Identification of Proteins Interacting with PA-X from H7N9 Avian Influenza Virus by GST pull-down Combined with Mass Spectrometry

Abstract

【Objective】In order to explore the function of PA-X protein from H7N9 avian influenza virus (AIV), the host proteins interacted with PA-X were screened and identified by GST pull-down and mass spectrometry technology.【Method】The recombinant expression vector pGEX-4T-PA-X was constructed and transformed into Escherichia coli BL21 (DE3) cells to express the recombinant protein, and GST-PA-X fusion protein was purified with GST affinity resin. With GST pull-down technique, GST-PA-X and GST control protein were respectively incubated with total proteins from the A549 cell lysate in vitro, the protein complexes were separated by SDS-PAGE and then analyzed by mass spectrometry.【Result】The mass spectrometry analysis demonstrated that 39 candidate proteins with a high credibility might interact with PA-X. Through the enrichment analysis of GO and KEGG pathway, it was found that these proteins were mainly involved in biological processes such as RNA metabolism and processing, mRNA translation and transport, peptide biosynthetic process, and posttranscriptional regulation of gene expression and so on. As one of the key candidate proteins, Hsp70-2 was further chosen to explore its interaction with PA-X. The immunofluorescent analysis demonstrated that Hsp70-2 and PA-X co-localized in the cytoplasm of the A549 cell line. This further confirmed the interaction between cellular Hsp70-2 and PA-X.【Conclusion】Thirty-nine candidate proteins were identified to interact with PA-X in A549 cells via the GST pull-down combined with mass spectrometry technology, and the colocalization between Hsp70-2 and PA-X was confirmed bythe immunofluorescence test. These results provide a basis for a further research on the role of PA-X in the life cycle of AIV.

Keywords

• h7n9 aiv
• pa-x
• gst pull-down
• mass spectrometry
• hsp70-2
• protein interaction