Background
Influenza A virus (IAV) is a critical respiratory pathogen that belongs to the Orthomyxoviridae family
, which causes severe morbidity and mortality rates every year [
1]. IAVs have negative-stranded RNA genomes consisting of eight RNA segments, and encode up to 17 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), polymerase basic protein 1(PB1), polymerase basic protein 2 (PB2), PB1-F2, PB1-N40, polymerase acidic protein (PA), PA-X, PA-N155, PA-N182, matrix protein 1 (M1), M2 and M42, nonstructural protein1 (NS1), NS2, NS3 [
2‐
8]. NP is a viral RNA genome-encapsulating structural protein which can associates with viral RNA and three polymerase proteins (PB1, PB2 and PA) to form viral ribonucleoprotein (vRNP) complex [
9]. NP also interacts with a plethora of cellular factors in viral RNA replication, virus assembly and intracellular trafficking of the virus genome [
10,
11]. Our previous study demonstrated NP interacted with histone deacetylase 1, which downregulated the acetylation level of NP. While the interaction between the NP and Histone Acetyltransferase (HATs) enzymes were largely unknown.
HATs are an enzyme family that regulates these processes by catalyzing the transfer of an acetyl moiety onto target proteins. TIP60 is a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) family of histone acetyltransferase that was found by interaction with the HIV-1 Tat protein [
12,
13]. It contains an N-terminal chromodomain and a C-terminal conserved MYST domain (HAT domain and Cys-Cys-His-Cys zinc finger). HAT domain binds to acetyl coenzyme A and the substrate, and the zinc finger is required for protein–protein interactions [
14]. Expression of TIP60 is observed in multiple cell lines and tissues, and its homologues have been identified in chicken, mouse, and human [
15].
TIP60 acts as a coactivator or a co-repressor involved in transcriptional regulation of a variety of factors, such as myelocytomatosis oncogene c (c-Myc), signal transducers and activators of transcription 3 (STAT3)[
16,
17]. In DNA damage response, TIP60 was activated and it interacts with the kinase ataxia telangiectasia-mutated (ATM), which activates and induces ATM autophosphory resulting in initiates a series of reparative reactions [
18]. TIP60 is a component of the p53 pathway. It was required for both cell growth arrest and apoptosis mediated by p53 and also induces its acetylation [
19]. It can regulate the stability of p53 by interfering with Mdm2-mediated p53 degradation to maintain a basal pool of p53 in normal growth conditions, but TIP60 functions as p53 co-activator following DNA damage [
20]. Moreover, a variety of cellular factors involved in the apoptosis mediated by p53 as cofactor. Programmed cell death 5 (PDCD5) binds to TIP60 and enhances HAT activity of TIP60 and induces TIP60-dependent K120 acetylation of p53, which advances expression of apoptosis-related genes [
21]. ING5 can as a cofactor of TIP60 to involved in the acetylation of p53 at K120 and it formed a complex with p53 and TIP60 [
22]. USP7 interacts with and deubiquitinates TIP60 to enhance its stability, which contribute to activated of the P53-mediated apoptotic pathway [
23]. In addition, TIP60 functions as a tumor suppressor was widely accepted and it lower expression was observed in multiple cancers [
24].
In addition to HIV type 1 (HIV-1) Tat, TIP60 also is a key target of several viral proteins during infection. Human T cell lymphotropic virus type 1 (HTLV-1) p30
II enhances Myc-associated transcriptional and transforming activities through interaction with the HAT of TIP60 [
25]. Furthermore
, TIP60 can interact with viral E6 and UL27 proteins, which encoded by human papillomavirus (HPV) and human cytomegalovirus (HCMV), respectively [
26,
27]. However, whether the TIP60 interacted with NP of IAVs is largely unknown.
In this study, we showed that the NP protein of IAV interacted with TIP60. We found that IAV infection upregulated the protein expression of TIP60 in a time- and dose-dependent manner. We further examined the effects of TIP60 on IAV replication. Overexpression of TIP60 inhibited viral replication through activation of TBK1-IRF3 signaling pathway.
Methods
Cells, viruses, and plasmids
A549 cells, 293 T cells and MDCK cells were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a 5% CO2 incubator of 37 °C. A/Qing Hai/environment/2005 (H5N1) strain were propagated in 10-day-old specific pathogen-free (SPF) embryonic chicken eggs and titrated on MDCK cells. All experiments with IAVs were carried out in biosafety level 3 containment laboratories approved by the Chinese Academy of Science.
Plasmids construction
The full-length TIP60 was cloned into the pCDNA3.0-HA vector. NP gene from A/Qing Hai/environment/2005(H5N1) influenza virus was cloned into pcDNA3.1-Myc and pHW2000 vectors as previously described [
28]. The sequence integrity of all plasmids was identified by gene sequenced.
Transfection and virus infection
Cells (4 × 105) were spread in 6-well plates and incubated for 18 h. Cells were transfected at 80–90% confluence with Lipofectamine 3000 reagent (Life Technologies) according to the manufacturer’s guidelines. After 48 h of incubation, the Cells were infected with the indicated virus at a multiplicity of infection(MOI) of 1. The DMEM supplemented for virus infection contained 2% FBS and 2.5% bovine serum albumin (BSA). Incubation 1 h at 37 °C and shaken at every 15 min, removed the inoculum and washed once with PBS. Then, added to fresh serum-free DMEM containing 2.5% BSA and incubated back at 37 °C.
TCID50
One day before infection, 3 × 104 MDCK cells were seeded in a 96-well plate with five repetitions. 24 h later, cells were infected with different dilutions of virus for 1 h at 37 °C, with shaking every 15 min. The inoculum were removed and washed once with PBS. Then, added to fresh serum-free DMEM containing 2.5% BSA and incubated back at 37 °C.The cytopathy was observed for one time every 12 h and confirmed by the hemagglutination assay. Calculated the TCID50 by the Reed-Muench method.
RNA extraction and quantification real-time PCR
Total RNA was extracted from infected cells using TRIzol reagent according to the manufacturer (Invitrogen), and 2 μg of RNA was reverse-transcribed into cDNA using the GoScript Reverse Transcription System (Promega). Primers used for real-time RT-PCR are TIP60 primers forward CAGGACAGCTCTGATGGAATAC and reverse AGAGGACAGGCAATGTGGTGAG. The primers of the corresponding virus mRNA, cRNA and vRNA are described in previous studies [
1]. Fold change in RNA levels were calculated using the delta
–delta threshold cycle (
ΔΔCT) method as described elsewhere [
29].
Western blotting
First, cells were washed PBS and harvested in lysis buffer (pH 7.6, 50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% Triton-100, and protease inhibitor [Roche]). Next, the total cell lysates were quantitated by using a BCA kit (APPLYGEN). Then, the equal amounts of proteins obtained were subjected to SDS- PAGE, and resolved proteins were transferred onto nitrocellulose membranes. Subsequently, after blocking with 5% nonfat milk, the membranes were incubated with primary antibody followed by secondary antibody. The membrane blots were visualized by using the Dura chemiluminescent kit (Millipore), and images were acquired on ImageQuant las 4000 imaging system (GE Healthcare).
Luciferase reporter assay
All polymerase complex component plasmids (PB2/pHW2000, PB1/ pHW2000, PA/ pHW2000, and NP/pHW2000) DNA (1 μg each) and a luciferase RNA expression (vNS-luc/pHH21) vector and pRL-TK (50 ng) were cotransfected into 293 T cells. At the same time, pcDNA3.0-HA empty vector or plasmid expressing TIP60 was also transfected into 293 T cells. The Renilla luciferase was used as an internal control. At 36 h post-transfection, cells were lysed and measured luciferase activity using the Dual Luciferase Assay System (Promega).
Co-immunoprecipitation analysis
293T cells were transfected with pCDNA3.1-MYC-NP and pCDNA3.0-HA-TIP60 or pCDNA3.0-HA. At 48 h post-transfection, cells were lysed in binding buffer containing protease inhibitor cocktail. The cell lysates were incubated with anti-MYC at 4 °C for 6–8 h. Then, added to protein G-Sepharose beads incubated at 4 °C overnight. Beads were washed three times with binding buffer, boiled with 2× SDS loading buffer for 10 min. Subsequently, the samples were assayed with MYC and HA antibody using western blot.
Immunofluorescence analysis
293T or A549 cells were grown on glass coverslip and transiently transfected with plasmids expressing TIP60. 48 h after transfection, the cells were infected with virus at a MOI of 3. At 12 h p.i., cells were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were then washed with PBS three times and blocked with 1% bovine serum albumin in PBS at RT for 10 min. Cells were incubated overnight at 4 °C with primary antibodies, then washed three times with PBS and incubated with the secondary fluorochrome-conjugated antibodies for 1 h. After several washes, cells were counterstained with DAPI (Sigma) for 3 min, and analyzed by confocal laser microscopy (Leica 780).
Statistic analysis
All date were analyzed with software GraphPad Prism 6 (GraphPad Prism, La Jolla California USA). The date were expressed as mean ± SD. Statistical comparisons were made by Student’s-test. AP value < 0.05 was considered significant.
Discussion
The infection of IAV induced the expression of many genes, suggesting that some may play essential or accessory roles in the viral life cycle or the host cell’s stress response [
31]. We found that IAV infection increased TIP60 mRNA as well as protein expression in A549 cells and 293 T cells. Increasing evidence suggests that TIP60 plays a role in viral life cycle. Recent research suggests that the human Papillomavirus (HPV) oncoprotein E6 interacts with and destabilizes the TIP60, and the degradation of TIP60 relieved cellular repression of the viral early promoter [
26]. Also, it has been reported that the human cytomegalovirus pUL27 interacts with the TIP60 and induce its degradation by proteasome, though the effect of this degradation to the viral life cycle was unknown [
27]. Another recent study showed that TIP60 binds to the early adenovirus promoter and depresses adenovirus EIA gene expression [
32]. Here, we show that TIP60 overexpression resulted in a considerable decrease in NP protein and gene levels, and it reduced the progeny viral titer in cells, which is the first reported the role of TIP60 in IAV infection. We also knockdown the TIP60 in cells using siRNA and shRNA, however, the endogenous TIP60 mRNA and protein level didn’t down regulated through we repeated three times both in A549 and 293 T cells.
The replication and transcription of viral genome and mRNA in the nucleus are completed through the influenza virus polymerase complex [
33]. Here, we found that the TIP60 overexpression reduced mRNA, vRNA and cRNA levels of IAV NP. In addition, we determined that TIP60 exerts its suppressive impact on the polymerase activity of IAV. Therefore, we speculated that TIP60 inhibits the replication of virus by reducing viral polymerase activity. Several proteins and compounds inhibit virus polymerase complex activity to depress the replication of influenza virus [
34‐
37]. Moreover, we also found that TIP60 enhances the production of type I IFN and pro-inflammatory cytokines during IAV infection.
The interplay between host factors and viral proteins play important roles in host regulation of influenza virus replication. In the present study, a cellular acetyltransferase protein, TIP60, was found to interact with NP of influenza A virus. In addition, immunofluorescence analysis showed that TIP60 and IAV NP colocalized in the nucleus, suggesting that the interaction between TIP60 and NP occurs in the nucleus. NP is highly conserved among different types IAVs and it was required for the transcription and replication of influenza virus [
9]. It is known to that NP protein interacts with several host factors to exhibit its diverse role in viral life cycle. Cytoskeleton-scaffolding protein actinin-4 interacts with NP to facilitate its intercompartmental localization and promotes viral replication [
38]. The host protein cluster (CLU) was found to interact with NP of IAV during virus infection, and CLU overexpression attenuated NP-induced cell death and depressed IAV replication [
39]. NF90 inhibits virus polymerase complex activity and replication through direct interaction with viral NP during IAV infection [
36]. Interaction between MOV10 and viral Nucleoprotein inhibited the polymerase activity and virus replication [
37]. The study of the interaction between host and NP can provide new ideas for the antiviral target sites [
40].