Identification of HCoV-229E Interacting Host Factor by Utilization of Proximity Labeling-Mass Spectrometry Technique
Objective Coronavirus is a class of long-standing pathogens,which are enveloped single-stranded positive-sense RNA viruses.The genome all encodes 4 structural proteins:spike protein(S),nucleocapsid protein(N),membrane protein(M),and envelope protein(E).The nucleocapsid protein(NP)serves as a key structural component of coronaviruses,playing a vital function in the viral life cycle.NP acts as an RNA-binding protein,with a critical role in identifying specific sequences within the viral genome RNA,facilitating the formation of ribonucleoprotein(RNP)complexes with viral RNA to stabilize the viral genome and contribute to viral particles assembly.The NP consists of two primary structural domains,the N-terminal domain(NTD)and the C-terminal domain(CTD).The NTD is primarily responsible for RNA binding,whereas the CTD is involved in polymerization.The N protein demonstrated to trigger the host immune response and to modulate the cell cycle of infected cells by interacting with host proteins.The NP,one of the most abundant protein in coronaviruses,is essential in understanding the pathogenic mechanism of coronaviruses through its interaction with host factors,which response for determining the virus pathogenicity.HCoV-229E is a widely distributed coronavirus that typically causes mild upper respiratory tract diseases,accounting for a significant portion of common cold cases.However,its pathogenicity is notably lower compared to other coronaviruses like MERS-CoV,SARS-CoV,and SARS-CoV-2.The exact molecular mechanism behind remains unexplained,and how HCoV-229E N protein influences virus replication,host antiviral immunity,and pathogenesis need to be further explored.Methods Proximity labeling-mass spectrometry technique and bioinformatics analysis were used to screen for potential host factors interacting with the NP of human coronavirus 229E(HCoV-229E).In this study,a recombinant adenovirus Ad-V5-NPHCoV-229E-TurboID was constructed to express the fusion protein of HCoV-229E NP and biotin ligase(TurboID).A549 cells were infected with the Ad-V5-NPHCoV-229E-TurboID.After 30 min biotin treatment,NP interacting proteins were labeled with biotin by biotin ligase,and subsequently isolated with streptavidin cross-linked magnetic beads.The potential interacting proteins were identified using label-free proteomic mass spectrometry and further validated through immunoprecipitation and immunofluorescence assays.Results We identified a total of 584 potential interacting proteins.Gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis highlighted the enrichment of glycogen synthase kinase(GSK)3A and GSK3B in the glycolysis/gluconeogenesis pathway,indicating HCoV-229E NP connection to diabetes through aberrant activity.Moreover,SARS-CoV-2 infection can exacerbate hyperglycemia and metabolic dysregulation in diabetic individuals by activating the ACE2 receptor.Moreover,SARS-CoV-2 was observed to cause potentially harm to pancreatic β-cells and leading to insulin deficiency,which not only worsens the condition of diabetic patients but also raises the possibility of new-onset diabetes in non-diabetic individuals.We demonstrated that GSK3 A and GSK3B interacted with NP of HCoV-229E,suggesting that the NP may engage in various coronavirus pathogenic processes by interacting with GSK3.Conclusion These findings suggest that proximity labeling-mass spectrometry technique is a valuable tool for identifying virus-host interaction factors,and lay the foundation for future investigations into the mechanisms underlying coronavirus replication,proliferation,and pathogenesis.
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