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.
Highlights
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]
To determine whether apoptosis was induced in the A549 cells transfected with influenza virus A/Beijing/501/2009(H1N1) non-structural protein 1 (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 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]
Summary
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]. 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]. Our finding suggested that NS1 affects cellular functions through interaction with b-Tubulin
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