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Endogenous pull-downs of human proteins ATP6V1A and AHNAK to validate interactions with influenza A virus proteins M2 and NEP by targeted mass spectrometry in virus-infected cells

ABSTRACT: Influenza A Virus (IAV) is a recurring respiratory virus with antiviral therapies of limited use. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry (AP-MS) and global phosphoproteomic and protein abundance analyses with three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we mapped 332 IAV-human protein-protein interactions (PPIs) and identified 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza revealed several genes, including the structural scaffold protein AHNAK, with predicted loss-of-function variants that were also identified in our proteomic analyses. Of our identified host factors, 54 significantly altered IAV infection upon siRNA knockdown, and two factors, COPB1 and AHNAK, were also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppressed IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT. This project includes endogenous IP data from A549 cells and NHBE cells infected with H5N1 IAV validating IAV-host PPIs M2-ATP6V1A and NEP-AHNAK, respectively. M2-ATP6V1A and NEP-AHNAK PPIs were first identified in AP-MS data that is submitted under its own dataset identifier (PXD036077).

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, Lung, Epithelial Cell, Cell Culture

DISEASE(S): Influenza,Avian Influenza

SUBMITTER: Kelsey Haas

LAB HEAD: Robyn Kaake

PROVIDER: PXD041663 | Pride | 2023-07-25

REPOSITORIES: pride

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Project description:E2 exposure significantly decreased peak viral titer in hNECs from female donors. We used microarray analyses to identify global gene expression patterns between E2 and vehicle exposed hNECs from female donors Influenza causes an acute infection characterized by virus replication in respiratory epithelial cells. The severity of influenza and other respiratory diseases changes over the life course and during pregnancy in women, suggesting that sex steroid hormones, such as estrogens, may be involved. Using primary, differentiated human nasal epithelial cell (hNEC) cultures from adult male and female donors, we exposed cultures to the endogenous 17Î²-estradiol (E2) or select estrogen receptor modulators (SERMs), then infected cultures with a seasonal influenza A virus (IAV) to determine whether estrogenic signaling could affect the outcome of IAV infection and whether these effects where sex-dependent. Estradiol, raloxifene, and bisphenol A decreased IAV titers in hNECs from female, but not male, donors. The estrogenic decrease in viral titer was dependent on the genomic estrogen receptor- 2 (ESR2) as neither genomic ESR1 nor non- genomic GPR30 were expressed in hNEC cultures and addition of the genomic ER antagonist ICI 182,780 reversed the antiviral effects of E2. Treatment of hNECs with E2 had no effect on interferon or chemokine secretion, but significantly downregulated cell metabolic processes, including genes that encode for zinc finger proteins, many of which contain estrogen response elements in their promoters. These data provide novel insights into the cellular and molecular mechanisms of how natural and synthetic estrogens impact IAV infection in respiratory epithelial cells derived from humans. Primary human nasal epithelial cells from females were exposed to E2 for 24h prior to infection, then infected with an H3N2 strain of influenza a virus for 2 hours. At 24 and 48h post infection, hNECs were collected in Trizol for RNA extraction and hybridization on Affymetrix Human Gene ST 2.0 microarrays.

Project description:Background: Acute respiratory distress syndrome (ARDS) is characterized by refractory hypoxemia caused by accumulation of pulmonary fluid, which is related to inflammatory cell infiltration, impaired tight junction of pulmonary epithelium and impaired Na, K-ATPase function, especially Na, K-ATPase α1 subunit. Up until now, the pathogenic mechanism at the level of protein during lipopolysaccharide- (LPS-) induced ARDS remains unclear. Methods: Using an unbiased, discovery and quantitative proteomic approach, we discovered the differentially expressed proteins binding to Na, K-ATPase α1 between LPS-A549 cells and Control-A549 cells. These Na, K-ATPase α1 interacting proteins were screened by co-immunoprecipitation (Co-IP) technology. Among them, some of the differentially expressed proteins with significant performance were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The protein interaction network was constructed by the related Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Several differentially expressed proteins were validated by Western blot. Results: Of identified 1598 proteins, 89 were differentially expressed proteins between LPS-A549 cells and Control-A549 cells. Intriguingly, protein-protein interaction network showed that there were 244 significantly enriched co-expression among 60 proteins in the group control-A549. while the group LPS-A549 showed 43 significant enriched interactions among 29 proteins. The related GO and KEGG analysis found evident phenomena of ubiquitination and deubiquitination, as well as the pathways related to autophagy. Among proteins with rich abundance, there were several intriguing ones, including the deubiquitinase (OTUB1), the tight junction protein zonula occludens-1 (ZO-1), the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complexes (CUL4B) and the autophagy-related protein sequestosome-1 (SQSTM1). Conclusions: In conclusion, our proteomic approach revealed targets related to the occurrence and development of ARDS, being the first study to investigate significant differences in Na, K-ATPase α1 interacting proteins between LPS-induced ARDS cell model and control-A549 cell. These proteins may help the clinical diagnosis and facilitate the personalized treatment of ARDS.

			Action	DRS
	LCandMS_Acquisition_Params.xlsx	Xlsx
	M2_ATP6V1A.fasta	Fasta
	Metadata_04182023.xlsx	Xlsx
	PRM_AHNAK.sky.zip	Other
	PRM_AHNAK_NEP.fasta	Fasta

Dataset Information

Endogenous pull-downs of human proteins ATP6V1A and AHNAK to validate interactions with influenza A virus proteins M2 and NEP by targeted mass spectrometry in virus-infected cells

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