tetano
Editor, Senior Moderator
PLoS One
. 2025 Oct 21;20(10):e0327939.
doi: 10.1371/journal.pone.0327939. eCollection 2025. Quantitative analysis of proteomic changes in two monoclonal suspension MDCK cell lines infected with human influenza A virus (H1N1)
Jan Küchler[SUP] 1 [/SUP], Tilia Zinnecker[SUP] 1 [/SUP], Patrick Hellwig[SUP] 1 2 [/SUP], Maximilian Wolf[SUP] 3 [/SUP], Dirk Benndorf[SUP] 4 [/SUP], Yvonne Genzel[SUP] 1 [/SUP], Udo Reichl[SUP] 1 2 [/SUP]
Affiliations
Suspension MDCK cells are a substrate for producing influenza A virus (IAV) and typically show very high virus yields compared to other animal cells. Due to the significant heterogeneity within cell populations, studying and comparing clonal cell lines with regard to specific properties, such as superior growth or higher productivity, could facilitate process optimization. In this study, we analyzed the expressed proteins of two clonal cell lines to identify intrinsic characteristics of effective IAV producers. We compared proteome changes in two human IAV PR8 (H1N1, A/PR/8/34) infected monoclonal suspension MDCK cell lines: C59, a low-yield IAV producer with fast cell growth and small cell diameter, and C113, a high-yield IAV producer with average cell growth and large cell diameter. We examined growth rate, size, metabolism and IAV production. A total of 5177 host cell proteins were detected in both cell lines using DIA-PASEF mode with a TimsTOFpro mass spectrometer. Analysis of the differentially expressed proteins revealed that fatty acid oxidation and branched-chain amino acid degradation were upregulated in highly productive cells. In contrast, steroid biosynthesis and DNA replication were more active in faster-growing cells. Following infection, 122 proteins were significantly upregulated (p < 0.05, log2-fold change ≥1) in the high-producing cell line. These proteins were associated with membrane trafficking, interactions with the IAV-NS1 protein and virus production. Additionally, 98 proteins associated with antiviral pathways such as the proto-oncogenic receptor tyrosine kinase MET and tumor necrosis factor (TNF) signaling were downregulated (p < 0.05, log2-fold change ≤1). In the cell line that produced lower IAV PR8 titers, 77 proteins were downregulated and 57 were upregulated after infection. RNA metabolism appeared to be downregulated, while the tricarboxylic acid (TCA) cycle and the stress response were both upregulated. In the high-yield C113 clone, only proteins associated with apoptosis and the target of rapamycin kinase (TOR) were expressed following infection. This may indicate a more effective release of virus particles. A comparison of intracellular IAV PR8 protein levels demonstrated that M1 and NA levels were 4-fold and 8-fold higher, respectively, for the high-yield C113 cell line. These findings again suggest an improved virus release.
. 2025 Oct 21;20(10):e0327939.
doi: 10.1371/journal.pone.0327939. eCollection 2025. Quantitative analysis of proteomic changes in two monoclonal suspension MDCK cell lines infected with human influenza A virus (H1N1)
Jan Küchler[SUP] 1 [/SUP], Tilia Zinnecker[SUP] 1 [/SUP], Patrick Hellwig[SUP] 1 2 [/SUP], Maximilian Wolf[SUP] 3 [/SUP], Dirk Benndorf[SUP] 4 [/SUP], Yvonne Genzel[SUP] 1 [/SUP], Udo Reichl[SUP] 1 2 [/SUP]
Affiliations
- PMID: 41118376
- DOI: 10.1371/journal.pone.0327939
Suspension MDCK cells are a substrate for producing influenza A virus (IAV) and typically show very high virus yields compared to other animal cells. Due to the significant heterogeneity within cell populations, studying and comparing clonal cell lines with regard to specific properties, such as superior growth or higher productivity, could facilitate process optimization. In this study, we analyzed the expressed proteins of two clonal cell lines to identify intrinsic characteristics of effective IAV producers. We compared proteome changes in two human IAV PR8 (H1N1, A/PR/8/34) infected monoclonal suspension MDCK cell lines: C59, a low-yield IAV producer with fast cell growth and small cell diameter, and C113, a high-yield IAV producer with average cell growth and large cell diameter. We examined growth rate, size, metabolism and IAV production. A total of 5177 host cell proteins were detected in both cell lines using DIA-PASEF mode with a TimsTOFpro mass spectrometer. Analysis of the differentially expressed proteins revealed that fatty acid oxidation and branched-chain amino acid degradation were upregulated in highly productive cells. In contrast, steroid biosynthesis and DNA replication were more active in faster-growing cells. Following infection, 122 proteins were significantly upregulated (p < 0.05, log2-fold change ≥1) in the high-producing cell line. These proteins were associated with membrane trafficking, interactions with the IAV-NS1 protein and virus production. Additionally, 98 proteins associated with antiviral pathways such as the proto-oncogenic receptor tyrosine kinase MET and tumor necrosis factor (TNF) signaling were downregulated (p < 0.05, log2-fold change ≤1). In the cell line that produced lower IAV PR8 titers, 77 proteins were downregulated and 57 were upregulated after infection. RNA metabolism appeared to be downregulated, while the tricarboxylic acid (TCA) cycle and the stress response were both upregulated. In the high-yield C113 clone, only proteins associated with apoptosis and the target of rapamycin kinase (TOR) were expressed following infection. This may indicate a more effective release of virus particles. A comparison of intracellular IAV PR8 protein levels demonstrated that M1 and NA levels were 4-fold and 8-fold higher, respectively, for the high-yield C113 cell line. These findings again suggest an improved virus release.