Introduction
Influenza is one of the most common infectious diseases, which mainly caused by the infection of influenza virus in seasonal epidemics [
1,
2]. There are four types of influenza viruses (A to D), belonging to the RNA virus family Orthomyxoviridae [
3,
4]. Avian influenza A virus mainly infects poultry, and some subtypes also infect mammals and humans. Different subtypes of influenza A virus come from different combinations of surface proteins, haemagglutinin (HA) and neuraminidase (NA) [
3]. Among them, a subtype of influenza virus of influenza A, such as H9N2, induces avian influenza, which is a form of respiratory infectious disease [
5,
6]. The H9N2 subtype avian influenza virus has been reported to be low-pathogenic. Infection of H9N2 virus displays no clinical illness or only mild defects, such as a slight drop in egg production or mild respiratory signs, unless the infection is mixed with other pathogens, which will lead to severe respiratory syndromes and a high mortality rate [
7]. Therefore, H9N2 avian influenza is a severe public health risk and causes a huge economic loss to poultry production.
It is known that the genome of influenza A virus consists of eight ribonucleoproteins (RNPs), in which includes the genomic RNA, nucleoprotein, and an RNA polymerase [
8]. The RNA polymerase in influenza A virus is a heterotrimer that composes of polymerase acidic (PA), polymerase basic 1 (PB1) and PB2. Accumulating studies have revealed that the polymerase complex is necessary for the viral host selection, replication, pathogenicity, and transmission [
9‐
14]. Moreover, PA subunit of the RNA polymerase was reported to contribute to overcome the host species barrier and adapt to the new host species [
15]. In addition, the polymorphisms in PA protein has also been identified to affect the pathogenicity and host adaptation of influenza A virus [
16]. The PA subunit of the RNA polymerase is an endonuclease and a protease that induces proteolytic degradation of co-expressed proteins [
17,
18]. PA Subunit was reported to interact with a cellular protein with homology to a family of transcriptional activators [
19]. However, other bind targets of PA subunit still need to be elucidated.
In the present study, mass spectrometry was employed to screen for the binding protein with PA subunit of viral RNA polymerase. We identified that programmed cell death protein 7 (PDCD7) is a new binding target of PA subunit of H9N2 virus. Gain- and loss-of-function experiments demonstrated that PA subunit interacting with PDCD7 can activate apoptosis pathway and induce cell apoptosis, which may provide new insights in the role of PA subunit during H9N2 influenza virus infection.
Material and methods
Cells and cell culture
A549 cells were purchased form American Type Culture Collection (ATCC® CCL-185™). Cells were cultured with McCoy’s 5A Media (Modified with Tricine) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution in a 37 °C incubator with an atmosphere of 5% CO2 and 95% air. Cells were passaged by trypsinization.
Mass spectrometry
For mass spectrometry analysis, lysates from A549 cells transfected with Flag-tagged PA or vector control cells were prepared and incubated with Flag-M2 agarose beads (Sigma) overnight at 4 °C. The beads were then washed 3 times with IP wash buffer (150 mM NaCl, 10 mM HEPES pH 7.4, 0.1% NP-40), followed by elution with 1 M glycine (pH 3.0) twice. The eluted proteins were denatured and separated on SDS–polyacrylamide gels and stained with Coomassie blue; the indicated bands were subjected to mass spectrometry analysis.
Co-immunoprecipitation
Firstly, PA subunit of H9N2 polymerase were overexpressed in A549 cells and cultured for 48 h, and then were collected via trypsinization. Total cell lysates were made with NP-40 lysis buffer and was quantitated by bicinchoninic acid (BCA). The overexpressed protein was precipitated by adding Flag-M2 beads and incubating at 4 °C overnight, then was isolated via centrifugation. The immunoprecipitation samples were analysed by SDS-PAGE and western blot.
Pull-down assays
Total protein lysates from A549 cells were as aforementioned. The purified GST-tagged protein was added into the cell lysates to bind interacted partners at 4 °C, GST beads were added to enrich the tagged complex, and the unspecific binding proteins were removed by multiple wash with washing buffer containing Tris–HCl and detergent. The samples were analysed by SDS-PAGE and western blot.
Cell Viability assay
A549 cells were plated in 96-wells plated with complete medium with FBS and antibiotics, and the proliferation was analysed by Cell Counting Kit-8 (CCK-8). Briefly, 20 μl CCK-8 reagent was added into each well and incubated at 37 °C for 1 h, absorbance at 450 nM was then collected by the Molecular Device M2. Data was analyzed by GraphPad Prism 6.0.
Flow cytometry analysis
Cell apoptosis was analyzed by Annexin V and PI staining. Briefly, A549 cells were trypsinized and washed with PBS. Annexin V and PI reagent were added and incubated for 30 min form light. The fluorescence signals were analysed by BD Accuri™ C6.
Quantitative real-time PCR.
The total RNA of A549 cells was extracted by Trizol method. Briefly: add 1 mL Trizol to the cell culture dish to lyse and collect the cells, then add chloroform at the ratio of 200 μL/1 mL Trizol, shake vigorously for 15 s, stand for 5 min and then centrifuge; take the upper aqueous phase into a new centrifuge tube and add equal volume of isopropanol to precipitate the RNA. The precipitate was collected by centrifugation after standing for 30 min at 4 °C. The precipitate was washed with 75% ethanol without RNase and DNase and centrifuged again. The supernatant was discarded and dried and then dissolved in ultrapure water without RNase and DNase. cDNA was synthesized by reverse transcription form 2 μg of RNA, and 1 μL of the synthesized cDNA was used as the template for real-time fluorescence quantitative PCR detection. PCR primers were as followed: GAPDH sense 5′- CAA GTT CAA CGG CAC AGT CA-3′, GAPDH antisense 5′- ATC TCG CTC CTG GAA GAT GG-3′; PDCD7 sense primer: 5′- GGG ATC CAG ATG AGT TCC CA-3′, PDCD7 antisense primer: 5′- ACG AAG TTG CCT TTG GGA TG-3’.
Western blot
Proteins samples were firstly separated with SDS-PAGE and were transferred onto PVDF membranes. Sequentially, the membranes were blocked with 5% non-fat milk and then were incubated with Primary antibodies overnight at 4 °C, Horseradish Peroxidase (HRP) conjugated secondary antibody was added and incubated for 1 h at room temperature. Chemiluminescence signalling was collected via adding ECL reagents and the images was processed and analyzed with photoshop.
CRISPR/Cas9 gene editing technology
Guide RNA targeting the PDCD7 coding sequence was design on websites (Benchling.com) and was synthesized by micro-helix. PX459 vector containing both gRNA backbone and spCas9 coding sequence was used to clone the designed gRNA and was confirmed via sanger sequencing. The gene editing plasmid was transfected into A549 cells using Lipo3000 and then were selected with puromycin at 1ug/ml for 3 days. The survived cells were plated at low concertation allowing them into single clones after 2 weeks culture, and the knockout alleles were confirmed by Sanger sequencing and western blot.
Virus preparation
Isolation of the A/Swine/HeBei/012/2008/(H9N2) virus was previously described [
20]. For virus propagation, chicken embryos of 10-day-old which were free of pathogens were infected (Beijing Laboratory Animal Research Center, Beijing, China) and passaged for about 3 generations. The virus were then isolated by centrifugation and store at 80 °C.
Statistical analysis
All the data were expressed as mean ± standard deviation. T test and a one-way ANOVA was used for the statistical analysis was performed by GraphPad 6.0.
Discussion
The avian H9N2 influenza virus belongs to the influenza A virus and was first isolated from the turkeys in Wisconsin in 1966 [
21,
22]. Since then, H9N2 virus becomes one of the most prevalent avian influenza viruses, causing repeated poultry and human infection [
2,
7,
23]. Although the H9N2 influenza virus has diversified into multiple lineages, H9N2 virus was reported to be low-pathogenic unless the viral infection is complicated with other pathogens [
7,
24]. Avian H9N2 viruses can overcome the host species barrier and infect other mammals and humans [
15,
25,
26]. Accumulating studies have revealed that PA subunit of the RNA polymerase in influenza A virus are necessary in overcoming the host species barrier and host adaptation [
15‐
19,
27‐
31], indicating that PA subunit is critical for the infection of avian H9N2 influenza virus. In the present study, combining MS with pull-down assay, we identified that PA subunit is capable of nuclear transport and binding with PDCD7 to activate apoptotic pathway in A549 lung cells.
The PA subunit of the RNA polymerase in influenza A virus is an endonuclease and a protease that induces proteolytic degradation of co-expressed proteins [
17,
18,
26]. It has been reported that amino acid residues in the N-terminus of the PA subunit is critical for the protein stability, endonuclease activity, cap binding, and protein binding [
22,
32]. Mutations in the PA subunit has been found to inhibit endonucleolytic cleavage of capped RNAs [
33,
34]. Moreover, PA subunit of the RNA polymerase was also revealed to contribute to overcome the host species barrier and adapt to the new host species [
15]. Mutations of PA subunit, such as PA-K356R, are necessary for viral adaptation to new hosts, and might render H9N2 viruses adapted for human infection [
35]. In addition, the polymorphisms in PA protein also affects the pathogenicity and host adaptation of influenza A virus [
16]. These evidences indicated that the PA subunit are critical for the function and infection of influenza A virus. A few binding proteins were identified to bind with PA subunit, such as a cellular protein with homology to a family of transcriptional activators [
19]. PA subunit can also be translocated into the nuclei and regulate cRNA/vRNA synthesis [
36]. Here, for the first time we demonstrated that PA subunit bind with PDCD7 in the nuclei and activated apoptosis pathway and induced cell apoptosis. The way how PA subunit bind with PDCD7 still needs to be further investigated.
PDCD7, also named as apoptosis-related protein ES 18, locates in chromosome 15 and encodes a 59 kDa protein that interacts with the U11 small nuclear ribonucleoprotein, a component of the minor U12-type spliceosome, and contributes to the pre-mRNA splicing of U12-type introns [
37‐
39]. Mouse PDCD7 was first identified using the screening approach in 1999 and characterized as an apoptosis-related protein that involves in apoptotic cell death of T-cells [
40]. However, the specific function of PDCD7 in human is still unknown, although several studies have reported that human PDCD7 may related to the acute myeloid leukemia (AML) [
41,
42]. Compared with the non-malignant samples, higher PDCD7 expression was found in AML patients and related to lower complete remission rate, shorter relapse-free survival, and overall survival, suggesting that overexpression of PDCD7 may serve as a molecular marker for prognosis estimation of AML [
41,
42]. By MS combining with Co-IP experiments, we found PDCD7 is a binding partner of PA subunit of avian H9N2 virus and mediates cell apoptosis after PA overexpression in A549 lung cells, indicating that the interaction between PDCD7 and PA subunit may contribute to the cell apoptosis during virus infection.
Conclusion
Collectively, the PA subunit of H9N2 virus might induce lung cell death through binding and activating PDCD7, which provide new insights in the role of PA subunit during H9N2 influenza virus infection and new treatment possibilities for H9N2 infected cases. Therefore, further study should be warranted.
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