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

Abstract

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.

C-terminal elongation of NS1 of H9N2 influenza virus induces a high level of inflammatory cytokines and increases transmission

“…NS1 also induces apoptosis during infection and plays an important role in the virulence of avian influenza virus (Han et al, 2012;Jackson et al, 2010;Lam et al, 2011;Zhang et al, 2010). The NS segment of influenza A viruses generally encodes an NS1 protein of 230 aa comprising an N-terminal RNA-binding domain (1-73 aa) and a C-terminal effector domain (74-230 aa).…”

Characterization of 18F-dipicolylamine (DPA) derivatives in cells infected with influenza virus

“…The fraction with the highest amount of radioactivity was dried and 3.1 mCi dry residue was received and then reconstituted with 10% DMSO. The procedure to synthesize [ 18 [13]. Briefly, NODA and DPA (molar ratio: 1.5:1) were mixed together in 200 μL 50% DMSO in water and pH was adjusted to 10.5 with 1 N NaOH.…”

“…The mixture was incubated at R.T. overnight and purified with semi-preparative HPLC. 18 F-H 18 O (20 mCi) was loaded onto an QMA cartridge, followed by 5 ml ion-free water wash, then eluted with 100 μL K 2 CO 3 (0.4 M). The pH was adjusted to 4.0 with acetic acid.…”

“…Based on the results obtained from the experiment using four different linkers to label DPA, 18 (5 mg), which was prepared as previously published [12], in 0.5 mL of anhydrous MeCN was added to the dry 18 F/K2.2.2/K 2 CO 3 residue and heated at 100°C for 10 min. Afterwards, TBAH (20 μL) in 0.5 mL MeCN was added and heated at 100°C for 5 min.…”

“…The mixture was reacted at 80°C for 15 min, then cooled down prior to HPLC purification. 18 F-FB-Cy7-DPA was collected at about 16 min.…”