key: cord-0793169-c2fynyq8
authors: C. Munday, Diane; Hiscox, Julian A.; Barr, John N.
title: Quantitative proteomic analysis of A549 cells infected with human respiratory syncytial virus subgroup B using SILAC coupled to LC‐MS/MS
date: 2010-11-26
journal: Proteomics
DOI: 10.1002/pmic.201000228
sha: 8dfe40027e45e429a3bc479875783f2ce3b8d61b
doc_id: 793169
cord_uid: c2fynyq8

Human respiratory syncytial virus (HRSV) is a leading cause of serious lower respiratory tract infections in infants. The virus has two subgroups A and B, which differ in prevalence and (nucleotide) sequence. The interaction of subgroup A viruses with the host cell is relatively well characterized, whereas for subgroup B viruses it is not. Therefore quantitative proteomics was used to investigate the interaction of subgroup B viruses with A549 cells, a respiratory cell line. Changes in the cellular proteome and potential canonical pathways were determined using SILAC coupled to LC‐MS/MS and Ingenuity Pathway Analysis. To reduce sample complexity and investigate potential trafficking both nuclear and cytoplasmic fractions were analyzed. A total of 904 cellular and six viral proteins were identified and quantified, of which 112 cellular proteins showed a twofold or more change in HRSV‐infected cells. Data sets were validated using indirect immunofluorescence confocal microscopy on independent samples. Major changes were observed in constituents of mitochondria including components of the electron transport chain complexes and channels, as well as increases in the abundance of the products of interferon‐stimulated genes. This is the first quantitative proteomic analysis of cells infected with HRSV‐subgroup B.

Human respiratory syncytial virus (HRSV) is a leading cause of serious lower respiratory tract infections in infants. The virus has two subgroups A and B, which differ in prevalence and (nucleotide) sequence. The interaction of subgroup A viruses with the host cell is relatively well characterized, whereas for subgroup B viruses it is not. Therefore quantitative proteomics was used to investigate the interaction of subgroup B viruses with A549 cells, a respiratory cell line. Changes in the cellular proteome and potential canonical pathways were determined using SILAC coupled to LC-MS/MS and Ingenuity Pathway Analysis. To reduce sample complexity and investigate potential trafficking both nuclear and cytoplasmic fractions were analyzed. A total of 904 cellular and six viral proteins were identified and quantified, of which 112 cellular proteins showed a twofold or more change in HRSV-infected cells. Data sets were validated using indirect immunofluorescence confocal microscopy on independent samples. Major changes were observed in constituents of mitochondria including components of the electron transport chain complexes and channels, as well as increases in the abundance of the products of interferon-stimulated genes. This is the first quantitative proteomic analysis of cells infected with HRSV-subgroup B.

Bioinformatics / Fluorescent labeling / Global protein analysis / Microbiology Western blots Human respiratory syncytial virus (HRSV) is a leading cause of serious lower respiratory tract infection in infants [1] . HRSV belongs in the Paramyxoviridae family (order Mononegavirales), which includes other viruses such as parainfluenza virus, human metapneumovirus and measles virus (MV). Two subgroups of HRSV have been identified (A and B), which generally share 81% genomic nucleotide homology and 88% aggregate proteome amino acid sequence identity. Between subgroup A and B, all viral proteins exhibit a degree of amino acid identity divergence, but some proteins exhibit this to a greater extent, such as M2-2 (72%), which is involved in modulating viral RNA synthesis [2] , the small hydrophobic protein (SH [76%]), which is a viroporin [3] , and the glycoprotein (G [53%]), which is responsible for receptor recognition and attatchment [4] . Arguably the best-studied variants are subgroup A viruses. No vaccine or effective therapeutic treatment currently exists, and anti-viral therapy is licensed only for the immunoprophylactic treatment of high-risk infants [5] . A better understanding of the interaction between HRSV and the host cell at the molecular level is essential for the development of new therapeutic strategies [6] . Two approaches for achieving this are transcriptomics and proteomics.

During infection of model cell lines with HRSV subgroup A, transcriptomic analysis revealed that the virus had multiple effects on the host cell including upregulation of immune response genes including antigen processing and interferon stimulated genes, upregulation of the urokinase plaminogen activator and urokinase plaminogen activator receptor system, apoptotic pathways and genes involved in the organization of the cytoskeleton [7, 8] . The onset of gene induction can be temporally regulated and in general gene upregulation was greater than downregulation [7] . Proteomics using 2-DE has been applied previously to study the interaction between HRSV subgroup A and the host-cell nuclear [9] and total cell proteomes [10] , where the abundance of 24 and 21 proteins, respectively, were shown to change. Areas of commonality included the induction of proteins involved in the stress response.

Specific canonical and signaling pathways have also been investigated in subgroup A-infected cells [6] , including cell cycle arrest through the upregulation of transforming growth factor b1 [11] , alteration of lipid raft membrane composition [12] , decreases in components of the interferon pathways such as TRAF3 and STAT2 [13] , activation of the NF-kB signal transduction pathway [14, 15] and activation of innate immunity through Toll-like receptor 2 [16] . Many of these processes are regulated by the induction of different cellular gene subsets highlighted in the transcriptomic analyses [8, 17] .

In contrast, very little is known about how subgroup B viruses interact with the host cell and this was the focus of this study. The elucidation of proteomic changes in cells infected with this subgroup would provide both a valuable data set, and more importantly, a point of comparison with the better characterized subgroup A viruses. Such studies may also help to identify common host-cell responses, and mechanisms used by viruses with different replication strategies, thus providing information on how the metabolic profile of a cell changes in response to infection and inform as to potential therapeutic targets.

To globally assess changes in the proteome of cells infected with HRSV subgroup B, SILAC coupled to LC-MS/ MS for protein identification and quantification was used [18, 19] . To reduce sample complexity and to study the interaction of HRSV with different cellular compartments, nuclear and cytoplasmic fractions were purified and analyzed separately. A549 cells, a human lung carcinoma cell line that retains properties of HRSV-permissive alveolar cells, were used in this study. Due to its respiratory origin, this cell line has been extensively used in the characterization of HRSV-infection and in the proteomic analysis of cellular and infectious respiratory diseases [9, 10, [20] [21] [22] . Mock-infected cells were grown in media labeled with R6K4 (Dundee Cell Products) and cells infected with subgroup B virus (at a multiplicity of infection of 1) were grown in media containing R0K0. Nuclear and cytoplasmic fractions were harvested 24 h postinfection. This time point was chosen to compare to other proteomic and transcriptomic analysis of HRSV-infected cells and also to ensure that the cells were approximately 75% confluent and not undergoing contact inhibition. In addition, at this multiplicity of infection and time point, little sign of cell death was apparent, probably reflecting that HRSV can delay apoptosis under certain conditions [23, 24] . Cell pellets were re-suspended in a cold cytoplasmic lysis buffer (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5 mM EDTA 0.5% NP-40, EDTA-free complete protease inhibitor mixture (Roche)) and incubated for 10 min on ice. The supernatant containing predominantly cytoplasmic proteins was collected after a 3-min centrifugation at 2000 Â g at 41C. The remaining pellet was re-suspended in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate 0.1% SDS, EDTA-free complete protease inhibitor mixture (Roche)) and incubated for 30 min at 41C. The supernatant containing predominantly total soluble nuclear protein was collected after a 2-min centrifugation at 13 000 Â g at 41C. Both fractions were incubated for 5 min at 41C in a sonicating water bath. The quality of the nuclear and cytoplasmic fractions was surveyed using specific markers to cellular and viral proteins (Supporting Information Fig. 1 ). The data indicated that enriched nuclear and cytoplasmic fractions were obtained, and suggested that potential changes in the abundance of cellular proteins occurred in HRSV-infected cells. For example, a decrease in the abundance of the nuclear/ nucleolar protein, nucleolin, was observed in the nuclear fraction (Supporting Information Fig. 1 ).

Each cytoplasmic and nuclear fraction from mock-infected and HRSV-infected cells was combined and the proteins separated by SDS-PAGE (4-12% Bis-Tris Novex mini-gel, Invitrogen). Ten gel slices per fraction were extracted and subjected to in-gel digestion using trypsin. Purified peptides were separated using an Ultimate U3000 (Dionex), trap-enriched nanoflow LC-system and identified using an LTQ Orbitrap XL (Thermo Fisher Scientific) via a nano ES ion source (Proxeon Biosystems) by Dundee Cell Products. Quantification was performed with MaxQuant version 1.0.7.4 [25] and was based on 2-D centroid of the isotope clusters within each SILAC pair. The generation of the peak list, SILAC and extracted ion current-based quantification, calculation of posterior error probability, as well as the false discovery rate (based on search engine results), peptide to protein group assembly, data filtration and presentation were carried out using MaxQuant. The derived peak list was searched with the Mascot search engine (version 2.1.04; Matrix Science, London, UK) against a concatenated database combining 80 412 proteins from the International Protein Index human protein database version 3.6 (forward database), and the reversed sequences of all proteins (reverse database). Full methodology for the SILAC coupled to the LC-MS/MS analysis to study virus/host interactions has been described previously [18, 19] .

For quantitative analysis, previous investigations using SILAC and LC-MS/MS have applied fold-change cutoffs ranging from 1.3-to 2.0-fold [26] . In this study, a 2.0-fold cutoff was chosen as a basis for investigating potential proteome changes between data sets using Ingenuity Pathway Analysis, and to provide a basis for comparing the current data set to previous HRSV and other virus studies that have used this delineator [17, 27] . Cellular and viral proteins were identified and quantified in the nuclear and cytoplasmic fractions and raw data sets were deposited in the PRIDE [28] using the PRIDE convertor tool [29] . In the nuclear and cytoplasmic fractions, 464 and 440 cellular proteins were identified and quantified, respectively. Of these, 123 proteins (Table 1 ) between the different fractions showed a difference in abundance of twofold or greater, which represented 112 unique proteins (as some proteins were present in both fractions). Mitochondrial proteins are a known contaminant of nuclear fractions [22] and are presented separately in Table 1 because of this. Several viral proteins were also identified in the nuclear (nucleoprotein (N), phosphoprotein (P), non-structural protein 1 (NS1), matrix (M) protein and M2-1 protein) fraction (Supporting Information Table 1 ) and the cytoplasmic fraction (N, P, NS1, M2-1, M and fusion (F) protein) (Supporting Information Table 2 ).

Ingenuity Pathway Analysis was used to examine the cellular protein data sets and to group proteins into similar functional classes. Pathway analysis highlighted several protein networks and canonical pathways that were potentially altered in HRSV-infected cells, based upon underlying biological evidence from the curated Ingenuity literature database. For the proteins that were differentially regulated in the nuclear (excluding mitochondrial proteins) and cytoplasmic fractions, the number of proteins assigned to different functional categories are shown in Fig. 1A . For example, 20 proteins involved in cell death showed a twofold or more decrease in the nucleus fraction in virus-infected cells (p-value 1.44 Â 10 À5 to 4.88 Â 10 À2 ). Other major changes were observed in pathways involved in cell morphology, cellular assembly and organization, protein degradation and gene expression (Fig. 1A) . This is similar to other quantitative proteomic analyses of virus-infected cells using SILAC coupled to LC-MS/MS. Such studies have focused on coronavirus- [18] , influenza virus- [20, 30] and HIV-1 [31] infected cells.

Several canonical pathways were potentially altered in HRSV-infected cells including interferon signaling (p-value 1.97 Â 10 À5 ) (Fig. 1B) . STAT1 was increased 6.3-fold. This protein mediates the expression of a variety of genes considered central to the host-cell response to infection or inflammation. Examples of such proteins identified in this study included interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) (increased 8.9-fold in HRSVinfected cells) and myxovirus (influenza virus) resistance 1 interferon-inducible protein p78 (MX1) (increased 11.2-fold in HRSV-infected cells) (Fig. 1B) . The observed increase in STAT1 in HRSV-infected cells has been shown previously in human diploid fibroblast 2fTGH cells, which were infected with HRSV subgroup A [32] . Likewise, the increased expression of MX1 mRNA and protein has been demonstrated in tissues isolated from cotton rats infected with HRSV subgroup A [33] . In the current data set, pathway analysis linked these molecules to NF-kB activated transcription and IFNa/b (e.g. Fig. 1B) , all of which have been described in HRSV subgroup A-infected cells [11, 14, 34] . Therefore, previously published data were reflected by the bioinformatic analysis of the quantitative proteomic data. However, there have been differing reports of the effect of different HRSV subgroup A viruses on inducing interferon type I. Similar to a micro-array analysis of A549 cells infected with HRSV subgroup A [7] , the quantitative proteomic analysis supports the activation of interferon stimulated genes in A549 cells infected with subgroup B virus.

One of the novel findings of this quantitative proteomic analysis was the alteration of mitochondrial proteins in HRSV-infected cells. The abundance of proteins associated with respiratory complexes 1, 3, 4 and 5 (Supporting Information Fig. 2 ), oxidative phosphorylation (Supporting Information Fig. 3 ), super-oxide dismutase, proteins involved in mitochondrial integrity (prohibitin) and transition pore complexes (voltage-dependent anion channels (VDACs)) were changed. As a result, Ingenuity Pathway Analysis predicted mitochondrial dysfunction in HRSV-infected cells (p-value 2.22 Â 10 À2 ). Although it is known that mitochondria play a central role in the host-cell response to microbial infection, the change in abundance of mitochondrial proteins in HRSVinfected cells has not been previously described, and may be linked to the induction of ROS [35, 36] .

The major responses to virus-infection can be directed by innate and adaptive immunity and clearly these pathways are activated in HRSV-infected cells [6, 7, 10] . More subtle specific host-cell proteins can exhibit anti-viral activity. One such protein is ADAR, an interferon inducible RNA editing enzyme, which functions to deaminate adenosine to inosine, and whose activity may depend on subcellular localization [37] . ADAR increases 2.8-fold in the nucleus in HRSV-infected cells (Table 1 ). ADAR has been reported to have a potential role in innate anti-viral immunity, including influenza A virus [38, 39] . Conversely, ADAR1 has been reported to act as a pro-viral, anti-apoptotic host factor in measles virus-infected cells [40] and also in cells infected with vesicular stomatitis virus [41] , which also belongs to the Mononegavirales. Similarly, 2 0 -5 0 -oligoadenylate synthetase 3 was shown to increase 5.0-and 3.5-fold in the nucleus and cytoplasm of HRSV-infected cells, respectively. This protein has anti-viral activity and is activated by interferon [42, 43] and has been shown to be a part of interferon-g-mediated inhibition of HRSV [44] .

Information from the Ingenuity database and an examination of the existing literature was used to prioritize the pathway-associated proteins of interest for validation. To that end, experiments using indirect immunofluorescence confocal microscopy were used, providing a complete and independent verification of the results as this technique does not rely on subcellular fractionation and purification of proteins from mock-or HRSV-infected cells. Also, the study would provide confidence in the proteomic data set as this was from a single experiment. Microscopy analysis of the subcellular localization of Tom22, VDAC1 and prohibitin in HRSV-infected cells (compared with mock-infected cells), 

The mRNA for OAS2 is upregulated 4.5-fold in HRSV subgroup A infected cells at 24 h p.i. [7] and also upregulated in infected mice [50] . This protein has anti-viral activity and can promote mRNA destabilization and rRNA cleavage. (Also discussed in text.)

IPI00152503. reflected the quantitative proteomic data analysis (Fig. 2) . Notably, in the immunofluorescence analysis of mockinfected cells, VDAC1 is present in the nucleus and cytoplasm but in HRSV-infected cells VDAC1 appeared to be absent from nuclear compartment by 24 h (Fig. 2) . This reflects the quantitative proteomic analysis, which measured an approximately 16-fold decrease of VDAC1 in the nuclear fraction and approximately twofold increase in VDAC1 in the cytoplasmic fraction prepared from HRSV-infected cells compared with mock-infected cells. Curiously, as discussed, many of the mitochondrial proteins were identified in the nuclear fraction. Independent reports of nuclear fractions obtained from A549 cells (prepared by a different method) also contained mitochondrial proteins, which were suggested to be a potential contaminant [22] , and has also been documented in the purification of nucleoli from the nucleus [45] . However, tubular structures that contain mitochondria can be found projecting into the nucleus [46] and may thus explain the presence of (some) mitochondrial proteins in nuclear factions. Several other proteins of interest were used to validate the data set and may also indicate that cut-off values lower than 2.0-fold could be considered. For example, in the quantitative proteomic analysis, nucleolin was shown to decrease 1.6-fold in the nuclear fraction prepared from HRSV-infected cells, compared with mock-infected cells, a result validated using immunoblot analysis (Supporting Information Fig. 1 ). Indirect immunofluorescence confocal microscopy revealed that nucleolin was absent from the nucleus/nucleolus of some infected cells at 24 h post-infection and from all infected cells at 44 h post-infection (example images are shown in Fig. 2) . Nucleolin was also reported to be decreased at 24 h post-infection in A549 cells infected with human metapneumovirus [10] .

In the quantitative proteomic analysis, caveolin was increased 1.7-fold in the cytoplasmic fraction prepared from HRSV-infected cells, compared with mock-infected cells.

Again, examples could be found using indirect immunofluorescence confocal microscopy where the relative fluorescence of caveolin was greater in HRSV-infected cells compared with mock-infected cells (Fig. 2) . No significant change in the abundance of myosin 6 or lamin B was identified by either the quantitative proteomic analysis or by indirect immunofluorescence confocal microscopy (Fig. 2) .

The quantitative proteomic analysis indicated that proteome changes in response to infection were not global, but confined to specific proteins or protein classes. This is similar to a recent temporal 2-DE comparison of the interaction of HRSV subgroup A and other respiratory viruses belonging to the Paramyxoviridae with A549 cells [10] . Here, based on this analysis, van Diepen et al. [10] proposed four processes in virus-induced apoptosis: virus uptake and infection, stress response, disruption of cellular structures and cell death by apoptosis. The quantitative proteomic analysis conducted here would support this hypothesis, particularly with regard to disruption of mitochondria and nucleoli, the latter of which has been observed in proteomic analysis of other virus-infected cells [18, 19, 27] . Such changes may have functional consequences for host-cell biology. For example, nucleolin is a major constituent of the nucleolus and functions as a possible hub protein [47] . Therefore, changes to the abundance of this protein may have consequences for nucleolar function [48, 49] .

Overall, the analysis demonstrates how the application of SILAC coupled to LC-MS/MS for identification and quantification, and bioinformatic analysis can be readily used to study the interaction of viruses with the cellular proteome. In this case, the relatively unstudied HRSV subgroup B virus has been shown to alter the abundance of proteins involved in the regulation of specific host-cell pathways.

Cellular and viral proteins were identified and quantified in the nuclear and cytoplasmic fractions and raw data sets were deposited in the Proteomics Identifications Database (PRIDE) using the PRIDE convertor tool. (Accession nos. 13270 for the cytoplasm and 13269 for the nucleus).

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The authors have declared no conflict of interest.