Proteomic analysis of mitochondria in respiratory epithelial cells infected with human respiratory syncytial virus and functional implications for virus and cell biology.

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Research Paper

Journal of Pharmacy And Pharmacology

Proteomic analysis of mitochondria in respiratory epithelial cells infected with human respiratory syncytial virus and functional implications for virus and cell biology Diane C. Mundaya, Gareth Howellb*, John N. Barrb and Julian A. Hiscoxa a Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool and bSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK

Keywords inhibition; mitochondria; proteomics; respiratory syncytial virus; siRNA Correspondence Diane C. Munday, Department of Infection Biology, Institute of Infection and Global Health, ic2 Building, Brownlow Hill, University of Liverpool, Liverpool L3 5RF, UK. E-mail: [email protected] Received May 6, 2014 Accepted October 12, 2014 doi: 10.1111/jphp.12349 *Present address: Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK.

Abstract Objectives The aim of this study was to quantitatively characterise the mitochondrial proteome of airway epithelial cells infected with human respiratory syncytial virus (HRSV), a major cause of paediatric illness. Methods Quantitative proteomics, underpinned by stable isotope labelling with amino acids in cell culture, coupled to LC-MS/MS, was applied to mitochondrial fractions prepared from HRSV-infected and mock-infected cells 12 and 24 h postinfection. Datasets were analysed using ingenuity pathway analysis, and the results were validated and characterised using bioimaging, targeted inhibition and gene depletion. Key findings The data quantitatively indicated that antiviral signalling proteins converged on mitochondria during HRSV infection. The mitochondrial receptor protein Tom70 was found to act in an antiviral manner, while its chaperone, Hsp90, was confirmed to be a positive viral factor. Proteins associated with different organelles were also co-enriched in the mitochondrial fractions from HRSVinfected cells, suggesting that alterations in organelle dynamics and membrane associations occur during virus infection. Conclusions Protein and pathway-specific alterations occur to the mitochondrial proteome in a spatial and temporal manner during HRSV infection, suggesting that this organelle may have altered functions. These could be targeted as part of potential therapeutic strategies to disrupt virus biology.

Introduction Human respiratory syncytial virus (HRSV) is the leading cause of virus-induced, acute lower respiratory infection (ALRI) in infants and children worldwide (reviewed in[1,2]). HRSV is responsible for over 30 million new ALRI cases each year and up to 199 000 deaths in children under 5 years old, and other high-risk groups include the elderly and immunocompromised.[3] HRSV belongs to the paramyxovirus family, which includes important human pathogens such as measles, mumps and human parainfluenza.[4] Ten HRSV genes encode 11 proteins: non-structural protein 1 (NS1), non-structural protein 2 (NS2), nucleo- (N), phospho- (P), matrix (M), small hydrophobic (SH), glyco(G), fusion (F), M2-1 and M2-2, and the large (L) protein.[5] HRSV replicates in the cytoplasm, and like all 300

paramyxoviruses the genome consists of a single-stranded, negative sense RNA; therefore, high mutation rates can lead to the emergence of resistance. HRSV causes disease in all ages and has been linked to the onset of asthma (reviewed in[6]), and infections recur throughout life. However, no approved vaccine is available, and few effective and safe antiviral compounds with a narrow spectrum of activity have emerged (reviewed in[2,7,8]). An urgent and unmet healthcare need therefore exists, and robust strategies are required to lower the current and future disease burden caused by this virus. Identifying those cellular proteins critical for HRSV biology and disputing their activity could lead to broader spectrum antiviral compounds being developed: high

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 300–318

Diane C. Munday et al.

throughput, quantitative proteomic analysis provides a powerful tool for identifying such intricate protein and pathway-specific alterations in virus-infected cells on a large scale.[9] Previous global proteomic analyses of HRSV–host cell interactions highlighted specific cellular proteome changes related to the antiviral response and demonstrated that host cell proteome alterations were confined to specific signalling pathways and processes.[5,10,11] Of particular interest were previously uncharacterised changes in the abundance of mitochondrial proteins quantified 24 h post-infection (p.i.) in comparison to mock.[5,10,11] Mitochondria are ubiquitous organelles that perform important metabolic functions, including ATP production, calcium homeostasis and regulation of cell proliferation, presenting an excellent target for pharmaceutical intervention. A range of stimuli regulate their distribution, shape and function, including viral infections[12,13] which can trigger host innate immune responses through the activation of transcription factors NFκB and interferon regulatory factor 3 (IRF3), which regulates the expression of type-I interferons, such as interferon-β (IFN-β).[14] Evidence suggests that mitochondria play a key role in co-coordinating this sustained response and that antiviral proteins may localise or change in abundance on mitochondria during virus infection. Alternatively, viruses can target mitochondria to control apoptosis, subvert the host cell defence[12,15,16] or aid replication.[17] Infection can also cause the restructuring of the endoplasmic reticulum (ER) and its association with mitochondria.[18] Thus, mitochondria represent an important stepping stone in virus infection with both negative and positive viral activity focused on these organelles. During infection with HRSV and viruses that are similar in genome replication and expression strategies, retinoic acid induced gene I product (RIG-I) has been implicated in detecting actively replicating Respiratory syncytial virus (RSV) (reviewed in[19]) as this pattern recognition receptor recognises ssRNA viral genomes bearing 5′-triphosphates.[20] RIG-I then interacts with an adapter, mitochondrial antiviral signalling (MAVS), which is targeted to the outer mitochondrial membrane (OMM).[14] This leads to the activation of TANK-binding kinase (TBK1), which phosphorylates IRF3, leading to its translocation to the nucleus.[21] This is followed by the early expression of IFNα/β and the subsequent establishment of the antiviral state.[22–24] The localisation of MAVS to the OMM is essential for signalling, as deletion or mutation of the MAVS mitochondrial targeting sequence causes disruption to the antiviral response.[14,25,26] As a subunit of the translocase of outer mitochondrial membrane (TOM), Tom70 has been proposed as an essential adaptor, linking the MAVS to antiviral signalling.[27] In non-infected cells, this major OMM import receptor

Mitochondrial proteome in HRSV-infected cells

recruits mitochondrial pre-proteins from the cytosol specifically through interactions with its chaperone, heat shock protein 90 (Hsp90).[28,29] However, the function of Tom70Hsp90 recruitment functions may alter during virus infection. In Sendai virus (SeV)-,[30] vesicular stomatitis virus and Newcastle disease virus-infected cells,[27] Tom70-Hsp90 interactions are essential for IRF3 activation[27] and the IRFmediated antiviral response,[30] and proteins associated with the innate antiviral immune response have been shown to re-localise to mitochondria in virus-infected cells.[27,30] Previous studies have also shown that during HRSV infection, the RIG-I/MAVS complex can activate the NFκB signalling pathway,[31–34] and the HRSV nucleoprotein (N) has been shown to co-localise with RIG-I and other molecules in virus-induced inclusion bodies.[35] However, the alterations to mitochondrial dynamics and the signalling events during HRSV infection, which could inform potential therapeutic targets, have not been fully characterised. Many studies focus on individual virus protein–cellular protein interactions or manipulate a particular signalling pathway to further understand virus–host cell interactions and the antiviral response. The aim of this study was to elucidate changes in the mitochondrial proteome of HRSV-infected cells that may inform function under physiologically relevant, unbiased conditions. Such studies can increase our understanding of virus–organelle interactions and provide important insights into immune responses[12] and the virus replication cycle, potentially indicating new therapeutic targets to inhibit virus-related functions. No such organelle-focused quantitative proteomics study has been applied to HRSV.

Materials and Methods Cells and virus HEp2 and A549 cells obtained from the Public Health England Culture Collections were grown at 37°C with 5% CO2 in Dulbecco’s modified eagle’s medium (DMEM) (Invitrogen) supplemented with 10% Feotal Bovine Serum (FBS) and 1% penicillin-streptomycin. For the stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomic analysis of mitochondria-enriched fractions, A549 cells were cultured in the following labelled media (Dundee Cell Products Ltd) supplemented with 10% dialysed Feotal calf serum (FCS) (Dundee Cell Products Ltd (DS1003)) and 1% penicillinstreptomycin: Mock-infected cells were grown in medium DMEM (R6K4: containing 13C-labelled arginine and 2D-labelled lysine). HRSV-infected cells harvested at 12 h were grown in heavy DMEM (R10K8: containing 13C- and 15 N-labelled arginine and lysine). HRSV-infected cells harvested at 24 h were grown in light DMEM (ROKO: labelfree). The HRSV A2 strain was propagated in HEp2 cells,


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purified/concentrated using a sucrose gradient,[36] and the titre was calculated as previously described.[5] A multiplicity of infection (m.o.i.) of 3 was used for all experiments.

Enrichment of mitochondrial proteins A Qproteome Mitochondria Isolation Kit obtained from Qiagen (37612) was used to enrich cytosolic, membrane and mitochondrial proteins from A549 cells (typically 1.5– 2 × 107 cells from two 500 cm2 dishes seeded at 8 × 106 48 h prior). Only mitochondrial proteins enriched from SILAClabelled cells were analysed by LC-MS/MS). Mitochondrial fractions from each condition were mixed 1 : 1 : 1 based on Bicinchoninic acid (BCA)-estimated protein concentration, prior to Liquid chromotography tandem mass spectrometry (LC-MS/MS) analysis.

Inhibition study

Quantitative proteomics LC-MS/MS was performed by Dundee Cell Products Ltd as described previously.[5,10,37] In brief, the mitochondrial fractions from HRSV-infected cells 12 h (heavy) and 24 h (light) p.i. in comparison to mock-infected (medium) cells were combined 1 : 1 : 1, and the proteins were separated by SDS-PAGE (4–12% Bis-Tris Novex Mini-Gel, Invitrogen). The gel lane was excised and cut into 10 gel slices which were subjected to in-gel digestion with trypsin.[38] Purified peptides were separated using an Ultimate U3000 (Dionex), trap-enriched nanoflow LC-system and identified using an LTQ Orbi-trap XL (Thermo Fisher Scientific) via a nano ES ion source (Proxeon Biosystems). The generation of a peak list, SILAC and extracted ion current-based quantification, calculation of posterior error probability and false discovery rate (based on search engine results), peptide to protein group assembly, data filtration, and presentation were performed with MaxQuant version 1.0.7.4.[39] The peak list was searched with the Mascot search engine (version 2.1.04; Matrix Science, London, UK). Raw datasets were deposited in the PRIDE using the PRIDE convertor tool.[40,41] Data were analysed using ingenuity pathway analysis (IPA) (Ingenuity Systems, www.ingenuity .com (Qiagen)) as previously described,[5,10,37] and the experiment was repeated using non-stable isotope-labelled cells.

Gene depletion Small interfering RNAs (siRNAs) (Qiagen) were transfected using Lipofectamine 2000. Cells were seeded at a density of 3 × 104 in antibiotic-free DMEM 24 h prior to transfection to achieve 30–40% confluent monolayers (24well plate). Of each siRNA sequence pair (20 pmol for the negative control), 10 pmol was prepared using 1 μl/well Lipofectamine 2000. Control wells of reagent only and media only were included. 302

For virus experiments, media containing transfection reagents were removed 20 h post-transfection, and HRSV (diluted in serum-free DMEM) was applied for 2 h at 37oC. The inoculum was removed and a minimum volume of fresh antibiotic-free DMEM was applied. Supernatant (for plaque assay assessment) and cell pellets were processed 21 h p.i., 40 h post-transfection. The siRNA sequence pairs were as follows: TOM70A-1 – AAGACAATAAGAAGGA ATGTT (SI00301973) and TOM70A-8 – CTGAATGACCTC TGACTTTAA (SI032 46824). The sequence of the nonspecific control was not disclosed (SI03650318). To determine the efficiency of protein knockdown, cell pellets were lysed in RIPA buffer and immunoblotted with the indicated antibodies.

A549 cells were seeded at a density of 1.5 × 104/well in 24-well plates 24 h prior to infection to achieve 70% confluency. HRSV was applied for 4 h at 37°C. Monolayers were washed with Phosphate buffered saline (PBS) prior to the addition of the geldamycin derivative 17-N-Allylamino17-demethoxygeldamycin (17-AAG) (Calbiochem/EMD Millipore (100068)) which was diluted in DMSO (vehicle control) and prepared in DMEM supplemented with 10% (v/v) FBS. Supernatant was removed 21 h p.i., 18 h post-17AAG treatment. Cytotoxicity was assessed using a colorimetric MTT assay, and a concentration range that maintained cell viability was established (0.0625–5 μm). Inhibitor activity was assessed via immunoblot analysis of cell division cycle protein 2 homolog (cdc2) depletion as the abundance of this client protein is dependent on Hsp90 chaperone activity[42] (data not shown). At the concentrations tested, there was no effect on the Hsp90 protein abundance (data not shown).

Plaque assay assessment HRSV titre was calculated for virus contained in the supernatant harvested from HRSV-infected Tom70-depleted or 17-AAG-treated cells. The supernatant was titrated on HEp2 cells and an antibody-based methylcellulose plaque assay was used as previously described.[5] The Kruskal– Wallis test was used to compare independent sets of triplicate plaque assay results using the Microsoft Excel software.

Indirect immunofluorescence confocal microscopy (IF) analysis Cell monolayers were fixed with formalin and permeabilised using PBS containing 0.1% Triton X-100. Primary antibodies were diluted 1 : 50 in PBS containing 2% FBS: MX1 (ab95925), TOMM20 (ab56783), IRE1



(Ab37073) and HSP90 (S88) (ab1429) were obtained from Abcam. OAS3 (H-130) (Sc-99100) and ISG15 (Sc-50366) were obtained from Santa Cruz. TOMM70 (N-terminal) (AP6745a), TOMM70 (C terminal) (AP6745b) and RIG-I (AP1900a) were obtained from Abgent and PMP70 (P0497) from Sigma . The following Alexa Fluor-conjugated secondary antibodies diluted 1 : 200 in PBS containing 2% FBS: Donkey anti-mouse far red 647 (A31571) (false coloured magenta for confocal images) and chicken anti-rabbit red 594 (A21442) were obtained from Molecular Probes (Invitrogen). HRSV proteins were detected by either a goat anti-HRSV primary antibody (ab20745) recognised by a FITC-conjugated secondary (ab6881) or via a FITCconjugated anti-HRSV A2-specific primary antibody (ab20391). Both were polyclonal antibodies raised against HRSV structural proteins. Stained cells were mounted using either Vectorshield (Vector Laboratories) or Prolong Gold antifade reagent (Invitrogen), both of which contained a DAPI nuclei counter stain. Confocal images were captured on either an inverted Zeiss LSM 510 META Axiovert 200M, an upright LSM 510 META Axioplan or on an LSM 700 microscope (Carl Zeiss Ltd). All microscopes were equipped with 40 × and 63 ×, 1.4 numerical aperture, and oil immersion lenses. Pinholes were set to allow optical sections of 1–2 μm to be acquired. Images were averaged either 8 or 16 times.

Immunoblot analysis The cellular fractions were obtained as detailed above. Total protein concentration was determined by BCA assay (Pierce). Proteins (2–5 μg) were detected with the same antibodies which were described for indirect immunofluorescence plus the following: tubulin (YOL1/34) (ab6161), GAPDH (6C5) (ab8245), ATP6V1C2 (Ab88444) and actin (ACTN05 (C4)) (Ab3280) were obtained from Abcam, cytochrome c (Ab-2) from Onogene, and calnexin (SP860) from Stressgen. Three Horseradish peroxidase (HRP)conjugated secondary antibodies (A5795, A4416 and A6154) were obtained from Sigma, and one (ab6741) was from Abcam. All were detected with enhanced chemiluminescence.

Results Resolution of the dynamic mitochondrial proteome in human respiratory syncytial virus-infected cells The mitochondrial proteome in HRSV-infected cells was analysed at two time points, 12 and 24 h p.i., which captured early and late events in the virus life cycle and allowed comparison with existing proteomic/ transcriptomic studies.[5,10,43] The mitochondrial proteome


at these time points was compared with the mitochondrial proteome taken from uninfected cells as a control. IF analysis indicated that ∼40% of cells were visibly infected with the HRSV-A2 strain at 12 h and that ∼80% of cells were visibly infected at 24 h (Figure 1a). As infection efficiency was not 100% at m.o.i. 3, changes in cellular protein abundance identified by the SILAC-based LC-MS/MS may be under-representative of those observed in individual cells. To enrich mitochondria, a multi-step process was used, including centrifugation to remove cytosolic components, pelleting to remove nuclei and cell debris, and separation of mitochondria through a gradient. Proteins were simultaneously identified and quantified, and SILAC allowed relative alterations in protein abundance between the three conditions (uninfected, 12 h and 24 h p.i.) to be differentiated (workflow shown in Figure 1b). Immunoblot analysis confirmed the presence of structural viral proteins (Figure 1c) and the enrichment of mitochondrial proteins in comparison to the cytosolic and membrane fractions (Figure 1c and 2), alongside whole cell lysate (Figure 2). For example, the abundance of Tom20, a mitochondrial TOM complex marker protein (OMM), was identified but not shown to change (more than twofold) in abundance in the mitochondrial fractions from HRSV-infected cells at 12 or 24 h, in comparison to mock-infected cells, and this was reflected in its abundance in whole cell lysates (Figure 2). Superoxide dismutase 2 (SOD2) is localised to the inner mitochondrial matrix and has been reported to increase in abundance in HRSV-infected cells.[44] This was used as a second marker protein and significantly increased in abundance in the mitochondrial fraction and whole cell lysate at 12 and 24 h p.i. compared with mock-infected cells (Figure 2). Cytochrome c was used as an inner mitochondrial membrane marker protein: transmembrane pores, such as voltage-dependent anion channels, may participate in the formation of the permeability transition pore complex, which is responsible for the release of mitochondrial products, such as cytochrome C, that trigger apoptosis. The proteomic analysis of mitochondria enrichments identified 16 different cytochrome subunits, precursors or isoforms. Six did not change in abundance over the course of infection, while 10 increased twofold at 24 h in HRSV-infected cells in comparison to mock-infected (Table S2), and this was reflected in the immunoblot analysis (Figures 2 and 4a). IF indicated that in mock-infected cells, cytochrome c fluorescence was low in comparison to HRSV-infected cells, and that in mock and infected cells cytochrome c co-localised to mitochondria (Tom20) (data not shown). Tubulin, a cytosolic marker protein and identified in whole cell lysates, was enriched in the cytosolic component compared with either the membrane fraction or the


303



(a)

(b)

M

Mock

12

24

Medium

Heavy

Light

Immunofluorescence

Subcellular enrichments = cytosolic, membrane and mitochondrial fractions

12

Immunoblotting

Mitochondrial enrichments 1:1:1 24 Protein identification and quantification by LC-MS/MS

(c)

Cytosolic components M

12

24

Membrane fraction M

12

24

Mitochondrial fraction M

12

24

G

F N P M

Figure 1 Stable isotope labelling with amino acids in cell culture-based proteomic analysis of mock-infected and human respiratory syncytial virusinfected cells over the course of infection. (a) Immunofluorescence confocal microscopy (IF) validation of infection efficiency at 12 h and 24 h postinfection in comparison to mock-infected cells (M). Human respiratory syncytial virus proteins are shown in green and the nuclei are stained blue with DAPI. Scale bars are 10 μM. (b) Diagram of the stable isotope labelling with amino acids in cell culture-based methodology: Subcellular fractionation was used to enrich proteins in cytosolic, membrane and mitochondria fractions from mock-infected cells (medium) and human respiratory syncytial virus-infected cells at 12 h (heavy) and 24 h post-infection (light (unlabelled)). Infection efficiency and the fraction purity were then validated prior to mitochondrial fractions being mixed 1 : 1 : 1 for LC-MS/MS analysis. (c) Immunoblot confirmation of human respiratory syncytial virus infection (and lack thereof in mock-infected (M) cells). The assignment of the structural virus proteins is indicated on the right.

mitochondrial fraction (Figure 2). GAPDH, a cytosolic marker also found in the ER and Golgi apparatus, was identified in whole cell lysates, but with less abundance in the membrane fraction and mitochondrial fraction compared with the cytosolic component (Figure 2). Overall, the data indicated that the mitochondrial fraction had been enriched in A549 cells infected with HRSV at 12 and 24 h p.i. and in mock-infected cells. 304

The SILAC-based approach resulted in the identification of five viral proteins (M, N, P, M2-1 and F proteins) (Figure 1c and Table 1; the raw dataset is presented in Table S1) and 1061 cellular proteins (the raw dataset is presented in Table S2). Of the cellular proteins, 519 were identified and quantified by two or more peptides (a commonly used benchmark for increasing confidence in identification and quantification[5,10,37,45–48]) and were used in further




Cytosolic components M

12

24

Membrane Mitochondrial fraction fraction

Whole cell lysate

M

M

12

24

M

12

24

12

24

Tom20

Outer memb

SOD2

Inner matrix

Cyt C

Inner memb.

Tubulin Controls GAPDH

Figure 2 Immunoblot confirmation of representative marker proteins from the cytosolic, membrane and mitochondrial fractions from mockinfected (M) and human respiratory syncytial virus-infected cells at 12 and 24 h post-infection (indicated). Membrane-immobilised proteins were detected with antibodies to Tom20 (outer mitochondrial membrane), SOD2 (inner mitochondrial matrix), cytochrome c (inner mitochondrial membrane) tubulin and GAPDH. Tubulin was predominately localised in the cytosolic fractions and mitochondrial markers Tom20, SOD2 and cytochrome c to the mitochondrial fraction.

Table 1

Viral proteins identified in mitochondrial fractions enriched from HRSV-infected cells at 12 and 24 h p.i.

Protein ID

Protein

12 h/M

24 h/M

Pep

Seq cov (%)

kDa

PEP

IPI00100006 IPI00100007 IPI00100004 IPI00100001 IPI00100009

Matrix protein Nucleoprotein M2-1 Fusion protein Phosphoprotein

1.1 1.1 1.4 1.0 –

19.9 19.6 14.4 7.3 –

7.0 6.0 2.0 6.0 1.0

48. 23.9 26.9 22.4 7.7

22.0 36.7 15.0 56.9 20.5

2.47 × 10−86 2.25 × 10−83 2.09 × 10−24 1.47 × 10−27 8.52 × 10−9

HRSV, human respiratory syncytial virus; p.i. The following are shown: protein ID (IPI accession number) and description; SILAC ratios of relative abundance at 12 h p.i. (heavy/medium) and 24 h p.i. (light/medium); the number of peptides that were detected and used to identify and semiquantify the protein and the % sequence coverage (% seq cov) of the total primary sequence of the protein these peptides represent. The posterior error probability (PEP) score is a measure of the probability of misidentifying a protein.

analyses (Table S3). Nevertheless, many of the proteins identified by single peptides were also identified as mitochondrial (Table S2). An arbitrary 2.0-fold cut-off formed the basis for investigating potential proteome changes between datasets through IPA, and to compare the current dataset with previous studies.[5,10] However, alterations in protein abundance under the cut-off value were also of interest in the context of pathway analysis and protein re-localisation. Gene ontology (http://www.geneontology.org/) was used to compare the 519 proteins (identified by two or more peptides) (Table S3) with available mitochondrial proteomes[49–51] and to classify the cellular proteins according to subcellular localisation (Figure 3a). Of these proteins, 200 could be specifically assigned as mitochondrial proteins. Proteins were also identified and quantified which localised to the cytoplasm (38 proteins), cytosol (32 proteins), Golgi (17 proteins), ER (26 proteins) and ER membrane (12 proteins). Identified mitochondrial proteins

could be further categorised by their localisation within this organelle (Figure 3b). Once organised, the datasets were uploaded to IPA, which we used previously to investigate proteome changes in HRSV-infected cells,[5,10] and relative increases or decreases in protein abundance were grouped into distinct functional categories. The data indicated that the mitochondriaenriched proteomes differed between the two time points in comparison to mock-infected cells.

Temporal changes in the mitochondrial proteome during human respiratory syncytial virus infection At 12 h p.i, 7% and 9% of proteins enriched in the mitochondrial fraction showed a twofold or greater decrease or increase in abundance, respectively. After 24 h, 0.4% and 49% of proteins showed a twofold or greater decrease or increase in abundance, respectively, in the


305



(a) Cell fraction Integral to membrane Other Golgi membrane Extracellular region Membrane Intracellular Chromosome Microtubule Kinetochore Endosome Centrosome

Cytoplasm Mitochondria Large ribosomal subunit Extracellular space Chromatin Lytic vacuole Cytoplasmic membrane Cell surface Macromolecular complex Histone acetyltransferase complex Condensed chromosome Annulate lamellae

Cytosol ER membrane Endoplasmic reticulum Plasma membrane Golgi apparatus Integrin complex Cytoskeleton Mismatch repair complex Lysosomal membrane ER-Golgi intermediate compartment Charperonin-containing T-complex Actin filament

Cell fraction Cytoplasm Cytosol

Mitochondrial proteins

(b)

Inner membrane

Mitochondrialgeneral assignment

Matrix Respiratory chain complex

Protontransporting ATP synthase complex Outer membrane

Respiratory chain complex InterPyruvate Inner memb. III memb. Outer memb. Respiratory presequence dehydrogenase space complex chain translocase translocase complex complex Figure 3 The proportional cellular localisation of proteins identified in the mitochondrial fractions from human respiratory syncytial virus-infected cells at both 12 h and 24 h. (a) The general assignment of cellular proteins in mitochondrial fractions to their localisation within the cell. A number of proteins originating from the cytoplasm, cytosol, Golgi, endoplasmic reticulum and endoplasmic reticulum membrane, for example, contributed to the mitochondrial enrichment. (b) Mitochondrial proteins could also be more specifically organised according to their localisation within the mitochondria.

mitochondrial fraction. Several canonical pathways were highlighted by IPA as being disrupted in HRSV-infected cells at 12 h p.i., including those involved in virus entry via endocytic pathways (12 molecules; P-value, 1.02 × 10−16), caveolar-mediated endocytosis signalling (10 molecules; P-value, 3.96 × 10−14), integrin signalling (10 molecules; P-value, 6.77 × 10−10) and NFκB activation by viruses (6 molecules; P-value, 1.84 × 10−7). Proteins involved in virus entry, integrin signalling and NFκB activation were all twofold or greater decreased in abundance at 12 h p.i. 306

The canonical pathways that were highlighted by IPA as being disrupted in mitochondrial fractions from HRSVinfected cells 24 h p.i. included mitochondrial dysfunction (7 molecules; P-value, 3.12 × 10−6), which was also described in our analysis of nuclear and cytoplasmic fractions from HRSV-infected cells.[5,10] Several proteins involved in the antigen presentation pathway were also identified (4 molecules; P-value, 4.35 × 10−5). A number of proteins that were increased in abundance at 24 h p.i. in HRSV-infected cells could be grouped into specific disease




(a) Membrane Mitochondrial Cytosolic fraction components fraction M 12 24 M 12 24 M 12 24

(b)

Whole cell lysate M 12 24 Cyt C

Mitochondria

Calnexin

ER

PMP70

Peroxisome

GAPDH

Control

Tom20

IRE1

HRSV

Merge

Tom20

PMP70

HRSV

Merge

M

24

M

24

Figure 4 Immunoblot and IF analysis of an endoplasmic reticulum or peroxisome marker, which were identified and quantified by LC-MS/MS in the mitochondrial fractions of human respiratory syncytial virus-infected cells at 12 h and 24 h post-infection in comparison to mock. (a) Immunoblot confirmation of the presence of endoplasmic reticulum marker calnexin and peroxisome marker PMP70. Protein localisation or pathway involvement is described on the right. (b) Subcellular localisation of endoplasmic reticulum marker, IRE1, and peroxisome marker, PMP70. IRE1 or PMP70 is shown in red, mitochondria are marked with Tom20 (magenta), human respiratory syncytial virus proteins green, and nuclei are stained blue with DAPI. A merge image is also presented. The colour pink indicates the co-localisation of proteins marked with magenta and red. The colour yellow indicates the co-localisation of proteins marked with green and red. Scale bars, 10 μM.

associations, including the inflammatory response (20 proteins; P-values, 6.85 × 10−5 − 1.96 × 10−2).

Human respiratory syncytial virus infection promotes mitochondrial associations with other double-membrane structures Several proteins associated with the ER and double membrane vesicles, such as peroxisomes (e.g. ATP-binding cas-

sette subfamily D, member 3 (Abcd3)) were identified as being increased in abundance in the mitochondrial fractions from HRSV-infected cells compared with mockinfected. For example, analysis of whole cell extracts by immunoblot indicated there was no change in the abundance of calnexin (ER-marker protein) and peroxisomal membrane protein 70 kDa (PMP70) (a component of peroxisomes) between mock- and HRSV-infected cells at 12 and 24 h p.i. (Figure 4a). However, in mitochondrial


307



(a)

Viral RNA

(b)

IRG

IFN TYPE 1

RNA sensor (DDX58)

MX1

DDX58

MSVS

OAS3 (includes EG:4940)

ISG15 IFN Beta Ifn NFkB (complex)

OAS2 Interferon alpha

Hsp90 Tom 70

IL 12 (complex)

Tom 20 TAP1

HLA-B*

MHC Class I (complex)

GARS

TAP2

Signalling complex

MHC CLASS I (family)

Nucleus

Tap

PDIA3

peptide-Tap1-Tap2

Antiviral genes

Figure 5 (a) Network pathway analysis of proteins associated with antiviral signalling 24 h post-infection. Proteins shaded grey represent a twofold or greater increase in abundance in the mitochondrial fraction from human respiratory syncytial virus-infected cells compared with mock-infected cells. The colour intensity corresponds to the degree of abundance. Proteins in white are those identified through the Ingenuity Pathways Knowledge Base. The shapes denote the molecular class of the protein. A solid line indicates a direct molecular interaction, and a dashed line indicates an indirect molecular interaction. (b) Diagram of the signalling pathway to which the antiviral components highlighted by the network analysis can be assigned: RIG-I (DDX58) detects viral RNAs in the cytoplasm and induces an antiviral response that involves its association with mitochondrial antiviral signalling protein, triggering a signalling cascade. Certain members of the Tom complex may have an altered function during RNA virus infection, playing an important role in linking mitochondrial antiviral signalling protein to the downstream signalling pathway. Subsequent activation of the signalling complex ultimately results in the early production of IFN-β and subsequent establishment of antiviral state. In addition, mitochondrial antiviral signalling protein signal complex could activate NFκB.

fractions from these time points, calnexin and PMP70 both increased in abundance in the mitochondrial fractions from HRSV-infected cells, compared with mock-infected cells (Figure 4a). The calnexin antibody used in the immunoblot analysis was found to be unsuitable for use in IF; therefore, another ER marker, inositol-requiring enzyme 1 (IRE1), was applied. IF analysis indicated that IRE1 was redistributed in HRSV-infected cells, and increased fluorescence was observed in areas of co-localisation with mitochondria (coloured pink). Viral proteins could also be co-located in discrete areas of the cytoplasm (coloured yellow in Figure 4b, arrowed).

Recruitment of innate antiviral immune response-associated proteins to mitochondria in human respiratory syncytial virus-infected cells IPA-mediated network pathway analysis identified several proteins associated with antiviral signalling, whose abun308

dance was increased in the mitochondrial fraction from HRSV-infected cells (compared with mock-infected) at 24 h p.i. (Figure 5a), which are presented alongside a diagram of the relevant signalling pathway (Figure 5b). IF analysis demonstrated that such components co-localised with mitochondrial marker proteins in HRSV-infected cells and by inference, on mitochondria (Figures 6b–7c). First in the pathway was the RNA detector, RIG-I (also known as DDX58) (refer to Figure 5a and 5b), which increased sixfold in mitochondrial fractions prepared from HRSV-infected cells at 24 h p.i., in comparison to mitochondrial fractions prepared from either mockinfected or cells-infected at 12 h p.i. RIG-I also increased in abundance in whole cell lysate at 24 h p.i. (Figure 6a). IF analysis confirmed that RIG-I co-localised with Tom20 in HRSV-infected cells, but not in mock-infected cells 24 h p.i. and reflected the immunoblot analysis (e.g. compare Figures 2 and 6b). Downstream in the signalling pathway (refer to Figure 5b), the abundance of Tom70 in the mitochondria




(a) Membrane Mitochondrial Cytosolic fraction components fraction M 12 24 M 12 24 M 12 24

Whole cell lysate M 12 24 RIG-I

RNA sensor

Tom70

Import receptor

HSP90

Chaperone

ISG15 OAS3

Type 1 IFN regulated

MX1 (b)

Tom20

RIG-I

Merge

M

10 µm

Tom20

10 µm

RIG-I

10 µm

10 µm

HRSV

Merge

24

10 µm

Tom70

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Figure 6 (a) Immunoblot analysis of proteins identified and quantified by the quantitative proteomics, and selected proteins further analysed by network pathway assignment. Mitochondrial fractions were compared with cytosolic and membrane fractions enriched at 12 h and 24 h in comparison to mock-infected cells (M) and whole cell lysate. Protein localisation/pathway involvement is described on the right. (b) Subcellular localisation of RIG-I, Tom70 and Hsp90 in mock-infected and human respiratory syncytial virus-infected cells 24 h post-infection: RIG-I and Tom70 were shown to increase in abundance in mitochondrial fractions and whole cell lysates from human respiratory syncytial virus-infected cells at 12 h and 24 h in comparison to mock. Here, mitochondria are marked with Tom20 (magenta), while RIG-I is red. Hsp90 was not shown to increase significantly by the proteomic analysis: Here, Tom70 is red and Hsp90 proteins are magenta. Human respiratory syncytial virus proteins are green, nuclei are stained blue with DAPI, a merge is presented, and the scale bar is 10 μM. The colour pink indicates the co-localisation of proteins marked with magenta and red. The colour yellow indicates the co-localisation of proteins marked with green and red. White indicates co-localisation of all proteins.

fraction at 12 and 24 h p.i. was unchanged (based on the twofold change cut-off value) and increased 1.9-fold, respectively (Table S3), and this was reflected in the analysis of the whole cell lysate (Figure 6a). However, there was no

change in the abundance of Tom70’s chaperone, Hsp90, in the whole cell lysate between 12 and 24 h p.i. compared with mock-infected cells. Hsp90 did, however, increase in abundance at 12 h p.i. in the cytosolic fraction and in the


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Figure 7 Subcellular localisation of interferon induced proteins, ISG15, OAS3 and MX1, which were shown to increase in abundance in mitochondrial fractions and whole cell lysates prepared from human respiratory syncytial virus-infected cells at 12 h and 24 h post-infection. Mitochondria are marked with Tom20 (magenta), ISG15, OAS3 and MX1 proteins are red, human respiratory syncytial virus proteins green, and nuclei are stained blue with DAPI. A merge image is presented. Scale bar is 10 μM. The colour pink indicates the co-localisation of proteins marked with magenta and red. The colour yellow indicates the co-localisation of proteins marked with green and red. White indicates co-localisation of all proteins.

mitochondrial fraction at 12 and 24 h (Figure 6a), suggesting an alteration in localisation. These observations were reflected in the IF analysis: Tom70 localised to distinct perinuclear regions and fluorescence intensity (although not quantitative) increased in HRSV-infected cells at 24 h compared with mock (Figure 6b, arrowed). Hsp90 localised to the nucleus in mock-infected cells but re-localised to the cytoplasm and mitochondria in HRSV-infected cells (Figure 6b, arrowed). Tom70, Hsp90 and viral proteins could potentially co-localise at specific sites in HRSVinfected cells (Figure 6b, arrowed). Downstream (refer to Figure 5b), immunoblot analysis confirmed that the following examples of interferon310

induced proteins (refer to Figure 5a) increased in abundance in mitochondrial fractions prepared from HRSVinfected cells at 12 and 24 h p.i and in whole cell lysate: interferon-induced GTP-binding protein Mx1, interferonstimulated gene 15 (ISG15) and 2′5′ oligoadenylate synthase 3 (OAS3) (Figure 5b), whose abundance increased twelve-, seven- and fivefold, respectively, at 24 h p.i. The subcellular localisation of ISG15 (Figure 7a) and OAS3 (Figure 7b) and MX1 (Figure 7c) in mock-infected and HRSV-infected 24 h p.i were confirmed by IF: co-localisation between these IFN-regulated proteins and mitochondria is indicated by the colour pink in Figure 7. OAS3, Tom20 and viral proteins could potentially co-localise in HRSV-infected cells as indicated the white areas (Figure 7b arrowed).

The results described above suggested that mitochondrial import mechanisms were altered in HRSV-infected cells. Proteins involved in antiviral signalling may have been recruited to mitochondria. Taken together with previous studies that have shown that Tom70 and its chaperone Hsp90 have altered recruitment functions in virus-infected cells,[27,30] we hypothesised that Tom70 may function to recruit IFN-regulated proteins to mitochondria. This would be part of the antiviral response or it may play a role in modulating HRSV biology, neither of which has been previously characterised. Hsp90, however, is a ubiquitous molecular chaperone fulfilling diverse roles in both cellular (reviewed in[52]) and viral systems (reviewed in[53]): expression of Hsp90 and other chaperones have been shown to increase in response to HRSV infection,[54] but have also been shown to be incorporated into HRSV virions and implicated in facilitating virus assembly.[55] Chaperones such as Hsp90 or Hsp70 have also been shown to play an important role in replication for a number of negative-strand viruses,[56–58] and Geller et al. recently showed that the long-term presence of the clinically relevant Hsp90 inhibitor, 17-AAG, did not incur resistance in HRSV-infected Hep2 cells.[57] The potential co-localisation of Hsp90 with viral proteins observed in Figure 6b, therefore, agreed with evidence from a previous study that identified Hsp90 in virus particles.[55] We, therefore, confirmed in our cell experimental system (A549 cells) that when the chaperone activity of Hsp90 activity was inhibited during the early stages of HRSV infection (4 h p.i.), virus replication could be affected. RNA interference (RNAi) was used to investigate whether Tom70 mediated an antiviral response in HRSV-infected cells or modulated HRSV biology, while targeted inhibition was used to investigate the possible role of Hsp90 in



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Figure 8 Immunoblot analysis of (a) viral and (b) cellular proteins present in whole cell lysate harvested 21 h post-infection from human respiratory syncytial virus-infected (I) and mock-infected (M) cells which were Tom70 depleted in comparison to non-treated cells and those transfected with a negative control siRNA. The locations of molecular mass markers (kDa) are indicated on the left, and the tentative assignments of viral proteins are indicated on the right. Protein localisation or pathway involvement is described on the right. (c) Plaque assay analysis of virus titre using supernatant harvested from Tom70 depleted cells 21 h post-infection in comparison to non-treated cells and those transfected with a negative control siRNA.

modulating HRSV biology. For Tom70, preliminary experiments indicated that Tom70 could remain depleted over an 18-h time window using siRNAs (data not shown), which was sufficient for infection with HRSV and subsequent comparison of progeny virus production (Figure 8a). Partial ablation of Tom70 did not affect overall abundance of viral proteins by immunoblot analysis (Figure 8a); comparison of progeny virus production, however, indicated that under conditions in which Tom70 depleted (Figure 7b), virus titre consistently increased approximately

twofold (Figure 8c). No change in the abundance of viral proteins or progeny virus production was observed when cells were treated with an siRNA of random sequence (Figure 8). To confirm whether Hsp90 played a role earlier in the virus life cycle prior to assembly,[55] 17-AAG was titrated on HRSV-infected cells 4 h p.i. (when viral mRNA transcription and protein synthesis is initiated[4]), and virus progeny production was assessed 21 h p.i. Hsp90 chaperone activity was monitored via the depletion of its client protein, cdc2, via immunoblot analysis (data not shown). A marked, dose-dependent decrease in progeny virus production in the presence of 17-AAG was observed (Figure 9), confirming that Hsp90 plays a role in HRSV replication as described by Geller et al.,[57] and this has formed the basis of further replication-focused studies.

Discussion To investigate alterations to the mitochondrial proteome in HRSV-infected cells, a combined approach of subcellular fractionation, organelle quantitative proteomics, bioimaging and functional assays were used. This general approach to identify and characterise cellular proteins that are critical for virus biology within a physiologically relevant infection model may open up new avenues of therapeutic intervention.


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Identification of viral proteins in mitochondrial fractions Five virus-encoded proteins (M, N, P, M2-1 and F) were identified in the mitochondrial fraction (Figure 1c and Table 1; the raw dataset is presented in Table S1). Through the use of a polyclonal antibody raised against structural virus proteins only, immunoblot analysis confirmed the presence of viral proteins in mitochondrial fractions from HRSV-infected cells (Figure 1). IF also showed viral proteins in close proximity to Tom70, Hsp90 and RIG-I (Figure 6). HRSV replicates in the cytoplasm and previous studies suggest that the generation of nascent genomic viral RNA occurs in discrete cytoplasmic inclusion bodies that contain the matrix protein and the viral polymerase proteins.[59,60] N and P may form ribonucleoprotein complexes with the genomic RNA in inclusion bodies, possibly with the M protein, then traffic to the apical cell surface where they coordinate with the glycoproteins which, following their translation, are trafficked through the secretory pathway to the apical surface.[61] Many host proteins have been implicated in the process of virus assembly and budding. It is, therefore, very possible that due to organelle remodelling during infection and close proximity of membranes, HRSV viral proteins would be observed in mitochondria-enriched fractions. Recently, a HRSV P and N co-expression study showed that HRSV N protein co-localises with RIG-I by 5 h p.i., and MAVS were also observed within inclusion bodies by 12 h p.i.[35] This study supports the identification of HRSV N in the mitochondrial fraction form HRSV-infected cells by LC-MS/MS and the observed close proximity of HRSV structural proteins with components of the antiviral immune response (Figures 4b, 6b–7c). In our previous analysis of nuclear and cytoplasmic proteomes from HRSV-infected cells, HRSV NS1 was identified in the nuclear and cytoplasmic fractions by LC-MS/MS and was found to be the most abundant viral protein in the cytoplasmic fraction.[5] Tagged NS1 proteins were also recently shown to localise to mitochondria, bind MAVS early in HRSV infection, and disrupt RIG-I– MAVS interactions and the downstream IFN antiviral/ inflammatory response.[62] HRSV possesses two unique non-structural proteins NS1 and NS2, which suppress IFNmediated innate immunity by degrading or inhibiting multiple cellular factors required for either IFN induction or response pathways, such as RIG-I, IRF3, TBK1 and STAT2, and Goswami et al. have recently suggested that to perform this function, the NS proteins assemble a large degradative complex on the mitochondria.[63] However, NS1 and NS2 were not detected in the current LC-MS/MS analysis, perhaps because our analysis was part of an HRSV infection study rather than an overexpression study of viral 312

proteins; perhaps low abundance or protein stability contributed to LC-MS/MS detection. In support of this study, however, no mitochondrial proteins were found to bind NS1 during a recent interactome study.[64] However, another overexpression study suggested that when in complex, NS1 and NS2 localise to the mitochondria.[65] It may, therefore, be necessary for NS1 and NS2 to be in complex during interactions with the mitochondria, or such interactions could be transient and may therefore not be detected during a time point-specific study.

Temporal changes in the mitochondrial proteome during human respiratory syncytial virus infection IPA analysis of the 12 h and 24 h p.i. datasets confirmed that specific pathways were disrupted in HRSV-infected cells in a time-dependent manner. For example, components involved in the activation of the NFκB were decreased in abundance more than twofold at 12 h p.i. Previous studies have shown that the RIG-I–MAVS complex can activate the NFκB signalling pathway during HRSV infection,[31–34] and that differential NFκB activation mechanisms between HRSV subgroups A and B may contribute to the differences in their pathogenesis.[33] At 24 h p.i. those pathways key to mitochondrial dysfunction and the inflammatory response were highlighted, and networks that included components of the innate antiviral response (Table S3 and Figure 5) were further investigated (Figures 6 and 7). The fact that different signalling cascades were triggered over the course of infection emphasises the idea that virus infections and the cellular antiviral activity mounted in response to infection are carefully orchestrated and occur in a temporal manner. The current and previous proteomic[5,11,54,66] and microarray studies[43] highlight the dynamic interplay between HRSV and the host cell, and present a comprehensive picture of the cellular proteome and genome changes that occur over the course of the HRSV replication cycle.

Human respiratory syncytial virus infection promotes mitochondrial associations with other double-membrane structures Proteins normally associated with other membrane-bound organelles were also identified and quantified in the mitochondrial fractions from HRSV-infected cells (Figure 3). For example, immunoblot analysis showed that the abundance of ER marker, calnexin, remained unchanged in the membrane fraction and at the whole cell level but increased in the mitochondrial fraction over the course of infection in comparison to mock (Figure 4a). IF analysis indicated that IRE1 redistributed in HRSV-infected cells and increased fluorescence were observed in areas of



co-localisation with mitochondria (coloured pink in Figure 4b, arrowed). Viral proteins could also be co-located in discrete areas of the cytoplasm (coloured yellow in Figure 4b, arrowed). Such changes only occurred during virus infection, and therefore their presence may represent intracellular alterations that occur in response to HRSV infection (Figure 4). Castanier and Arnoult recently showed that Rigi-like receptor(RLR) activation during SeV infection promoted elongation of the mitochondrial network to facilitate the mitochondria–ER association required for signal transduction.[16] In previous work, we also observed mitochondrial extensions penetrating the nucleus in HRSV-infected cells.[5] It is, therefore, feasible that changes in organelle proximity and associations induced by HRSV infection are responsible for the presence of ER and peroxisome proteins in the mitochondrial fraction. Peroxisomes originate from the ER and freely exchange proteins with mitochondria[67] but interact more specifically with MAVS, which anchor directly to mitochondria or peroxisomes.[13] Association between mitochondria and other membranebound structures may be a common cellular modification in virus-infected cells. For example, a quantitative proteomic analysis of cells infected with human cytomegalovirus demonstrated remodelling of ER–mitochondrial contacts.[18] Recent studies have also demonstrated that mitochondrial activity is modulated by contacts with ER subdomains known as mitochondrial-associated membranes,[68,69] which can be visualised by Electron Microscopy (EM).[70] The physical distance between the OMM and the ER is ∼10 nm, and the distance between the two organelles is larger (∼25 nm) when ribosomes are observed between the juxtaposed membranes.[71] Ribosomal proteins were also identified and quantified in mitochondrial fraction enriched from HRSV-infected cells in comparison to mock in this study (Table S2). Protein tethers, such as mitofusin 2 (Mfn2)[63,72–74] and mitostatin,[75] along with calcium signalling complexes, have also been implicated in stabilising contacts between OMM and ER.[76] A relationship between mitochondrial dynamics and HRSV-associated cell cycle arrest was recently observed,[63] and another recent study also suggested that RSV infection induces the IRE1 (identified in this study) stress pathway. This complex, evolutionarily conserved signalling cascade is known to play a role in apoptosis and inflammation, and is likely triggered by viral protein synthesis (reviewed in[77]) and suggest that IRE1 may inhibit RSV replication.[78] SILAC-based quantitative proteomics has been used to study virus–organelle interactions and alterations in organelle dynamics during infection, including the Golgi apparatus,[79] lipid rafts[80] and the nucleolus.[37,46–48] Certainly, this and previous work[5,10,63] suggest that changes in the mitochondrial proteome and organelle remodelling occur


as a result of HRSV infection. These complex processes will reflect both negative and positive activity, but the underlying mechanisms are currently unknown.

Recruitment of innate antiviral immune response associated proteins to mitochondria in human respiratory syncytial virus-infected cells Many virus-related studies focus on individual virus protein–cellular protein interactions or manipulate a particular signalling pathway to further understand virus–host cell interactions and the antiviral response. To add to such studies, using quantitative proteomics that allowed the study of virus–organelle interactions under physiologically relevant infection conditions and with relative quantification, showed that proteins associated with immune response localise to mitochondria during HRSV infection. In HRSV infection, activation of RIG-I induces an antiviral response[81] that involves its association with the MAVS proteins, allowing the recruitment of signalling adapters to the mitochondrial surface. Subsequent activation of the signalling complex is followed by translocation of NFκB into the nucleus and the activation of associated genes[82] (refer to Figure 5a and 5b), and HRSV induced RIG-I– mitochondria associations that were identified by the LC-MS/MS were confirmed by IF analysis (Figure 6b). The abundance and localisation alterations observed for the identified and quantified antiviral signalling marker proteins focused on those involved in RNA sensing and type 1 IFN-regulated gene products, which increased more than twofold in abundance in the mitochondrial fraction from HRSV-infected cells 24 h p.i. compared with mock-infected cells (Table S3 and Figures 5–7). These IFN-regulated proteins were also shown to co-localise with components of the Tom complex (e.g.Tom20) through indirect IF analysis (Figure 7). OAS3 has been shown to play a critical role in IFN-gamma (IFN-γ) inhibition of HRSV and is one of the first direct antiviral compounds released by virus-infected cells.[83] ISG15 has previously been shown to increase in abundance in HRSV-infected cells:[5,81,84] ISG15 is a ubiquitin-like protein that has been shown to subvert the proteosome-mediated degradation of IRF3 in infected cells[85] via ISGylation.[86] As discussed above, HRSV efficiently suppresses cellular innate immunity using NS1 and NS2, and in a search for their mechanism NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2.[65,87] Several studies have suggested that HRSV may target mitochondria during infection to subvert the innate immune response: NS1 has been detected in the mitochondria[65] and has been shown to co-localise with MAVS protein.[62] Tying this together,


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Goswami et al. suggest the formation of an NS1- and NS2driven multi-protein complex on the mitochondria with the specific function of suppressing innate immunity.[63] We hypothesise that such protein–protein interactions are key in HRSV subversion of the host immune response through mitochondrial-mediated activity, and that pharmacological protection of mitochondria during infection could allow a ‘reset’ of the innate antiviral response during HRSV infection and reduce the burden of disease.

Further examining the roles of Tom70 and Hsp90 during human respiratory syncytial virus infection Alterations in mitochondrial import functions indicated by the presence of IFN-regulated proteins in the mitochondrial proteome 24 h p.i may be related to specific alterations in TOM protein abundance/re-localisation: Tom70 increased 1.87-fold while certain Toms (e.g. Tom20) did not increase more than 1.5-fold in the mitochondrial fraction from HRSV-infected cells at 24 h compared with mock (Table S3). These results were reflected in the immunoblot analysis (Figure 3) and mitochondrial redistribution was shown by IF analysis: Previous analysis has shown the re-localisation of Tom20 to a distinct perinuclear in the HRSV-infected,[5] and in this study Tom70 re-localised in HRSV-infected cells and increased fluorescence was observed in HRSV-infected cells at 24 h compared with mock (Figure 6b). These results may be interpreted as demonstrating alterations in mitochondrial function during HRSV infection and that changes in protein abundance were specific to a subset of cellular proteins rather than overall generic changes, indicating defined activity. RNAi was used to deplete Tom70 and assess whether this protein mediated an antiviral response in HRSV-infected cells or modulated HRSV biology. Under conditions in which Tom70 was partially knocked down, viral progeny production was reproducibly increased approximately twofold, while no obvious change in viral protein abundance was observed by immunoblot analysis (Figure 8). Given that Tom70 has been shown to promote host antiviral responses during infections with other Mononegavirales[27] and from the results presented here, we hypothesise that this mitochondrial protein may also act in an antiviral manner during HRSV infection and perhaps be involved in recruiting components of the antiviral response to mitochondria in HRSV-infected cells. Immunoblot analysis of whole cell lysate in Tom70 depleted, HRSV-infected cells, however, showed that the abundance of its chaperone, Hsp90, and examples of IFN-regulated proteins, were not decreased in abundance 21 h p.i. (Figure 8b), and that further characterisation is required. 314

In contrast to Tom70, Hsp90 is a ubiquitous molecular chaperone fulfilling diverse roles in both cellular[52] and viral systems,[53] and further investigation of the proteomic analyses involved targeted inhibition of Hsp90’s chaperone function with a clinically relevant Hsp90 inhibitor, 17-AAG. Many chaperone client proteins are involved in cellular processes, such as transcription, translation protein metabolism[88] and mitochondrial function,[29] making them vital to cellular protein homeostasis.[89] Many chaperones are heat shock proteins as they tend to aggregate when non-functional proteins are increasingly denatured following cellular stress response activation which functions to maintain cellular balance.[90] Expression of Hsp90 and other chaperones have been shown to increase over the course of HRSV infection:[54] in the context of the host cell response to virus infection, Hsp90 has been implicated as an important modulator of the signal transduction pathways and transcription factors, such as NFκB,[91] STAT[92] and IRF3[30], which cumulate in the induction of antiviral state.[93] In the context of the viral life cycle and the structural and functional complexity of many viral proteins, it is not surprising that, like cellular proteins, many viruses are also dependent on chaperones.[53] A previous proteomics study of purified virus particles carried out by Radhakrishnan et al. identified Hsp90 in the virus preparation. 17-AAG was subsequently applied 8 h p.i., and although addition at this time point during infection did not alter virus protein expression, inhibition of virus particle formation and impaired virus transmission were observed in Hep2 cells.[55] In this study, however, HRSV titres were shown to decrease from infected A549 cells when they were treated with 17-AAG earlier during infection, at 4 h p.i. Such observations have added to the body of evidence that chaperones, such as Hsp90 or Hsp70, play an important role in the replication for a number of negative-strand viruses.[56–58] In addition, they strengthen the concept of repurposing specific Hsp90 inhibitors, such as 17-AAG and 17-DMAG, which are currently undergoing clinical trials for use in cancer treatment,[94–96] for use in a generic antiviral therapeutic strategy: Geller et al. recently showed that 17-AAG and 17-DMAG exhibited antiviral activity against laboratory and clinical isolates of RSV, and that continuous passage of the virus through Hep2 cells in the presence of 17-AAG did not select for resistant viruses.[57] In this study, we confirmed in our cell culture system that 17-AAG decreased virus titre in a dose-dependent manner when added p.i. but prior to virus release. In agreement with other studies,[53,57] we postulate that Hsp90 and other chaperone proteins may be required for maintaining the correct folding and functionality of viral proteins that are involved in replication, and this aspect of the study is being investigated further.




Conclusions

Declarations

Alterations to the mitochondrial proteome of HRSVinfected cells are protein and pathway-specific, and may also be temporally and spatially regulated during infection. The proteomic analysis quantitatively demonstrated that proteins involved in the innate antiviral immune response converge on mitochondria during HRSV infection. This is likely to be a cellular response to HRSV infection involving the coordination of both organelle orientation and signalling cascade induction. The gene knockdown and targeted inhibition studies suggest that Tom70 may act in an antiviral manner in HRSV-infected cells, whereas Hsp90 was confirmed to be a positive viral factor. The role of chaperones in HRSV infection and their potential as antivirals is currently being investigated with the view to developing a chemotherapy that targets a specific virus–host cell protein interaction.

Conflict of interest

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The Author(s) declare(s) that they have no conflicts of interest to disclose.

Funding This research was supported by an MRC training Account PhD studentship awarded to JNB and JAH to support DCM, and the award of a Leverhulme Trust Research Fellow to JAH.

Acknowledgements Dr Patricia cane at Public Health England is thanked for providing the RSV A2 strain used in this study.

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Table S1 Viral proteins identified in the mitochandrial fractions (raw dataset). Table S2 All cellular proteins identified in the mitochandrial fractions (raw dataset). Table S3 All cellular proteins identified and qualified by two or more peptides plus.

Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:


A Cross-Study Biomarker Signature of Human Bronchial Epithelial Cells Infected with Respiratory Syncytial Virus.

Human antibody-dependent cell-mediated cytotoxicity against target cells infected with respiratory syncytial virus.

Differential mucin expression by respiratory syncytial virus and human metapneumovirus infection in human epithelial cells.

Functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus.

CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells.

Leptin Is Oversecreted by Respiratory Syncytial Virus-Infected Bronchial Epithelial Cells and Regulates Th2 and Th17 Cell Differentiation.

Respiratory syncytial virus infections.

Respiratory syncytial virus can infect basal cells and alter human airway epithelial differentiation.

Respiratory syncytial virus in nurseries.

Human cathelicidin, LL-37, inhibits respiratory syncytial virus infection in polarized airway epithelial cells.

Polypeptides of respiratory syncytial virus.

Nosocomial respiratory syncytial virus infections.

Neonatal respiratory-syncytial-virus infection.

Non-typeable Haemophilus influenzae protects human airway epithelial cells from a subsequent respiratory syncytial virus challenge.

Respiratory syncytial virus, 1979: Scotland.

Neonatal respiratory syncytial virus infection.

Preventing hospitalizations for respiratory syncytial virus infection.

Nosocomial infection with respiratory syncytial virus.

Respiratory syncytial virus and reactive airway disease.

Ribavirin aerosols and respiratory syncytial virus infection.

Mechanical ventilation and respiratory syncytial virus infection.

Respiratory syncytial virus infection and bronchiolitis.

A novel host factor for human respiratory syncytial virus.

Recovery of infectious proviral DNA from mammalian cells infected with respiratory syncytial virus.