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Impact of influenza A virus infection on the proteomes of human bronchoepithelial cells from different donors Samuel T Mindaye, Natalia A Ilyushina, Giovanna Fantoni, Michail A Alterman, Raymond P. Donnelly, and Maryna C. Eichelberger J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00286 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 27, 2017

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Journal of Proteome Research

Impact of influenza A virus infection on the proteomes of human bronchoepithelial cells from different donors

Samuel T. Mindaye,*,†,1 Natalia A. Ilyushina,‡ Giovanna Fantoni,†,1 Michail A. Alterman,ǁ,1 Raymond P. Donnelly‡ and Maryna C. Eichelberger*,†

†

Division of Viral Products, OVRR, CBER, Food and Drug Administration, Silver Spring, MD

20993, USA ‡

Division of Biotechnology Research and Review II, CDER, Food and Drug Administration,

Silver Spring, MD 20993, USA ǁDivision

of Cellular and Gene Therapies, OTAT, CBER, Food and Drug Administration, Silver

Spring, MD 20993, USA

1

Current address: STM – Division of Bacterial, Parasitic and Allergenic Products, OVRR,

CBER, FDA; GF – Center for Human Immunology, National Heart, Lung and Blood Institute, NIH; MA – Office of Product Quality, CDER, FDA

*Corresponding authors E-mail: [email protected]. Phone 240 402 9633; E-mail: [email protected]. Phone: 240 402 9505.

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ABSTRACT (200 words) Susceptibility to influenza A virus is determined by a balance of viral and host factors. The genetic background of the host contributes to the severity of disease, but the influenza-related proteome of cells from different individuals have not been compared. We used high resolution mass spectrometry to identify proteins in normal human bronchial epithelial (NHBE) cells isolated from three different donors. Infection of each NHBE cell culture with influenza A/California/07/2009 (H1N1) resulted in expression of viral proteins and a variety of host proteins, including interferons, interferon-stimulated genes, and secreted chemokines/cytokines. The expression level of viral proteins corresponded with the level of host proteins that support influenza infection (i.e., pro-viral proteins), however, production of infectious virus was inversely related to the levels of antiviral proteins, suggesting that a balance of pro-viral proteins and the antiviral response controls virus replication. In summary, our results demonstrate that expression levels of pro-viral as well as antiviral factors are different for each donor and suggest that relative quantitation of these factors may provide a way to identify individuals or population groups who are susceptible to severe influenza disease.

Keywords: influenza, proteome, human airway epithelial cells, interferons, cytokines, ubiquitin, metallothionein, superoxide dismutase, trypsin


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INTRODUCTION Susceptibility to influenza is determined by a balance of viral and host factors. Since influenza virus replication requires interaction with a number of host proteins and is moderated by antiviral proteins that are induced following infection, the genetic background of the host can impact consequent disease.1 For example, data from in vitro studies showed that interferon (IFN)inducible transmembrane (IFITM) protein family members restrict the replication of many different viruses.2 The impact of IFITM3 on influenza replication was demonstrated in knock-out mice and in examining the predominance of an IFITM3 gene variant in hospitalized influenza cases in China; these studies suggest this protein has a significant effect on disease severity.3 However, the association of the IFITM3 variant with severe disease is not evident in all populations.4 The complexity of factors that impact susceptibility to influenza makes it difficult to assess individual and population risk of disease on genetic background alone. In vitro studies are critical for identifying both host and viral factors that play a role in influenza pathogenesis. Influenza virus uses its hemagglutinin (HA) glycoprotein to bind to sialylated glycoproteins on the host cell surface. It is then endocytosed into coated pits containing clathrin, coat protein I (COP-I) and coat protein II (COP-II). Release of the coated pits from the plasma membrane is controlled by dynamin, a GTPase which also serves to uncoat the vesicle, allowing fusion with early endosomes.5 Endosomes are transported along actin and microtubule cytoskeletons, with motor proteins providing the force for this movement.6 The endosomes become acidified when they fuse with lysosomes, causing HA to undergo a conformational change that allows fusion of the viral and lysosomal membranes. The viral matrix protein (M1) and ribonucleoprotein (RNP) complex then enter the cell’s cytoplasm and the RNP is transported into the nucleus where the vRNA is duplicated using the viral polymerase



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but also taking advantage of the host cell’s transcriptional machinery. Newly formed RNP complexes are exported into the cytoplasm where they are encapsidated and then transported along microtubules to the cell surface where it associates with HA and neuraminidase (NA) that are studded into the cell’s plasma membrane at lipid rafts.7 There are consequently a large number of host cell proteins which play essential roles in the cellular processes that support virus replication; these are considered “proviral factors”. These factors are usually required for normal cell function. To guard itself from infecting agents, the host cell utilizes mechanisms that either directly or indirectly limit the virus’s ability to replicate. An example of an antiviral protein is type I interferon (IFN) which inhibits influenza virus replication indirectly by upregulating a number of effector proteins – one of these is myxovirus (Mx) resistance protein I that destabilizes newly formed RNP complexes.8 The influenza virus non structural (NS) protein, NS1, counteracts the activity of IFN type I through multiple mechanisms.9 Successful virus replication therefore utilizes many different cellular pathways and is a complex interplay between virus and host cell proteins. Transcriptomic and proteomic studies have resulted in the identification of a number of proviral factors. Watanabe et al.10 reviewed the results of studies using predominantly RNAi gene silencing technique that had been conducted in osteosarcoma cells (Brass et al 2009),11 MDCK canine kidney cells,12 a human lung cell line, A54913-14 and human bronchoepithelial cells15 to identify specific host cell proteins and cellular pathways that support the influenza virus life cycle. The proteins identified largely differed between studies, suggesting the sensitivity of assays conducted in different laboratories may impact the findings and/or that the pathways used for different hosts and/or cell types vary. Proteomic studies have been conducted using mass spectrometry to identify host proteins associated with the influenza replication include studies


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performed with infected human embryonic kidney 293T cells,15 quantitative global analysis of proteins in lungs of dogs experimentally infected with a canine H3N2 influenza A virus,16 comparison of changes in protein expression following infections of macaques with influenza viruses that differ in pathogenicity17 and examination of the human cellular response to a model influenza virus, A/PR/8/34 (H1N1) in undifferentiated normal human bronchial epithelial (NHBE) cells. 5-18 While these studies have identified a number of important findings that can be applied to drug discovery or toward investigating reasons for severe human disease, none has examined person-to-person differences in the influenza-related proteome of differentiated NHBE cells cultured at an air interface. The types of cells that grow under these conditions are representative of cells present in human bronchioles that are the target of infection with influenza virus. In this study, we addressed the hypothesis that bronchial epithelial cells from different human donors vary in their support of, and response to, influenza A virus infection. Our objective was to compare the proteomes of individuals before and after influenza infection in order to determine the extent of differences in the responses to influenza. NHBE cultures isolated from 3 donors were infected with A/California/7/2009 (H1N1). We used multidimensional separation (ultraperformance liquid chromatography, UPLC) and high definition/resolution mass spectrometry (HDMSE) to identify and quantify proteins in the cell cultures before and after infection. We then compared the relative amounts of viral, pro-viral and antiviral proteins at different time points post-infection and between cultures. The results show that cultures from different individuals are unique at baseline and in their response to infection. Comparison of viral and host protein concentrations in each culture suggests that the ratio of pro- to antiviral factors determines the extent of influenza virus replication.



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EXPERIMENTAL PROCEDURES NHBE cells and virus preparation NHBE cells from human donors were obtained from Lonza (www.lonza.com, USA). Donor 1 was a male Caucasian 43 years old; Donors 2 and 3 were black females, 56 and 65 years old respectively. Cell cultures of passage 2 were grown at similar density on membrane supports (6.5-mm Transwell, Corning Inc., USA) at the air-liquid interface in serum-free and hormoneand growth factor-supplemented medium as described previously.19 Fully differentiated 4 to 8 week-old cultures were used for all experiments. At this stage the monolayers were confluent, with a static number of cells through the 48 hr infection period.20 Influenza virus A/California/7/09 (H1N1) (abbreviated A/H1N1pdm) was kindly provided by Dr. Robert G. Webster at St. Jude Children’s Research Hospital, Memphis, TN. Stock virus was grown in 10day-old chicken eggs for 48 h at 37°C following standard procedures.21 Aliquots of the allantoic fluid were stored at −80°C until used. All experimental work was performed in a biosafety level 2 laboratory under protocols approved by the Institutional Biosafety Committee. In vitro infection of NHBE cells and determination of infectious units NHBE cell cultures were infected as previously described.19 The fully differentiated cultures were washed extensively with sterile phosphate-buffered saline (PBS) to remove mucus secretions on the apical surface and then inoculated via the apical side with A/H1N1pdm at a multiplicity of infection (MOI) of 3. Since the MOI was determined from a titer measured on MDCK cells, it may not accurately reflect the number of infectious units per susceptible NHBE cells. Approximately 80% of the cells in the NHBE culture were infected,19 with a similar proportion of infected cells in cultures from different donors. After 1 h of incubation at 37°C, the 6 ACS Paragon Plus Environment

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inoculum was removed and the infected cells were cultured at 37°C until cells or supernatants were sampled for further analysis. Periodic sampling was performed to establish virus growth curves, to quantify the apical and basal levels of chemokines/cytokines produced in response to infection and to compare relative amounts of host and viral proteins. Prior to sampling from the apical compartment that interfaces with air, 300 μl serum-free medium was added to the culture. This medium was harvested 30 min later. The virus titers were determined as log10 plaque forming units (PFU)/ml in Madin-Darby canine kidney (MDCK) cells as described previously.22 Briefly, confluent MDCK cell monolayers were inoculated with 10fold serial dilutions of the cell supernatant and then incubated at 37°C for 1 h. The cells were then washed and overlaid with minimal essential medium (MEM) containing 0.3% bovine serum albumin (BSA), 0.9% Bacto agar, and 1 μg/ml l-[tosylamido-2-phenyl]ethylchloromethylketone (TPCK)-treated trypsin. After 3 days of incubation at 37°C, cells were stained with 0.1% crystal violet in 10% formaldehyde solution, and the number of plaques counted. The average PFU per milliliter of culture supernatant is reported. Quantitative real-time polymerase-chain reaction (qPCR) Changes in IFNL1, IFNL2/3, and IFNL4 gene expression were quantified by qPCR. Total cellular RNA was isolated from virus-infected NHBE cell cultures using RNeasy Minikit (Qiagen, Germany) and treated with DNase. Purified RNA (1 μg) was reverse-transcribed to cDNA with Quantiscript reverse transcriptase (Qiagen). The cDNA was mixed with RT2 SYBR green qPCR Mastermix (Qiagen) and qPCR was performed using the ViiATM 7 system (Applied Biosystems, USA). Changes in gene expression levels were analyzed using ViiATM 7 software v.1.2.2 (Applied Biosystems). Gene expression levels were normalized to that of the housekeeping gene, GAPDH. The results are expressed as the mean-fold increase in normalized



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gene expression relative to uninfected cells from the same donor. Quantitative values represent the mean ± standard deviation (SD) of at least triplicate determinations. Determination of chemokine/cytokines levels in apical and basal supernatants For chemokine/cytokine analysis, medium was periodically collected from the basal and apical compartments. Since the apical surface interfaces with air, 300 µl of medium was added to the apical compartment and allowed to incubate at 37 °C for 30 min before collecting it and an equal volume from the basal compartment for cytokine analysis. Chemokine/cytokine concentrations were measured in the supernatants using a multiplex chemokine/cytokine mesoscale discovery (MSD) electrochemiluminescence kit following the protocols recommended by the manufacturer (MSD, USA). IFN-γ, IL-13, IL-1β, IL-8, TNF-α, eotaxin, eotaxin-3, IL-8, IP-10, MCP-1, MCP4, MDC, and TARC were measured after infection of NHBE cell cultures with A/H1N1pdm. The MSD plates were analyzed using a MESO QuickPlex SQ 120 instrument and the data processed using Discovery Workbench version 4.0 (MSD, www.mesoscale.com). Each target concentration (pg/ml) was measured in quadruplicate wells; the average and SD are reported. Sample preparation and LC-HDMSE analysis The detailed protocols for sample preparation, nanoLC-HDMSE acquisition, Protein Lynx Global Server (PLGS)-based data processing, database searching, and additional bioinformatic analyses are provided in Supplement 1. Briefly, NHBE cells harvested from 3 wells that were infected independently were pooled, washed using PBS, and then lysed by suspension in buffer containing 1.25% sodium dodecyl sulfate, sonication and pressure treatment. The total protein concentration was determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA) and a trypsin digest was prepared as described in the Supplemental Information. For every LC-HDMSE run, 4 µL digest (corresponding to 20 µg total protein) was


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loaded and eluted on a 2D nanoAcquity UPLC system (Waters Corp., USA). The eluate was loaded onto a SynaptG2 (Waters Corp.) mass spectrometer (MS) by electrospray via a PicoTip Emitter (New Objective Inc., Woburn, MA). The MS was operated in data-independent acquisition mode (MSE). Ions in gas phase were resolved both by their time of flight (TOF analyzer) and their drift time (ion mobility cell). Data were processed using ProteinLynx Global Server (PLGS) software (version 2.4, Waters Corp.). The precursor and product ion outputs were used to identify proteins in the SwissProt human database as well as a laboratory-assembled database of all A/H1N1pdm proteins. The search included tolerance limitations of 20 ppm for the precursor and 0.1 Da for fragment ion mass. Up to 3 missed trypsin cleavages were allowed. The search considered carbamidomethylation of cysteine residues as a fixed modification and oxidation of methionine and histidine residues as variable modifications in the protein sequence. Protein identification was limited to those with at least 3 product ions per peptide and 7 ion matches per protein, 100 ion counts for low and 75 ion counts for high energy acquisitions, and a 1,000 count intensity threshold. A one-time randomized target database was used as decoy to calculate the false discovery rate (FDR) of protein identification. The confidence in identification was further ensured by applying a FDR filter of 2 or < -2 are considered significant. The supporting data for Figure 4 are provided in Supplementary Table S8. The complexity of the changes and differences between cultures were evident even from early time points after infection. For example, at 4 h p.i., D1 had at least 29 canonical pathways with Z-score > 2, whereas D2 and D3 had 3 and 18 canonical pathways with Z-score > 2, respectively. Notably, the RhoA-associated signaling pathway increased and the RhoGDI signaling pathway decreased very rapidly in cultures D1 and D3. The Hippo signaling network decreased dramatically in all cultures. The Hippo network suppresses virus-induced interferon production31, and therefore it is expected that the decrease of this pathway observed in infected cells facilitates the antiviral response. Impact of A/H1N1pdm infection on chemokines and cytokines secreted from NHBE cells from different donors


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To evaluate differences in soluble factors secreted from the infected cells, cytokines and chemokines were quantified in supernatants collected from the apical and basolateral compartments of NHBE cultures obtained from the different donors (Supplementary Figure S1). The cytokine concentrations were often higher in supernatants collected from the basolateral compartment of the cell culture than the apical supernatant. At 48 h p.i. the concentration of IFNγ in the apical compartment was greater in D2 than in the other cultures; this difference was not apparent in supernatant collected from the basolateral compartment. This trend was also noted when comparing other cytokine/chemokine levels; for example, at 48 h p.i., the apical but not basolateral supernatant of D2 had significantly higher concentrations of IP-10, IL-8, MCP-1 and TARC than D1 and D3 (P < 0.05). We also measured interferon (IFN)- gene (IFNL1, IFNL2/3, and IFNL4) expression in the A/H1N1pdm-infected NHBE cell cultures. The results showed that IFNL2/3 and IFNL4 mRNA levels were comparable between the three cell cultures at all tested time points (Supplementary Figure S2). However, a much greater level of IFNL1 mRNA was present in D1 than D2 and D3 at 24 and 48 h p.i. (P < 0.05). IFN-λ1 restricts influenza replication, particularly when acting synergistically with other antiviral drugs32, and therefore IFN-λ1 may suppress A/H1N1pdm replication in cells from D1 to a greater degree than those from D2 and D3. Many intracellular antiviral proteins are induced in response to interferons that are produced during influenza infection.33-34 We compared the relative levels of IFN-induced protein with tetratricopeptide repeats (IFIT) and IFITM family member proteins encoded by ISGs, as well as the Mx1 and myxovirus resistance 2 (Mx2) proteins. Figure 5 shows the results for IFITM1, IFITM2, IFITM3, IFIT1, IFIT3 and Mx1. The relative amounts of ISG proteins was greatest after infection of culture D2; the amount of IFIT1 measured at 48 h p.i. in D2 was at



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least 7-fold higher than in D1 or in D3 (P < 0.0001). The relative amount of IFIT3 was also greatest in D2 (10-fold higher than in D1 and 2.4-fold higher than in D3 (P < 0.0001). The expression of Mx1, a GTPase with potent anti-influenza activity, was significantly greater in D2 than in D1 and D3 (P < 0.0001) at 24 and 48 h post-infection.

DISCUSSION Our goal was to determine whether the proteomes of cells from the respiratory tracts of different donors are distinct, and to identify proteomic changes after influenza A virus infection. Primary NHBE cells from three donors were cultured, and changes in protein expression were evaluated following inoculation with A/H1N1pdm at a MOI that ensured infection of all cells. Our results showed that the proteome of each of the influenza virus-infected cultures was unique. Proteins known to support viral replication were present at higher relative concentrations in D2, a culture in which A/H1N1pdm proteins were expressed at higher relative concentrations than in cultures from the other two donors. As described earlier, these included proteins such as metallothionein and superoxide dismutase that are associated with protecting the cell from a stress response. Proteins that play a role in cell differentiation or maintain epithelial cell polarity such as cdc42 and ezrin35 were also present at higher levels in D2 cultures. Other pro-viral proteins that were present at higher concentrations in D2 than in the other cultures included myosin regulatory light chain which maintains the stability of myosin II. Myosin II contributes to successful influenza virus replication by allowing transport of viral cores to the budding site through an association between M1 and the actin-myosin network.36 Talin is another protein that was present at higher concentrations in D2 than in D1 and D3; talin is associated with focal adhesion kinase (FAK), an intracellular protein tyrosine kinase.37 FAK regulates phosphatidyl16 ACS Paragon Plus Environment

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inositol-3-kinase activation and actin reorganization, contributing to influenza replication by supporting virus entry and virus replication.38 In contrast, cyclophilin, a protein that destabilizes virus proteins, was expressed at lower levels in D2 than D1 and D3. Cyclophilin limits influenza virus replication through degradation of the M1 protein.30 The proviral and antiviral proteins identified in our study match well with proteins identified in other studies39 but were not universally identified. Talin, ATPases and ribosome binding proteins are examples of proteins identified following influenza infection in our as well as other studies. As expected, some cellular proteins that are regulated in response to influenza were not identified in our study. As stated by others,40 differences in proteins identified may reflect the cell type, culture conditions, virus strain, and specific methods used in each laboratory. Since our study examined proteins expressed in replicate cultures that were pooled rather than independent analysis of replicate cultures, some proteins may not have been identified in the single pooled sample, especially if they were present at low concentrations. An example of a proviral protein that we did not identify is the putative ubiquitin ligase (gene symbol UBR4) that was shown to complex with the viral M2 protein, facilitating its transport to the apical cell surface.39 Although this specific ubiquitin ligase was not identified in NHBE cultures used in our study, other E2 and E3 ubiquitin ligases were upregulated. Interestingly, E3 ubiquitin ligase (RNF135) that activates retinoic acid inducible protein I (RIG-I) and thereby promotes type I IFN induction,41 was upregulated in the NHBE cultures from donor 3. Since the relative amounts of pro-viral proteins were greater in D2 than the other cultures and the concentrations of proteins that destabilize virus production were less, one might expect that influenza would replicate to higher titers in D2 than the other cultures. Indeed, viral proteins were expressed at higher levels in D2 than in D1 and D3, and higher virus titers were indeed



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measured in supernatants collected at 24 and 48 hr from D2 than D3. However, the infectious titers of supernatants collected from D1 and D2 cultures were similar. The lack of correlation between viral protein expression levels and infectious virus production (Figure 3) suggests that the full replication cycle is restricted by the least abundant viral protein. Alternatively, the lack of increased infectious virus titers in D2 may be due to the greater antiviral response measured in this culture: D2 had the greatest expression of ISGs, including Mx1, a potent inhibitor of viral replication.42 It is also possible that the supernatants harvested from infected D1, D2 and D3 cultures contain distinct cellular factors which differentially affect the infection and/or replication of influenza virus in MDCK cells when the plaque assay is performed. Given the large virus dilutions used in the plaque assay, this hypothesis is unlikely, but we cannot rule it out at this time. These results, therefore, provide strong support for the idea that productive virus replication is a balance between pro-viral and antiviral factors. Proteins that support virus fitness have a range of functional and structural properties. Many of the proteins that were upregulated in D1, D2 and D3 following influenza infection are linked to intracellular trafficking, generation of energy, and protein synthesis. While changes in host protein expression generally serve to protect the host, influenza virus has exploited some of these changes to enhance virus replication. An example is RhoA, a small GTPase that regulates the actin cytoskeleton and as a result impacts a number of cellular processes. Upregulation of RhoA is recognized as a stress response that induces the formation of disorganized actin stress fibers.43 This may protect the cell to some degree because it leads to inefficient endosome and exosome movement. Interestingly, another study the examined the impact of influenza infection on cellular pathways in NHBE cells grown in liquid medium showed through mRNA profiling that RhoA was upregulated during A/PR/8/34 infection and that silencing of this gene resulted in


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an increase in virus titer (Shapira et al., 2012). This supports the idea that RhoA is upregulated as a cellular defense following infection and limits virus replication.

CONCLUSIONS In this study we show that the proteomes of uninfected NHBE cells from different donors, and their responses to influenza A virus infection, are unique. Global protein analysis and relative protein quantitation identified a number of pathways that have previously been linked to influenza replication and the antiviral response. Importantly, the data support the idea that a balance between pro-viral and antiviral proteins controls influenza virus replication. Proteomic analysis of NHBE cells may therefore help determine individual susceptibility to infection and risk of disease in a population. Together with full genetic analysis of a large number of donors, this may facilitate identification of genes associated with influenza susceptibility. Further studies that examine the proteomes of large numbers of donors with known predisposition to severe influenza disease are warranted to expand the applicability of our global proteomic analyses.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website http://pubs.acs.org. Supplementary Information (PDF) Table S1: Proteins identified in Donor 1 NHBE cell culture (also XLS sheet 1) Table S2: Proteins identified in Donor 2 NHBE cell culture (also XLS sheet 2) Table S3: Proteins identified in Donor 3 NHBE cell culture (also XLS sheet 3) 19 ACS Paragon Plus Environment


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Table S4: Comparison the proteomes of D1, D2 and D3 NHBE cell cultures prior to influenza infection (also XLS sheet 4) Table S5: Relative concentrations of host factors in D1 NHBE cells (also XLS sheet 5) Table S6: Relative concentrations of host factors in D2 NHBE cells (also XLS sheet 6) Table S7: Relative concentrations of host factors in D3 NHBE cells (also XLS sheet 7) Table S8: Activation Z-scores of canonical pathways in different donor cells at time intervals post influenza A/H1N1pdm infection (also XLS sheet 8) Figure S1: Expression profiles of chemokines and cytokines from apical and basal supernates of D1, D2 and D3 NHBE cultures (also XLS sheet 9) Figure S2: IFNL1, IFNL2/3, and IFNL4 gene expression in A/H1N1pdm-infected NHBE cells (also XLS sheet 10)

ACKNOWLEDGMENTS We thank Lianlian Jiang and Jin Gao for technical support and Dino Feigelstock and Yanming An for suggestions in preparing the manuscript. The project was supported in part by CBER Panflu funds awarded to MCE, and in part by Grant No. 2011094 from the FDA Medical Countermeasures Program to RPD. SM’s support was administered by the Oak Ridge Institute for Science and Engineering.

ORCID ID numbers: RPD: 0000-0002-0695-5276, http://orcid.org/0000-0002-0695-5276 MCE: 0000-0002-3219-4140, http://orcid.org/0000-0002-3219-4140 STM: 0000-0001-6514-3342, https://orcid.org/0000-0001-6514-3342 20 ACS Paragon Plus Environment

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REFERENCES

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Table 1. Common proteins present in culture D2 at 3-fold or greater relative concentration than in D1 and D3. Gene symbol

ACON

Protein accession number P30443, P04439, P13746, P30447, P05534 Q04826, P30481, P30485, P30495, P18465 Q99798

ANXA5 CDC42

P08758 P60953

COF1 COMT

P23528 P21964

COR1B CYB5 EF1D

Q9BR76 P00167 P29692

ERO1A

Q96HE7

EZRI GBB4

P15311 Q9HAV0

GDIB

P50395

GDIR1

P52565

GELS GNA12

P06396 Q03113

GRN HN1

P28799 Q9UK76

HNRPF

P52597

1A01, 1A03, 1A11, 1A23, 1A24, 1A36 1B4, 1B44, 1B47, 1B56, 1B57

Protein name/description

Protein NISa Culture D1 Culture D2 Culture D3

HLA class I histocompatibility antigen A1, 3, 11, 23, 24, 36 alpha chain

0.022

0.126

0.019

HLA class I histocompatibility antigen B alpha chain

0.021

0.114

0.020

Aconitate hydratase mitochondrial Annexin A5 Cell division control protein 42 homolog Cofilin 1 Catechol O methyltransferase Coronin 1B Cytochrome b5 Elongation factor 1 delta ERO1 like protein alpha Ezrin Guanine nucleotide binding protein subunit beta 4 Rab GDP dissociation inhibitor beta Rho GDP dissociation inhibitor 1 Gelsolin Guanine nucleotide binding protein subunit alpha Granulins Hematological and neurological expressed 1 protein Heterogeneous nuclear ribonucleoprotein F

0.081

0.329

0.059

0.270 0.020

0.919 0.365

0.184 0.037

0.212 0.026

1.026 0.156

0.252 0.051

0.066 0.039 0.044

0.205 0.165 0.365

0.042 0.039 0.035

0.045

0.203

0.074

0.677 0.110

1.872 0.097

0.402 0.099

0.072

0.237

0.064

0.138

0.542

0.057

0.524 0.010

1.624 0.435

0.039 0.015

0.128 0.035

1.917 0.154

0.429 0.003

0.082

0.242

0.032


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HSP76

P17066

HSP77

P48741

IPYR

Q15181

ITB1 K1C23

P05556 Q9C075

K1C9

P35527

K2C1

P04264

ML12A

P19105

ML12B

O14950

MOES MT1E MT1M MT1X NACAM

P26038 P04732 Q8N339 P80297 E9PAV3

NPC2

P61916

PDLI1

O00151

PDXK PGAM1

O00764 P18669

PP1B

P62140

PTTG

P53801

PUR9

P31939

RAB8B

Q92930

RADI RL27A

P35241 P46776

RL40

P62987

ROA3

P51991

Heat shock 70 kDa protein 6 Putative heat shock 70 kDa protein 7 Inorganic pyrophosphatase Integrin beta 1 Keratin type I cytoskeletal 23 Keratin type I cytoskeletal 9 Keratin type II cytoskeletal 1 Myosin regulatory light chain 12A Myosin regulatory light chain 12B Moesin Metallothionein 1E Metallothionein 1M Metallothionein 1X Nascent polypeptide associated complex subunit alpha muscle specific form Epididymal secretory protein E1 PDZ and LIM domain protein 1 Pyridoxal kinase Phosphoglycerate mutase 1 Serine threonine protein phosphatase PP1 beta catalytic subunit Pituitary tumor transforming gene 1 protein interacting protein Bifunctional purine biosynthesis protein PURH Ras related protein Rab 8B Radixin 60S ribosomal protein L27a Ubiquitin 60S ribosomal protein L40 Heterogeneous nuclear ribonucleoprotein A3

0.218

0.649

0.114

0.109

0.457

0.050

0.072

0.523

0.098

0.043 0.060

0.144 0.41

0.027 0.157

0.193

2.020

0.091

0.453

4.118

0.483

0.036

0.531

0.037

0.032

0.531

0.040

0.152 0.074 0.075 0.077 0.058

0.806 0.858 0.862 0.861 0.231

0.244 0.032 0.032 0.054 0.017

0.031

0.188

0.042

0.171

0.620

0.105

0.025 0.092

0.216 0.313

0.013 0.049

0.042

0.147

0.008

0.022

0.172

0.019

0.039

0.159

0.011

0.118

0.321

0.051

0.179 0.019

0.778 0.157

0.153 0.016

0.471

1.634

0.244

0.110

0.781

0.068 25

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

RS27

P42677

RS27A

P62979

SODC

P00441

STOM

P27105

TGM2

P21980

THIO TLN1 TMED9

P10599 Q9Y490 Q9BVK6

UBB UBC

P0CG47 P0CG48

a

40S ribosomal protein S27 Ubiquitin 40S ribosomal protein S27a Superoxide dismutase Cu Zn Erythrocyte band 7 integral membrane protein Protein glutamine gamma glutamyltransferase 2 Thioredoxin Talin 1 Transmembrane emp24 domain with protein 9 Polyubiquitin B Polyubiquitin C

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0.042

0.257

0.068

0.422

1.798

0.245

0.066

0.377

0.085

0.334

1.193

0.033

0.170

1.298

0.360

0.029 0.117 0.017

0.883 0.556 0.187

0.244 0.055 0.010

1.245 4.086

5.544 17.986

0.065 2.324

NIS, normalized intensity sum. When multiple proteins within a group are listed, the average

NIS is shown.


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Table 2. Common proteins present in culture D2 at 3-fold or lower relative concentration than in D1 and D3. Gene symbol

Protein Protein name accession number H2A1J Q99878 Histone H2A type 1 J KCRB P12277 Creatine kinase B type MIF P14174 Macrophage migration inhibitory factor PAL4A Q9Y536 Peptidyl prolyl cis trans isomerase A like 4A B C PAL4D F5H284 Peptidyl prolyl cis trans isomerase A like 4D RS4Y2 Q8TD47 40S ribosomal protein S4 Y isoform 2 TRY1 P07477 Trypsin 1 TRY2 P07478 Trypsin 2 TRY6 Q8NHM4 Putative trypsin 6 a

Protein NISa Culture D1 Culture D2 Culture D3 0.502 0.006 0.538 0.027 0.074 0.037 0.292 0.081 0.242 0.569

0.078

0.284

0.706

0.073

0.236

0.211

0.047

0.378

0.139 0.141 0.139

0.038 0.019 0.027

0.135 0.581 0.240

NIS, normalized intensity sum.



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Figure legends Figure 1. Comparison of baseline proteomes of NHBE cell cultures from D1, D2 and D3. (A) Percent of all proteins identified that are present in all, some or only one culture. (B) The correlation of relative concentrations of proteins in cultures from different donors. The graph generated by a linear regression analysis using the average NIS from multiple LC/MS runs is shown with the corresponding correlation coefficient.

Figure 2. Viral protein concentrations and infectious virus titers in D1, D2 and D3 cell cultures. The identities and relative concentrations of influenza M1, NS1 and NP proteins in cell lysates were determined by LC-MSE. The graphs show fmol viral protein ± SD per 20 µg total protein on column or infectious virus titer (log10PFU/ml ± SD) for D1 (blue bars), D2 (red bars) and D3 (green bars) at 4, 8, 24 and 48 h p.i.

Figure 3. Proportion of differentially expressed proteins identified in NHBE cultures following influenza infection. The bar graph shows the proportion of proteins with increased (blue) and decreased (red) concentrations at different times after infection. The number of proteins that were differentially expressed (had either increased or decreased concentration) in each culture is shown above each bar. Proteins that remained at approximately the same concentration were not included in the calculation.


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Figure 4. A heat map showing activation Z-scores of canonical pathways identified in D1, D2 and D3 cultures at 4, 8, 24 and 48 h after A/H1N1pdm infection using the IPA platform. The data used to generate the heat map is provided in Supplementary Table S8.

Figure 5. The expression profiles of representative host proteins encoded by interferon stimulated genes IFIT1, IFIT3, IFITM1, IFITM2, IFITM3, and Mx1 at 24 and 48 h after A/H1N1pdm infection of NHBE cultures D1 (blue), D2 (red) and D3 (green). Each bar shows the average NIS for the identified protein, with a cross-hatched bar indicating SD. At 48 h postinfection, the expression levels of all IFN-stimulated gene products were statistically greater (ANOVA, p

Impact of Influenza A Virus Infection on the ... - ACS Publications

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