Influenza Virus A/Beijing/501/2009(H1N1) NS1 Interacts with β-Tubulin and Induces Disruption of the Microtubule Network and Apoptosis on A549 Cells

NS1 of influenza A virus is a key multifunctional protein that plays various roles in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways. These functions rely on its ability to participate in a multitude of protein-protein and protein-RNA interactions. To gain further insight into the role of NS1, a tandem affinity purification (TAP) method was utilized to find unknown interaction partner of NS1. The protein complexes of NS1 and its interacting partner were purified from A549 cell using TAP-tagged NS1 as bait, and co-purified cellular factors were identified by mass spectrometry (MS). We identified cellular β-tubulin as a novel interaction partner of NS1. The RNA-binding domain of NS1 interacts with β-tubulin through its RNA-binding domain, as judged by a glutathione S-transferase (GST) pull-down assay with the GST-fused functional domains of NS1. Immunofluorescence analysis further revealed that NS1 with β-tubulin co-localized in the nucleus. In addition, the disruption of the microtubule network and apoptosis were also observed on NS1-transfected A549 cells. Our findings suggest that influenza A virus may utilize its NS1 protein to interact with cellular β-tubulin to further disrupt normal cell division and induce apoptosis. Future work will illustrate whether this interaction is uniquely specific to the 2009 pandemic H1N1 virus.


Introduction
Influenza A viruses are globally important human and animal respiratory pathogens that are responsible for both seasonal, endemic outbreaks, and periodic unpredictable world-wide pandemics [1]. Three human pandemics occurred during the last century [2].The worst influenza A pandemic on record in 1918 killed approximately 50 million people worldwide [3]. In 2009, a novel swine-origin H1N1 influenza virus emerged in Mexico and quickly spread to other countries, including China. According to the WHO statistics, the virus has killed more than 18000 people. Influenza A virus belongs to the orthomyxoviridae family along with influenza viruses B and C. The influenza A virion is an enveloped RNA virus of spherical to ovoid shape measuring 80-120 nm in diameter. They contain a single-stranded, negative sense, segmented RNA genome consisting of eight segments of viral RNA (vRNA), which encode 11 known proteins [4]. The non-structural protein 1 (NS1) is the most important viral regulatory factor during infection. It is translated from a transcript of the segment eight and plays various roles in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways. All of these functions of NS1 rely on its ability to participate in a multitude of protein-protein and protein-RNA interactions [5]. To date, over twenty cellular factors have been described which interact with NS1. These include retinoic acid-inducible gene I (RIG-I) [6], poly(A)-binding protein I (PABPI) [7], p85beta [8], importin-a, nucleolin [9], NS1-Binding protein (NS1-BP) [10], eukaryotic initiation factor 4GI (eIF4GI) [7], hStaufen [11], NS1-I [12], protein kinase R (PKR) [13], PKR activator (PACT), the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30) [14], poly(A)-binding protein II (PABPII) [15], cellular adaptor protein Crk/CrkL [16], PDZ domain-containing proteins [5], the viral polymerase, and components of the cellular mRNA nuclear export machinery (E1B-Adaptor Protein 5 (E1B-AP5), p15, nuclear export factor 1 (NXF1), and nuclear/cytoplasm shuttling factor Rae1) [17] However, in view of the extreme multifunctional nature of NS1, more cellular factors maybe need to associate to this protein so as to fulfill different functions. It is reasonable to assume there are other unidentified interaction partners of NS1 protein.
Despite our substantial knowledge of this amazing and fascinating protein, much still remains to be learnt of its roles in the virus replication cycle. To gain further insight into the role of NS1, we made efforts to utilize influenza Virus A/Beijing/501/ 2009(H1N1) NS1 to find novel cellular factors that interact with NS1. To this end, a tandem affinity purification (TAP) system was chosen for this study. The key feature of TAP system is the use of two different affinity purification tags, they have gentle washing and elution conditions that allow the protein-protein interactions to remain intact, this not only allow for isolation of exceptionally clean proteins without disrupting the targeted complex, but increase the amount of the resulting purified protein complex. Moreover, as the histopathological and virological finding in fatal cases of 2009 H1N1 revealed that the 2009 H1N1 virus infected type II pneumocytes and caused diffuse alveolar damage (DAD), and potential infection in alveolar epithelial cells is also the main feature that differentiates it from seasonal influenza strains [18,19]. Human lung adenocarcinoma cell line A549 was used. By use of these, we identified a cellular factor, b-tubulin, as new interaction partner of NS1 protein. In addition, the disruption of the microtubule network and apoptosis were also observed on NS1transfected A549 cells. Our finding suggested that NS1 affects cellular functions through interaction with b-Tubulin.

Materials and Methods
Cell and the Viral Total RNA

Plasmid Constructions
The NS1 cDNA was amplified by RT-PCR using primers 59-CGC GGATCC ATG GAC TCC AAC ACC ATG TCA AGC T-39 (BamHI site, underlined) and 59-CCG GAATTC TCA TTT CTG CTC TGG AGG TAG TGA A-39 (EcoRI site, underlined) and were ligated between the BamHI and EcoRI sites of pnTAP vector (Stratagene); The correct sequence of all constructs described above was verified by sequencing.

NS1-TAP Expression and Purification of the Protein Complexes
Ten T175-cm 2 cell culture flasks of 90% confluence A549 cells were transfected with pnTAP-NS1 plasmids by using Lipofectamine TM 2000 Reagent (Invitrogen) according to the manufacturer's protocol. In parallel, A549 cells were transfected with pnTAP vector as control. Approximately 48 hours post-transfection, the cells were washed three times with PBS, then 5 ml of ice cold PBS was added to each flask to prepare the cell suspensions, the cells were harvested by centrifuging for 10 minutes at 1500 6 g. After removing the PBS, the protein complexes were purified by using InterPlay TAP Purification Kit (Stratagene, catalog #240107) according to the manufacturer's instructions. To detect the purified proteins, the protein preparation were resolved on 15% SDS-PAGE gels and stained with Coomassie Blue solution.

Peptide Mass Fingerprinting Analysis
To characterize the TAP-purified protein, the protein bands were excised from the Coomassie Blue-stained SDS-PAGE gel, ingel digested by trypsin, and analyzed by MALDI-TOF mass spectrometer AXIMA-QIT (Beijing Genomics Institute). Proteins were identified from peptide fragments by comparison to theoretical digests of the human proteome using MASCOT search tools.

Co-immunoprecipitation Analysis
To exclude the possibility that the interacting partner might represent unspecific factor, and further confirm the specific interaction, co-immunoprecipitation experiments were performed. The b-tubulin cDNA was amplified by RT-PCR using primers 59-GGA ATTC CATATG ATG AGG GAA ATC GTG CAC ATC CAG G-39 (NdeI site, underlined) and 59-TCC CCCGGG TTA GGC CTC CTC TTC GGC CTC CTC-39 (SmaI site, underlined). After being digested with NdeI and SmaI, the cDNA fragment was cloned into a NdeI-SmaI digested pCMV5-HA vector. A549 cells were co-transfected with expression plasmids pnTAP-NS1 and pCMV5-HA-b-tubulin or pnTAP vector and pCMV5-HA-b-tubulin. Approximately 24 hours post-transfection, the cells were harvested after washing with PBS, and then resuspended inA549 cells were lysed in lysis buffer A (20 mM Tris-  HCl (pH7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Trion X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerol phosphate, 1 mM sodium orthovandate, 10 ml/ml Protease Inhibitor Cocktail (Sigma) and 1 mM PMSF). After centrifugation at 10000 g for 15 min, the supernatant was collected and used as whole cell extract for later co-immunoprecipitation analysis. By using protein A/G beads (Santa Cruz Biotechnology) in the presence of antibody against HA (Santa Cruz Biotechnology), coimmunoprecipitation experiment was performed as described previously [20]. The co-immuoprecipitated products obtained were then subjected to SDS-PAGE and western blot analysis.

Immunofluorescence Staining
A549 cells were transfected with the expression plasmids pCMV5-HA-NS1 and control vector pCMV5 in suspension by using Lipofectamine 2000 (Invitrogen) respectively, then plated in a 24 well dish containing glass cover slips. Cells were fixed with 4% paraformaldehyde in PBS for 1 h, washed once with PBS and permeablized with 0.2% Triton in PBS for 30 min. After washing with PBS, cells were incubated with 10% goat serum in PBS for 1 h at 37uC to block unspecific binding of antibodies. Next, the primary antibody-mix (polyclonal anti-HA 1:100; monoclonal anti-b-tubulin 1:100 (Santa Cruz Biotechnology) in PBS containing 1% BSA was added for 1 h at RT. Cells were washed three times with PBS before the fluorescence-labeled secondary antibody mixture (FITC linked anti-mouse IgG 1:200 and TRITC linked anti-rabbit IgG 1:200 in PBS containing 1% BSA) was added for 1 h at 37uC. After cells were washed three times with PBS and rinsed with water, the cover slips were mounted with Fluorescent Mounting Medium on slides and observed with OLYMPUS IX81 microscope.
A cell that is undergoing apoptosis demonstrates nuclear condensation and DNA fragmentation, which can be detected by staining with Hoechst 33342 and fluorescence microscopy [21]. To determine whether apoptosis was induced in the A549 cells transfected with influenza virus A/Beijing/501/2009(H1N1) NS1, coverslips with adherent transfected A549 cells were collected at 24 h post transfection and washed, and the A549 cells were fixed with 4% paraformaldehyde and stained with Hoechst 33342 (100 ng/ml, Sigma, USA) for 20 min at room temperature. The coverslips were washed, mounted on glass slides, and stored at 4uC until quantification by fluorescence microscopy could be performed.   vector. The proteins were induced by 0.2 mM IPTG overnight at 16uC in LB and purified with glutathione Sepharose beads (GE Healthcare), free glutathione was removed by dialysis.

Mapping of the b-tubulin-binding Domain in the NS1 Protein
A549 cells were lysed with buffer A for 30 min on ice and then centrifuged at 16000 6 g for 30 min at 4uC. The supernatant was collected and used as the total cell lysate. Each GST-fused NS1 was added into the total cell lysate, then the mixture was incubated with glutathione Sepharose beads for 1 h at 4uC. After extensive washing, the bound materials were eluted with 20 mM glutathione in 50 mM Tris-Cl pH 8.0. The eluate was resolved on SDS-PAGE (14% gel) and analyzed by immunoblotting using anti-btubulin (Santa Cruz Biotechnology).

b-tubulin was Pulled Down Together with NS1 Using the N-terminal TAP Affinity Tags and Identified as a Novel NS1-binding Protein
The representative result of the purified protein complexes is shown in Figure 1A. As confirmed by western blot, NS1 and interacting partners were major in the soluble portion of whole cell extract ( Figure 1B). When the pattern of protein bands was compared between TAP-NS1 complexes and TAP-Null complexes, one major band (about 55 kDa, indicated by asterisk) was specific to the TAP-NS1 complexes on Coomassie Blue-stained gel. Although there are some other minor bands, we focus on the 55 kDa band. To identify this protein, the observed 55 kDa band was analyzed by using a MALDI-TOF mass spectrometer. The peptide mass fingerprinting pattern of the band was obtained successfully and applied to query a database search engine, MASCOT. The best match of the 55 kDa protein was with btubulin, a multifunctional cytoskeletal protein (Figure 2A). Overall, 12 peptides, ranging in size from 8 to 19 amino acids, were matched, representing a 33% sequence coverage of b-tubulin (141 of a total of 426 amino acids). By immunoblotting using an anti-btubulin monoclonal antibody, we confirmed that the detected 55 kDa band was b-tubulin in the TAP-NS1 complexes ( Figure 2B). b-tubulin was not detected in parallel analysis with the TAP-Null complexes (mock) ( Figure 2B).
The association of b-tubulin and NS1 in A549 cell lysate was further confirmed by conventional co-immunoprecipitation using an anti-HA rabbit polyclonal antibody. As expected, TAP-NS1 was detected in the precipitates of A549 cells co-tranfected with pnTAP-NS1 and pCMV5-HA-b-tubulin by the anti-calmodulin binding peptide (CBP) antibody ( Figure 2C), whereas in control co-immunoprecipitation using pnTAP vector and pCMV5-HA-btubulin co-transfected cells, no TAP-NULL was detectable.

The N terminal Domain of NS1 is Responsible for Binding with b-tubulin
Among the three fragments of influenza virus A/Beijing/501/ 2009(H1N1) NS1, as seen in the results Figure 2D-F, b-tubulin was pulled down with GST-NS1 full or GST-NS1 N but not with GST-NS1 C ( Figure 2F).

Co-localization of NS1 and b-tubulin in Cells
A549 cells were transfected with pCMV5-HA-NS1, NS1 was apparent from 24 h post-transfection, mainly in nucleus (green color) ( Figure 3E). On the other hand, b-tubulin was stained in nucleus and cytoplasm (red color) ( Figure 3F).The signals of NS1 and b-tubulin clearly overlapped in nucleus ( Figure 3G).

Apoptotic and Cytoskeleton Cell Morphology Observation
A549 cells were transfected with influenza virus A/Beijing/ 501/2009(H1N1) NS1 for 24 h and stained with Hoechst 33342. As illustrated in in Figure 3D and 3H, the tranfected cells exhibited a stronger blue fluorescence and condensated and fragmented nuclear after expression NS1 for 24 hours.
In addition, an intriguing phenomenon was observed in immunofluorescence staining test. Transfecting A549 cells with plasmid pCMV5-HA-NS1 induced visible changes in microtubule structure in accordance with the disassembly of tubulin polymers at 24 h post transfection ( Figure 3F), whereas mock transfection of A549 cells with pCMV5 vector did not induce perceptible changes in microtubule structure.

Discussion
The b-tubulin is the main constituent of microtubules (MTs), MTs are dynamic, polarized polymers composed of a/b-tubulin heterodimers, and ubiquitous cytoskeleton components that play a key role in various cellular processes relating to cell shape and division, motility, and intracellular trafficking [22,23]. MTs have important functions in the life cycle of most viruses [24,25]. In the present study, we identified b-tubulin as a novel interaction partner of influenza A virus NS1 protein, the two proteins colocalize in the nucleus of A549 cell transfected with NS1. As btubulin was generally regarded as a cytosolic protein, only b(a)-tubulin was found be present in few normal cells and a variety of cancerous cell lines [26,27]. Therefore we presumed it should be b(a)-tubulin which interacts with NS1 in A549 cells. NS1 consists of two functional domains, the C-terminal effector domain and the N-terminal RNA-binding domain. Here we determined that the RNA-binding domain of NS1 is responsible for binding with the b-tubulin. In addition, we also observed the depolymerization of the MT network on NS1-transfected human A549 Cells. For many anticancer compounds such as taxanes, isochaihulactone and the Vinca alkaloids, interfere with tubulin polymerization and microtubule depolymerization by binding to b-tubulin, and there is no evidence that interaction of NS1 with other known cellular factors induce depolymerization of MT on cells, therefore we assume that the interaction influenza virus A/Beijing/501/ 2009(H1N1) NS1 with b-tubulin induces disruption of the MT network on NS1-transfected human A549 Cells.
Apoptosis plays an important role in the pathogenesis of many infectious diseases, including those caused by viruses [28,29]. Influenza viruses have been reported to induce apoptosis in numerous cell types, both in vivo [30,31] and in vitro [32]. Several viral proteins (M1, NS1, and PB1-F2) from different strains of human influenza viruses have been shown to induce or inhibit apoptosis in human cells [33,34,35] [36,37,38]. Though the exact signaling pathway that influenza virus A/Beijing/501/2009(H1N1) NS1 induce apoptosis on A549 cells is not clear, progress made in the mechanism that microtubule depolymerization agents activate apoptosis may provides some helpful information. Previous studies have showed that microtubule depolymerization agents interfere with tubulin polymerization and microtubule depoly-merization, thereby arrest the cell cycle in G2/M phase and disrupt normal cell division, further act through several types of kinases, leading to phosphorylation cascades and the activation of cyclin B1/cdc2 complex and Bcl-2 phosphorylation, finally initiates the apoptotic cascade [39,40,41]. Our observation indicated influenza virus A/Beijing/501/2009(H1N1) NS1 caused G2-M cell cycle arrest (data not shown), moreover caspase 3-dependent apoptosis was showed to be involved in the homologous strain A/Wenshan H1N1-induced A549 cell and CNE-2Z cell death. Taken together, we presumed that the interaction of influenza virus A/Beijing/501/ 2009(H1N1) NS1 with b-tubulin depolymerized MT network and thereby disrupt normal cell division and commit the cell to apoptosis, thereby facilitate virus replication and indirectly contribute to virus pathogenicity. However, the exact role of NS1 on apoptosis induced by the 2009 pandemic H1N1 virus needs further investigation.
In summary, the present study provides evidence that b-tubulin represent a novel interaction partner of influenza A virus NS1 protein. The RNA-binding domain of NS1 is responsible for binding with b-tubulin. The interaction of NS1 with b-tubulin disrupts the cellular microtubule network and induces apoptosis on human A549 cells.