratory tract infections and reduced egg production in poultry and has caused global economic losses [1
]. aMPV was first reported in South Africa in 1978 [3
] and was subsequently reported in many countries worldwide. Both aMPV and human metapneumovirus (hMPV) belong to the new Pneumoviridae
family, genus Metapneumovirus
. There are four subtypes of aMPV, termed A, B, C and D, which are based on genetic and antigenic differences [4
]. There is a high similarity between subtype C aMPV and hMPV [5
]. The infection of subtype C aMPV (aMPV/C) in Muscovy ducks has been reported in France and China [8
] as well as in turkeys and wild birds in the USA [10
], forming two distinct genetic lineages, one North American and the other Eurasian. In July 2010, an outbreak with severe respiratory symptoms occurred in meat chickens in southern China. Subsequently, we isolated a subtype C aMPV (aMPV/C), designed strain JC, from the infected chickens and showed that it was the causative agent. This was the first report of chickens infected with aMPV/C [11
]. Subsequently, aMPV/C was reported to experimentally infect mice and induce strong inflammation in the lungs [12
]. Another research study showed that aMPV/C could induce a complete autophagic response, which was partially regulated by ER stress [13
]. However, analysis of proteomic profiles in aMPV/C-infected cells has not yet been described.
Currently, high-throughput proteomic approaches are effectively used for the comprehensive analysis of virus infection. Isobaric tags for relative and absolute quantification (iTRAQ) combined with liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis is a sensitive and accurate proteomic approach that has been widely employed to investigate proteomic profiles of host cells during viral infections, including highly pathogenic avian influenza (HPAI) virus [14
], hepatitis B virus (HBV) [15
], transmissible gastroenteritis virus (TEGV) [16
], porcine circovirus type 2 (PCV2) [17
], classical swine fever virus (CSFV) [18
], porcine reproductive and respiratory syndrome virus (PRRSV) [19
] and porcine epidemic diarrhea virus (PEDV) [20
]. These studies have quantified cellular proteins and identified potential biomarkers in the infected cells, which also help improve our understanding of viral pathogenesis.
PLK2 (also called Snk), which belongs to a highly conserved mammalian serine/threonine protein kinase family, has an N-terminal serine/threonine kinase domain and a C-terminal polo-box domain (PBD). PLKs play multiple roles throughout the cell cycle and have emerged as anticancer therapeutic drug targets [22
]. PLK2 was originally regarded as an immediate–early response gene and cell cycle regulator [23
] and has been reported to display a complex functional response by acting as a cell survival factor [25
] but occasionally as an apoptotic effector [28
] in tumorigenesis and certain diseases. PLK2 signaling via p53 is involved in checkpoint signaling and tumor growth [31
]. Studies have also revealed that the PLK2 response to oxidative stress is associated with p53 signaling [38
To date, no study has been carried out to assess the protein profiles of aMPV/C-infected cells. In the present study, iTRAQ combined with LC-MS/MS was used to reveal the global proteomic changes occurring in Vero cells in response to aMPV/C infection for the first time and will help understand the molecular mechanisms underlying viral infection. We found that PLK2 expression was upregulated and further examined its biological function in aMPV/C infection. Suppression of PLK2 by small interfering (si)RNA attenuated aMPV/C-induced p53-dependent apoptosis and reactive oxygen species (ROS) generation. We also determined the effects of the PLK2 signaling pathway on aMPV/C replication, and found that inhibiting PLK2 decreased virus release at 72 h pi. Our results demonstrate that high PLK2 expression is correlated with induction of apoptosis in aMPV/C-infected Vero cells and facilitates viral release.
2. Materials and Methods
2.1. Cells and Virus Infection and Determination
Vero cells were grown at 37 °C with 5% CO2 using Dulbecco’s modified Eagle’s minimal essential medium (DMEM; Gibco, NY, USA) supplemented with 5% heat-inactivated fetal bovine serum and 1% penicillin–streptomycin solution. The avian metapneumovirus subgroup C JC strain was isolated from meat-type commercial chickens in southern China, as previously reported [11
], and propagated in Vero cells. CPEs was observed under a light microscope. All the cells infected with aMPV/C followed this process. Cells were incubated with the JC strain at a multiplicity of infection (MOI) of 1 for 2 h of adsorption. After the virus was removed, the infected cells were washed in PBS and then maintained in DMEM supplemented with 2% FBS. Cells at the indicated time points were harvested and stored at −80 °C prior to analysis. Then, the viral titer was determined using a TCID50
assay on a monolayer of Vero cells in 96-well plates. A total of 100 μL of 10-fold serial dilutions of the supernatant samples were cultured with Vero cells in 96-well plates. At 4–5 days postinfection, CPEs were quantified and virus titer was determined as the 50% tissue culture infective dose (TCID50
) per 0.1 mL.
2.2. Protein Extraction, Digestion and Labeling with iTRAQ Reagents
Vero cells at 80% confluence in 75 cm2 flasks were inoculated with the JC strain at an MOI of 1 for 48 and 72 h. Uninfected cells were used as controls. Cells from three independent experiments were collected with a cell scraper, washed in cold PBS and centrifuged at 500× g for 10 min. The proteins were extracted, and the pellets were resuspended in lysis buffer with 1 mM PMSF, 2 mM EDTA and 10 mM DTT. We then obtained solubilized protein and removed the cellular debris by sonicating and centrifuging at 4 °C and 30,000× g. A Bradford protein assay was used to quantify the protein concentration in the supernatants. One hundred grams of each sample was digested at 37 °C overnight with sequencing grade modified trypsin (Promega, Madison, WI, USA). Each sample was processed according to the manufacturer’s protocol for 8-plex iTRAQ reagent labeling. The labeled digested proteins were then pooled and vacuum dried.
2.3. SCX Chromatography and LC-MS Analysis
Strong-cation exchange (SCX) chromatography was performed with a Thermo EASY-nLC High-Performance Liquid Chromatography (HPLC) System. iTRAQ-labeled peptide mixtures were loaded onto Thermo scientific EASY columns in buffer A (10 mM KH2PO4 pH 3.0, 25% CAN). Peptides were eluted with a gradient of 0%–40% buffer B (10 mM KH2PO4 pH 3.0, 500 mM KCl, 25% ACN) for 85 min, 40%–100% buffer B for 3 min and then 100% buffer B for 2 min using a flow rate of 250 nl/min. The reconstituted samples were analyzed using a Thermo Finnigan Q Exactive Mass Spectrometer. The MS scanning conditions included 90 min in positive ion mode; 300–1800 m/z; 70,000 resolution at m/z 200; AGC target: 3e6; maximum IT: 10 ms; number of scan ranges: 1; dynamic exclusion: 40.0 s; MS2 activation type: HCD; isolation window: 2 m/z; 17,500 at m/z 200; microscans: 1; maximum IT: 60 ms; normalized collision energy: 30 eV; and underfill ratio: 0.1%.
2.4. Data Analysis
Relative quantification and protein identification were both performed with Proteome Discoverer 1.4 (Thermo). Trypsin was chosen due to its enzyme cleavage specificity, and there was a maximum allowed number of two missed cleavages. Carbamidomethylation and oxidation were set as fixed modifications for the iTRAQ labeling approach. The analysis was performed using a peptide tolerance of 20 ppm and a fragment tolerance of 0.6 Da using the iTRAQ method. The mass spectrometry proteomics data were deposited into the ProteomeXchange Consortium via the PRIDE [40
] partner repository with the dataset identifier PXD017827.
Each MS/MS Ion search was based on the UniProt Macaca mulatta proteome database (UP000006718). The score threshold for peptide identification was set at a 1% false discovery rate (FDR). To designate significant changes in protein expression, fold changes >1.33 or <0.76 were set as cutoff values.
2.5. Knockdown of PLK2 by RNA Interference (RNAi)
Three following siRNAs were designed: siRNA-PLK2-739 (sense, 5′-CCACUACUUUGAGGACAAATT-3′; antisense, 5′-UUUGUCCUCAAAGUAGUGGTT-3′), siRNA-PLK2-1214 (sense, 5′-GCUCCUGCCAAGCACUUAATT-3′; antisense, 5′-UUAAGUGCUUGGCAGGAGCTT-3′), siRNA-PLK2-1355 (sense, 5′-CCAGAUUUCCACUUAUCAATT-3′; antisense, 5′-UUGAUAAGUGGAAAUCUGGTT-3′) and siRNA-NC (NC) (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT-3′). These siRNAs were designed by the GenePharma Company (Suzhou, China) and used to silence the expression of PLK2 protein in Vero cells. The monolayer cell cultures were transfected with siRNA using Lipofectamine RNAiMAX reagent according to the manufacturer’s protocol.
2.6. Western Blot Analysis
Western blotting was employed to analyze changes in the expression levels of proteins from the cell lysates. All protein concentrations were determined. Samples were loaded with 1 × sample loading buffer, boiled for 10 min at 100 °C, separated by SDS-PAGE and then transferred to a PE membrane. The membrane was washed and blocked for 2 h in TBST with 5% nonfat dried milk, incubated with the indicated primary antibody overnight at 4 °C and diluted in TBST according to the manufacturer’s protocol. The membrane was washed three times in TBST for 10 min and incubated for 45 min in secondary antibody diluted in PBS at room temperature. The membrane was washed three times; the target proteins were detected using a SuperSignal West Femto Substrate Trial Kit (Thermo Scientific, 34096) according to the manufacturer’s instructions and then exposed to a chemiluminescence apparatus (Proteinsample, Santa Clara, CA, USA). The following primary antibodies as following: mouse anti-F monoclonal antibodies was prepared in our laboratory; rabbit anti-PLK2 antibody (ab137539) was obtained from Abcam; mouse anti-Bcl-2 antibody (sc-7382), mouse anti-β-actin (c-4) antibody (sc-47778), mouse anti-Bax (B-9) antibody (sc-7480), mouse anti-p53 (A-1) antibody (sc-393031), mouse anti-caspase-3 (E-8) antibody (sc-7272), mouse anti-caspase-3 p11 (C-6) antibody (sc-271759), mouse anti-BIN3 (C-10) antibody (sc-514396), mouse anti-Annexin II (3D5) antibody (sc-47696) and mouse anti-Rab 8A antibody (63-BJ) (sc-81909) were purchased from Santa Cruz Biotechnology. The following secondary antibodies were obtained from Sigma-Aldrich, including horseradish peroxidase (HRP)-conjugated goat anti-mouse (A9044), anti-rabbit (A0545).
2.7. Apoptosis Analysis
Vero cells were transfected with PLK2-siRNA-739 and siRNA-NC or treated with NAC (100 µM) before virus infection. After 12 h of transfection, the cells were infected with aMPV/C for 48 h. After this, the treated cells, mock-infected cells and cells infected with aMPV/C alone were collected and incubated with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for flow cytometry analysis.
2.8. Reactive Oxygen Species Measurement
Vero cells were plated in 96-well plates. The cells were transfected with PLK2 siRNA-739 or siRNA- NC and 12 h later infected with aMPV/C for 48 h. After this, the treated cells, mock-infected cells and cells infected with aMPV/C alone were incubated with DCFH-DA in accordance with the manufacturer’s procedures (Beyotime Biotechnology, Shanghai, China) and analyzed by a Multiscan Spectrum. For the ROS assay, the cells were incubated with 10 µM DCFH-DA fluorescent probe for 20 min in the dark at 37 °C.
2.9. Measurement of Virus Release in Vero Cells
Vero cells treated with NAC or siRNA were infected with aMPV/C at MOI of 5, and cells supernatant cultures were collected at 72 hpi. The virus titer was determined as the 50% tissue culture infective dose (TCID50) per 0.1 mL.
Viral infections often induce host cell environment modifications, which prevent or favor viral replication. To reveal the mechanism of aMPV/C infection, we need more detailed information. Proteomics, especially iTRAQ combined with LC and tandem MS analysis, is a powerful technology used to quantitatively analyze and identify proteins and can be used to find disease-specific targets in a high-throughput manner. To date, there is no report on the cellular proteome changes after aMPV/C infection. Cultured Vero cells are the best available substrate for aMPV and have been used to study fuctions of aMPV proteins, entry to cells, mediation to membrane fusion, replication, induction of autophagy and vaccine preparation in previous reports [42
]. aMPV/C is more closely related to hMPV and may have larger cellular host ranges potentially including mammalian than other subtypes [12
]. In this study, iTRAQ was used for the first time to analyze Vero cells infected with aMPV/C. We identified 921 DE proteins at two time points post infection. Three selected DE proteins were confirmed using Western blotting as an independent analytical method. The proteomic results provided valuable data for the further analysis of aMPV/C infection. Bioinformatics indicated that the DE proteins are involved in a variety of processes, including metabolism, biosynthesis, signal transduction, the host response to stress, metabolic pathways, the PI3K-Akt signaling pathway, the AMPK pathway, cell death and the cell cycle (Figure 3
and Figure 4
, Table 1
). Our data identified several previously uncharacterized key proteins possibly involved in aMPV/C infection of Vero cells.
The cell cycle consists of DNA replication, mitosis and cytokinesis (S, M, G1 and G2 phases). Many viruses that interact with the cell cycle can subvert host-cell functions and increase viral replication. Quiescent cells are referred to as being in the G0 phase. Many host molecules participate in the regulation of the cell cycle. Cyclin-dependent kinase (CDK) 4/6 complexes and phosphorylation of the downstream retinoblastoma (Rb) protein initiate and regulate G1-phase progression. Cyclin E-Cdk2 activity is important in the G1/S transition and S phase. CDK1 is required for entry into mitosis and plays an essential role in S phase. The cell cycle is negatively regulated by cyclin-dependent kinase inhibitors (CKIs). Many viruses, including herpesviruses [49
], murine hepatitis virus (MHV) [50
], severe acute respiratory syndrome coronavirus (SARS-CoV) [51
], influenza A virus [52
], human respiratory syncytial virus [53
], murine norovirus (MNV) [54
] and porcine circovirus type 2 [55
], have been reported to induce cell cycle arrest for efficient replication. In our study, the expression of cyclin-dependent kinase inhibitor 1B (CDN1B), cyclin-dependent kinase 6 (CDK6) and cyclin-dependent kinase 1 isoform 1 (CDK1) was found to be downregulated during aMPV/C infection in Vero cells (Table 1
). These DE proteins might be responsible for cell cycle arrest during aMPV/C infection. The specific mechanism requires further investigation.
Various cell signal transduction pathways have been shown to facilitate virus survival or the antiviral response in previous studies. The PI3K/Akt and mTOR pathways have been well documented to stimulate cell proliferation and inhibit apoptosis. Many viruses can activate the PI3K/Akt pathway to increase the viral titer. Respiratory syncytial virus (RSV), belonging to the same family as aMPV, has been shown to increase the survival of epithelial cells by activating PI3K/Akt, which increases replication before Fas-mediated apoptosis [56
]. The proteins in the VTNC, 14-3-3 and RHEB pathways were found to be differentially regulated during aMPV/C infection (Table 1
). The MAPK cascade pathways are part of the protein kinase cascade, which involves transcription factors and modulates cell growth, differentiation and inflammation. Some human respiratory viruses, such as RSV and H1N1 influenza virus, have been shown to utilize protein kinases, such as MAPK, for replication [57
]. The expression of the STAT1, PAK2 and HSPB1 proteins was downregulated in aMPV/C-infected Vero cells (Table 1
). Both up- and downregulated proteins are shown. One protein processing pathway, the heat shock response, is a multifunctional mechanism for cell survival following pathogen infection [58
]. In this study, Hsp90 and Hsp70 were identified as DE proteins in Vero cells 48 h after aMPV/C infection. By regulating these pathways, the virus utilizes specific mechanisms for its infection and replication.
Apoptosis regulates the pathogenesis of many infectious diseases [59
]. Apoptosis induced by virus infection can inhibit or favor viral replication. Blocking apoptosis may lead to an increased viral titer because of the increased survival of infected cells. In this study, apoptosis-related genes were identified among the DE proteins following aMPV/C infection in Vero cells. The 14-3-3 proteins represent a family of adaptor and scaffold proteins in eukaryotic organisms. They can interact with multiple cellular proteins and might be involved in modulating the cell cycle, apoptosis and cell death at multiple steps [60
]. The 14-3-3 proteins have been reported to be involved in the hepatitis C virus-mediated activation of Raf-1 [61
] and HIV-1-induced cell cycle regulation [62
]. As shown in Table 1
, the decreased expression of three 14-3-3 proteins (eta, beta/alpha, gamma) was found at 48 hpi. We speculate that the inhibition of apoptosis during early infection might contribute to the rapid replication of progeny virions before cell death. More studies are required to examine the function of the 14-3-3 proteins in aMPV/C-infected Vero cells.
In the aMPV/C proteomic data, the proteins with decreased expression were the majority. However, we discovered significant upregulation of PLK2 expression in aMPV/C-infected cells, which was further confirmed by Western blotting. Polo-like kinases are related to serine/threonine protein kinases, which are well-defined cell cycle regulators. PLK2 has been reported to act as a tumor suppressor gene but sometimes plays an oncogenic role in tumors. PLK2 can affect cell cycle regulation and cell survival, which is closely associated with the p53 family and ROS signaling pathway. We found that aMPV/C infection triggers apoptosis and ROS generation in Vero cells, which is inhibited by PLK2 siRNA. Considering that the p53 signaling pathway is involved in aMPV/C infection, the measurement of Bcl-2, Bax, p53 and caspase-3 was performed in aMPV/C-infected Vero cells. The BCL-2 family is a key regulator of mitochondrial apoptosis that comprises proapoptotic and antiapoptotic members. BCL-2 belongs to antiapoptotic proteins. Bax is a proapoptotic effector protein. Specific cellular signaling can modulate the expression of these two kinds of proteins to promote cell survival or death. A family of cysteine proteases called the caspase cascade can mediate the process of apoptosis. Caspase-3 is the final common molecule involved in apoptosis. A decreased Bcl-2/Bax ratio can damage the integrity of mitochondria and lead to caspase-3 activation during apoptosis. In our report, suppressing PLK2 inhibited ROS production and reversed the apoptosis marker expression changes, including an increased Bcl-2/Bax ratio and decreased cleaved caspase-3 and p53 expression. In the current study, our data collectively suggest for the first time that PLK2 expression contributes to p53-mediated apoptosis and ROS generation in aMPV/C-infected Vero cells. This result demonstrates that PLK2 plays an important role in aMPV/C-induced apoptosis through ROS production and activation of the p53 signaling pathway. To a certain extent, this result is consistent with those of previous reports showing the proapoptotic function of PLK2 but not its antioxidant function [39
]. Through further investigation, we demonstrated that the inhibition of PLK2 expression decreases virus production in the supernatant at 72 h pi. We speculate that PLK2 might promote apoptosis for viral release. The findings of this study suggest that PLK2 can regulate p53 and ROS and plays an essential role in apoptosis and viral replication in Vero cells after aMPV/C infection.
aMPV/C infection has recently emerged in China and has been gaining increasing attention due to its high morbidity rates and pathogenicity. In general, this is the first report of the proteomic profiles of Vero cells following aMPV/C infection using iTRAQ combined with LC-MS. These data provide us comprehensive information about several DE proteins in cellular responses regarding cell cycle, endocytosis, and PI3K-Akt, mTOR, MAPK and p53 signaling pathway in the cultured aMPV/C-infected Vero cells. Furthermore, a high expression of PLK2 by aMPV/C infection promoted apoptosis through ROS accumulation and p53 signaling activation, and release of the virus. However, in-depth analysis of host cellular responses to aMPV/C infection in natural host-chickens will be needed to better understand the pathogenic mechanisms for the development of vaccine strategies and antiviral therapeutic targets for aMPV/C infection in the future.