HeLa cell response proteome alterations induced by mammalian reovirus T3D infection
© Coombs; licensee BioMed Central Ltd. 2013
Received: 19 February 2013
Accepted: 14 June 2013
Published: 21 June 2013
Cells are exposed to multiple stressors that induce significant alterations in signaling pathways and in the cellular state. As obligate parasites, all viruses require host cell material and machinery for replication. Virus infection is a major stressor leading to numerous induced modifications. Previous gene array studies have measured infected cellular transcriptomes. More recently, mass spectrometry-based quantitative and comparative assays have been used to complement such studies by examining virus-induced alterations in the cellular proteome.
We used SILAC (stable isotope labeling with amino acids in cell culture), a non-biased quantitative proteomic labeling technique, combined with 2-D HPLC/mass spectrometry and reciprocal labeling to identify and measure relative quantitative differences in HeLa cell proteins in purified cytosolic and nuclear fractions after reovirus serotype 3 Dearing infection. Protein regulation was determined by z-score analysis of each protein’s label distribution.
A total of 2856 cellular proteins were identified in cytosolic fractions by 2 or more peptides at >99% confidence and 884 proteins were identified in nuclear fractions. Gene ontology analyses indicated up-regulated host proteins were associated with defense responses, immune responses, macromolecular binding, regulation of immune effector processes, and responses to virus, whereas down-regulated proteins were involved in cell death, macromolecular catabolic processes, and tissue development.
These analyses identified numerous host proteins significantly affected by reovirus T3D infection. These proteins map to numerous inflammatory and innate immune pathways, and provide the starting point for more detailed kinetic studies and delineation of virus-modulated host signaling pathways.
KeywordsRNA virus Virus infection Host cell alterations Proteomics Mass spectrometry Liquid chromatography Bioinformatics
The cellular proteome (the total protein repertoire, which includes how each protein may be co-translationally or post-translationally modified) is affected by numerous stresses, including infection by viruses. Numerous previous microarray studies have determined how cellular transcriptomes respond after virus infection (see for example: [1, 2]). However, since mRNA levels cannot provide complete information about levels of protein synthesis or the types and degrees of post-translational modifications, there frequently is little concordance between microarray and protein data [3–5]. Therefore, quantitative and comparative proteomic analyses are also being used to provide additional information about host alterations to virus infection (reviewed in: [6, 7]). Commonly used methods include 2-dimensional difference in gel electrophoresis (2D-DIGE (see for examples: [8, 9]), isotope coded affinity tags (ICAT; [10, 11]), isobaric tags for relative and absolute quantitation (iTRAQ; [12, 13]), and stable isotope labeling by amino acids in cell culture (SILAC; [14–18]). The SILAC method involves labeling cells with “light” (normal; L) and “heavy” (H) isotopic forms of amino acids. Advantages of this particular technique include: experimental set-up is relatively simple, L and H samples are mixed together early in this process, thereby reducing sample-to-sample variability, and, if 12C6-Lys and 12C614N4-Arg (L), and 13C6-Lys and 13C615N4-Arg (H) amino acids are used, virtually every tryptic peptide should contain a labeled amino acid, thereby providing increased protein coverage. Indeed, several early comparative studies suggested SILAC provided more identifications than the other commonly used methods (reviewed in ). We have been using SILAC to measure comparative proteomic alterations induced by influenza virus in A549  and in normal human bronchial epithelial (NHBE) airway cells . We have also begun similar analyses with reovirus-infected cells, focusing initially upon reovirus serotype T1L-infected HEK-293 cells .
The mammalian reoviruses (MRV) are non-enveloped viruses with a genome that consists of 10 segments of double-stranded (ds)RNA. The dsRNA genome is enclosed in a double-layered concentric protein capsid composed of 8 viral structural proteins. For reviews, see [22–24]. MRV is the prototype member of the Orthoreovirus genus in the family Reoviridae. The Ortheoreoviruses include nonfusogenic MRV and fusogenic avian reovirus. The Reoviridae family also contains rotaviruses , orbiviruses , and at least 9 other genera, several of which can infect animals, insects and/or plants [23, 24]. MRV infections are generally mild in humans but many of the other family members are highly pathogenic in their hosts. MRV currently consist of 4 identified serotypes, with each represented by a prototype strain: strain Lang (T1L) for serotype 1; strain Jones (T2J) for serotype 2, strain Dearing (T3D) for serotype 3 and strain Ndelle for serotype 4 . The reoviruses have long served as models for understanding viral pathogenesis  and they have also been identified as potential oncolytic agents [28–30] because of their capacity to selectively kill cancer cells that contain activated Ras pathway and functional p53 [28, 31].
MRV are capable of infecting a wide range of cells, including mouse L929 cells, often used for stock preparation and titration , and various human cells, including HEK-293 . Numerous reovirus studies have also been performed in HeLa cells (see for examples: [33–36]). Global microarray analyses of MRV-infected cellular transcriptomes detected activation of numerous cellular genes, including many related to apoptosis [37–41]. These microarray assays have recently been complemented by quantitative and comparative proteomic analyses. For example, Li and colleagues recently demonstrated, using 2D-DIGE, that MRV-infected murine myocytes regulate several proteins, including heat shock proteins and interferon-response proteins . We have also shown, using SILAC and 2D-HPLC/MS, that proteins involved in cell death, cell growth and proliferation, molecular transport, gene expression, and inflammatory response pathways are affected in MRV T1L-infected HEK-293 cells . Similar pathways were also found regulated in a preliminary analysis of reovirus T3D-infected HeLa cells that concluded that inclusion of Proteominer® bead-based non-biased enrichment did not significantly improve proteomic coverage of unpurified cell extracts .
Thus, as part of an ongoing systematic delineation of reovirus-induced comparative host protein responses, we are examining how reoviruses T1L and T3D, two of the most commonly used MRV strains, affect various permissive cells. We have extended our previous T1L-infected HEK-293 cell study  to T3D-infected HEK-293 cells (Berard, in preparation). Our previous proteomic analysis of HeLa cells infected with T3D concluded that inclusion of Proteominer® bead-based non-biased enrichment did not significantly improve proteomic coverage of unpurified cell extracts . Therefore, the current study extends these previous studies by examining proteomic alterations in purified cytosolic and nuclear fractions in order to globally assess sub-cellular protein distribution. Reciprocal labeling also was incorporated to identify probable contaminants which were removed from the analyses. We also extended previous protein identification by more detailed follow-up kinetic studies of some selected important proteins. This study identified and measured 2856 cytosolic proteins by 2 or more peptides at >99% confidence and 884 nuclear proteins. DAVID™ and IPA™ ontological analyses identified significantly up- and down-regulated proteins as well as significantly affected canonical pathways.
Materials and methods
Cells and viruses
Cell lines and media
Mouse L929 fibroblast cells (L929) were grown in suspension in Joklik’s modified minimal essential medium (J-MEM) (Gibco, Grand Island, NY) supplemented to contain 5% fetal bovine serum (FBS) (Invitrogen Canada Inc., Burlington, Ontario), and 2 mM L-glutamine as described . Cells were sub-cultured daily.
Human HeLa cells were cultured as monolayers in Dulbecco’s modified MEM (D-MEM) supplemented with 0.2% (w/v) glucose, 10% FBS (Invitrogen), 2 mM l-glutamine, non-essential amino acids, and sodium pyruvate. Cells were sub-cultured 2 – 3 times each week.
Reovirus strain Type 3 Dearing (T3D) is a laboratory stock. There are two commonly-used clones of T3D; the Cashdollar strain (T3DC) and the Fields strain (T3DF). We elected to use the T3DF strain because a large number of genetic reagents, including temperature-sensitive mutants (reviewed in [44, 45]) and T1L/T3DF intertypic reassortants [46–48] were generated from this clone. Virus stocks were usually grown in L929 cell monolayers in J-MEM in the presence of 5% CO2 at 37°C as above, but with 3% FBS, 100 U/ml of penicillin, 100 μg/ml streptomycin sulfate and 100 μg/ml amphotericin-B as previously described .
Large quantities of reovirus T3D were grown in suspension L929 cell cultures and purified by routine procedures that make use of Vertrel-XF™ extraction and cesium chloride ultracentrifugation . Purified virions were harvested and dialyzed against D-Buffer (150 mM NaCl, 15 mM MgCl2, 10 mM Tris, pH 7.4). Virus concentration was measured by optical density at 260 nm, using the relationship 1 ODU = 2.1 × 1012 particles per milliliter  and infectivity was titrated.
Sets of HeLa cells were infected with gradient-purified T3D at a multiplicity of infection (MOI) of 7 PFU per cell. For routine infections not destined for SILAC analysis (i.e. for photomicrography, virus growth kinetic determinations, or Western blot analyses – see below), cells were harvested at various time points (0 – 72 hours post infection; hpi), then fractionated as described below.
For SILAC labeling, HeLa cells were adapted through 3 passages (=6 doublings) into D-MEM media provided in a SILAC™ Phosphoprotein Identification and Quantification Kit (Invitrogen Canada Inc.; Burlington, Ontario), supplemented as above (except without non-essential amino acids), and with 10% dialyzed FBS (Invitrogen), plus 100mg each of normal (L; 12C6-lysine and 12C6-/14N4-arginine) or “heavy” (H; 13C6-lysine [6.0Da difference] and 13C6-/15N4-arginine [10.0Da] difference) per liter of D-MEM. Once HeLa cells had grown through six doublings in appropriate SILAC media, sets of cells were infected with gradient-purified T3D or were mock infected with diluent. Cells were overlaid with appropriate SILAC media and cultured for 24 hours. In one experiment (Run #1) the L cells were infected and the H cells were mock infected, whereas H cells were infected and L cells were mock infected in the reciprocal labeling experiment (Run #2).
HeLa cells were allowed to attach to 6-well culture dishes or onto sterile glass cover slips in 6-well culture dishes and incubated overnight at 37°C. Cells, at approximately 70% confluency, were washed twice with 1X PBS and T3D was added to each culture at an MOI of 7. Virus was adsorbed to cells on ice for one hour to ensure infection synchronization. Mock-infected cells received only diluent. Cells were overlaid with complete pre-warmed media and cells were then incubated for various periods of time from 0 to 72 hours at 37°C.
Infections were monitored at various times and aliquots taken for cell viability determinations, using trypan blue and ensuring > 200 cells were counted at each time point, and for virus titrations. Cell monolayers were also examined with a Nikon TE-2000 and cells were photographed with a Canon-A700 digital camera. Images were imported into Adobe and slight adjustments made in brightness and contrast, but which did not alter image context with respect to each other.
Infected and mock-infected cells were harvested at various times post-infection and counted. Aliquots of each culture were saved for virus titration to verify infection status. Non-SILAC-labeled cells were individually processed. For comparative SILAC assays, equivalent numbers of L and H cells in each experimental run were confirmed to contain equivalent amounts of protein by BCA™ Protein Assay (Pierce; Rockford, IL) and were mixed together 1:1. Harvested cells were washed 3× in >50 volumes of ice-cold Phosphate Buffered Saline (PBS). Washed cells were resuspended in 250 μl of ice-cold PBS supplemented with 1.5× complete™ Protease Inhibitor (Pierce) and lysed by the addition of 1/10th volume of 5% NP-40. Cells were incubated for 30 min with periodic mixing then centrifuged at 500×g for 10 min to pellet nuclei. The supernatants (cytosol and soluble membranes) were transferred to fresh microfuge tubes and the nuclear pellets were resuspended in 250 μl of PBS supplemented with 1× complete™ Protease Inhibitor + 10% sucrose + 0.44% NP-40 and nuclei re-pelleted, with the 2nd supernatants added to the first ones. The nuclei were then washed 4 times with 1ml of PBS + 0.25× complete™ Protease Inhibitor + 10% sucrose. Washed nuclei were extracted by a new 2-step MS-compatible procedure . Briefly, nuclei were resuspended in 150 μl of High Salt Extraction Buffer (620 mM NaCl, 1 mM DTT, 1 mM MgSO4, 10 mM HEPES, pH 8.0), freeze-thawed, sonicated, insoluble material pelleted at 17,000×g for 10 min, and the supernatants transferred to fresh microfuge tubes. The insoluble pellets were resuspended in 50 μl of 8 M urea, freeze-thawed, sonicated, insoluble material pelleted at 17,000×g for 10 min, and the 2nd supernatants combined with the first.
Protein content in each fraction was determined by BCA Protein Assay (Pierce) and bovine serum albumin standards. The cytosolic and nuclear fractions were stored at -80°C until further processing took place.
Equivalent cytosolic and nuclear fractions were resolved by either 10% linear mini sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, (8.0 × 6.5 × 0.1 cm)) at 180 V for 60 min. or in 4-16% gradient SDS-PAGE (8.0 × 16.0 × 0.1 cm) at 20 mA per gel for 5.5 hours (or overnight for a cumulative total of 110 mAhr per gel). Proteins were transferred to 0.2 μm polyvinylidene difluoride (PVDF) membranes at 20 V for 40 min with a Semi-dry apparatus (BioRad), and protein transfer was confirmed by Ponceau-S staining. The membranes were blocked with 5% (w/v) skim milk in Tris-buffered saline with Tween-20 (TBST; 50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4) and probed with various primary antibodies. Primary antibodies were: in-house produced rabbit anti-reovirus, α- GAPDH (Cell Signaling, cat#2118), α-Mx1 (Ori-Gene # TA308496), α-PARP (Cell Signaling, cat#9541), α-ISG15 (Rockland, cat#200-401-438), α-IFIT (Abcam, cat#ab55837), and α-SAMD9 (Sigma, cat#HPA021318); and mouse α-Actin (Sigma, cat#A5441) and anti-STAT1 (Cell Signaling, cat#9176). Appropriate secondary horseradish peroxidase (HRP)-conjugated rabbit anti-mouse or goat anti-rabbit (Cell Signaling, cat#7076, cat#7074, respectively) were used to detect immune complexes. Bands were developed by enhanced chemiluminescence and imaged with an Alpha Innotech FluorChemQ MultiImage III instrument.
Comparative SILAC analyses
Protein digestion and peptide fractionation
After protein concentration determinations, SILAC-labeled samples were diluted with freshly made 100 mM ammonium bicarbonate to concentrations of ~1 mg/ml and pH ~8. Three hundred μl of each sample (~300 μg of protein) were reduced with dithiothreitol (DTT), alkylated with iodoacetic acid, quenched with additional DTT, and trypsin-digested overnight at 37°C with 6 μg of sequencing grade trypsin (Promega, Madison, WI) as previously described . Digests were dried.
Tryptic peptides were fractionated by an orthogonal 2-dimensional reverse-phase (RP) high pH – RP low pH procedure [50, 51]. Lyophilized tryptic digests were dissolved in 200 μl of 20 mM ammonium formate pH 10 (Buffer A), injected onto a 1×100 mm XTerra (Waters, Milford, MA) column and fractionated with a 0.67% acetonitrile per minute linear gradient (Agilent 1100 Series HPLC system, Agilent Technologies, Wilmington, DE) at a flow rate of 150 μL/min. Sixty 1-min fractions were collected (covering ~40% acetonitrile concentration range) and concatenated [51, 52], with the last 30 fractions combined with the first 30 fractions in sequential order (i.e. #1 with #31; #2 with #32, etc.). Combined fractions were vacuum-dried and re-dissolved in buffer A for the second dimension RP separation (0.1% formic acid in water).
A splitless nano-flow Tempo LC system (Eksigent, Dublin, CA) with 20 μL sample injection via a 300 μm×5 mm PepMap100 pre-column (Dionex, Sunnyvale, CA) and a 100 μm×200 mm analytical column packed with 5 μm Luna C18(2) (Phenomenex, Torrance, CA) were used in the second dimension separation prior to MS analysis. Both eluents A (water) and B (acetonitrile) contained 0.1% formic acid as an ion-pairing modifier. A 0.33% acetonitrile per minute linear gradient (0-30% B) was used for peptide elution, providing a total 2-hour run time for each of the 30 concatenated samples.
Mass spectrometry, bioinformatics, and data mining
A QStar Elite mass spectrometer (Applied Biosystems, Foster City, CA) was used in a data-dependent MS/MS acquisition mode. One-second survey MS spectra were collected (m/z 400-1500) followed by MS/MS measurements on the 3 most intense parent ions (80 counts/sec threshold, +2 - +4 charge state, m/z 100-1500 mass range for MS/MS), using the manufacturer’s “smart exit” (spectral quality 5) settings. Previously targeted parent ions were excluded from repetitive MS/MS acquisition for 60 sec (50 mDa mass tolerance) and the bias correction option was used to correct for small pipetting errors. Raw data files (30 for each of the 4 experimental run samples) were submitted for simultaneous search using standard SILAC settings for QStar instruments and were analyzed by Protein Pilot®, version 4.0, using the non-redundant human gene database (NCBInr, released March 2011, downloaded from http://ftp.ncbi.nih.govrefseqH_sapiensmRNA_Prot, containing 37,391 entries). Proteins, their confidences, and their expression ratios, expressed as infected: mock (I:M), were returned with gi accession numbers. Only proteins for which at least 2 fully trypsin digested L and H peptides were detected at >99% confidence were used for subsequent comparative quantitative analyses. The false discovery rate (FDR), defined as the percentage of reverse proteins identified against the total protein identification, was determined to be < 0.8%.
where “b” represents an individual protein in a dataset population a….n, and z-score is the measure of how many standard deviation units (expressed as “σ”) that protein’s log2 I:M ratio is away from its population mean. Thus, a protein with a z-score > 1.960σ indicates that protein’s differential expression lies outside the 95% confidence level, > 2.576σ indicates outside the 99% confidence level, and 3.291σ indicates 99.9% confidence. Z-scores >1.960 were considered significant. Proteins that obtained significant positive z-scores in one labeling experiment, but that also obtained significant negative z-scores in the reciprocal labeling experiment were assumed to be contaminants, were removed from analysis, and z-scores iteratively re-calculated. Gi numbers of all significantly regulated proteins were converted into HGNC identifiers by Uniprot (http://www.uniprot.org/) and HGNC terms were submitted to and analyzed by STRING [54, 55] and by the DAVID bioinformatic suite at the NIAID, version 6.7 [56, 57] and gene ontologies examined with the “FAT” datasets. The gi numbers were also submitted to, and pathways constructed with, Ingenuity Pathway Analysis software (IPA™).
Results and discussion
T3D replicates in HeLa cells
Identification of altered host proteins
Number of peptides, proteins, log 2 Infected: Mock (I:M) ratio means and standard deviation, and Z-scores of SILAC-measured HeLa cell proteins after T3D infection
Total number of peptide pairs1
Total number of proteins2
Number of proteins analysed3
Mean Log2 I:M ratios
Standard deviation of Log2 I:M ratios
Number of proteins at Z-score cutoff of: ± 1.960σ (95%)
± 2.576σ (99%)
± 3.291σ (99.9%)
Each protein’s infected-to-mock (I:M) H:L or L:H ratio was converted into a z-score to normalize the data and facilitate comparisons of each dataset as described in Materials and Methods (and in ). A number of proteins were found to have significantly high or low log2 values and corresponding z-scores in either the H:L or L:H labeling scheme, but z-scores that were significantly regulated in the opposite direction in the reciprocal labeling experiment. These included hornerin, keratins, some S100 calcium-binding proteins, and some other proteins (Additional file 2: Table S1; rows 3111-3243). These significant reciprocal values could arise if, for example, exogenous unlabeled (= L-labeled) proteins were introduced during processing. Thus, such proteins were assumed to represent possible contaminants and were removed from further analysis. Z-scores were iteratively recalculated until all probable contaminants had been removed.
Significantly-regulated HeLa cell proteins after T3D infection
Run # 1 (H:L)
Run # 2 (L:H)
Inf / Mock
Measured more than once
myxovirus resistance protein 1
coiled-coil domain containing 56
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide RIG-I
interferon-induced protein with tetratricopeptide repeats 3
ISG15 ubiquitin-like modifier
interferon-induced protein with tetratricopeptide repeats 1 isoform 2
PPAR-alpha interacting complex protein 285 isoform 1
interferon-induced protein with tetratricopeptide repeats 2
signal transducer and activator of transcription 1 isoform alpha
2'-5'oligoadenylate synthetase 3
quinoid dihydropteridine reductase
sterile alpha motif domain containing 9
eukaryotic translation initiation factor 2-alpha kinase 2
interferon-induced protein 35
guanylate binding protein 1, interferon-inducible, 67 kDa
galectin 3 binding protein
zinc finger antiviral protein isoform 1
ras homolog gene family, member T2
bone marrow stromal cell antigen 2
polyribonucleotide nucleotidyltransferase 1
transcription factor B1, mitochondrial
apolipoprotein A-I preproprotein
myxovirus resistance protein 2
complement component 9 precursor
interferon-induced protein 44-like
2',5'-oligoadenylate synthetase 1 isoform 3
2'-5'-oligoadenylate synthetase 2 isoform 1
epsin 1 isoform c
Yes-associated protein 1, 65 kD
DEAD (Asp-Glu-Ala-Asp) box polypeptide 60
tripartite motif protein 21
hect domain and RLD 5
C-terminal binding protein 2 isoform 1
proteasome beta 10 subunit proprotein
transporter 2, ATP-binding cassette, sub-family B isoform 1
SAM domain- and HD domain-containing protein 1
low density lipoprotein-related protein 1
endothelial cell growth factor 1 (platelet-derived) precursor
interferon-induced protein with tetratricopeptide repeats 5
interferon, gamma-inducible protein 16
unc-93 homolog B1
transporter 1, ATP-binding cassette, sub-family B
major histocompatibility complex, class I, C precursor
Measured more than once
solute carrier family 4, sodium bicarbonate cotransporter, member 7
PTPRF interacting protein binding protein 1 isoform 1
RAS p21 protein activator 1 isoform 1
ADP-ribosylation-like factor 6 interacting protein 5
cell division cycle protein 27 isoform 1
anti-silencing function 1B
phospholipase C gamma 1 isoform a
interferon regulatory factor 2 binding protein 2 isoform B
aldo-keto reductase family 1, member C1
hect domain and RLD 4 isoform a
DNA directed RNA polymerase II polypeptide E
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6, 14 kDa
retinol dehydrogenase 14 (all-trans and 9-cis)
hypothetical protein LOC79169
ubiquitin-like with PHD and ring finger domains 1 isoform 1
adaptor-related protein complex 1, gamma 1 subunit isoform a
Measured more than once
ISG15 ubiquitin-like modifier
nuclear antigen Sp100 isoform 2
interferon, gamma-inducible protein 16
promyelocytic leukemia protein isoform 1
bone marrow stromal cell antigen 2
defender against cell death 1
N-myc and STAT interactor
interferon-induced protein 35
DnaJ (Hsp40) homolog, subfamily B, member 1
desmoplakin isoform I
zinc finger antiviral protein isoform 1
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide
Sm protein F
protein phosphatase 1, catalytic subunit, alpha isoform 1
Measured more than once
G protein-binding protein CRFG
endothelial differentiation-related factor 1 isoform alpha
thymopoietin isoform alpha
proteasome 26S non-ATPase subunit 7
programmed cell death 8 isoform 1
T-complex protein 1 isoform a
ribosomal protein S23
tetratricopeptide repeat domain 11
HeLa cell proteins up-regulated by T3D infection are associated with defense responses, immune responses, macromolecular binding, regulation of immune effector processes, and responses to virus
Up-regulated proteins were assigned to 42 GOTERM biological processes at 95% confidence (Figure 3, upper), that included responses to viruses, immune responses, defense responses, regulation of immune effector processes and numerous related processes. Up-regulated proteins were also assigned to 23 functional groups (Figure 3, middle) including primarily macromolecular binding and transcription cofactor and nucleotidyltransferase activities.
HeLa cell proteins down-regulated by T3D infection are associated with cell death, macromolecular catabolic processes, and tissue development
DAVID assigned down-regulated proteins to 18 biological processes at 95% confidence (Figure 3, lower), that included ubiquitination regulation, cell adhesion regulation, macromolecular complex assembly and catabolic processes. IPA indicated down-regulated proteins were enriched in translation regulation and transporter factors (Figure 4A). Many down-regulated proteins also are related to mitochondrial dysfunction (Additional file 3: Figure S2). Many other proteins, such as EIF4G3, FERMT1, CDC27 and COIL were mapped to the cytoplasmic fractions (Figure 4E, F).
An apparent down-regulation in protein levels in either of the sub-cellular fractions examined could represent reduced protein biosynthesis, protein degradation, redistribution of proteins from one fraction to another, or various combinations of the above. Protein redistribution could be manifested by the same protein apparently being up-regulated in one compartment at the same time it appeared down-regulated in the other compartment. While there were numerous proteins that were detected as either up-regulated or down-regulated in one compartment and unaffected in the other compartment (i.e. IFIT3, MX2, PML, RASA1, XPO6) there were very few proteins detected that were up-regulated in one compartment and simultaneously down-regulated in the other. For example coilin (COIL) was found up-regulated about 2.5-fold in purified nuclear fractions, based upon 2 peptides; however, it appeared down-regulated by about 2-fold, but only based upon a single peptide (Additional file 2: Table S1). This molecule is an integral component of nuclear suborganelles called Cajal bodies that play roles in small RNA post-transcriptional modifications [68, 69]. More detailed analyses of fates of various proteins, with regards to the possible altered biosynthesis, degradation and redistribution as a result of virus infection, are warranted.
Similarities and differences between HeLa cell and HEK293 cell protein responses to reovirus infection
Transcriptomic responses to T3D infection have been previously reported . That study identified more than 100 interferon- and NF-κB-responsive genes that were either positively or negatively regulated by T3D infection. We identified and measured 30 of these genes’ proteins and assessed correlation between the mRNA and protein levels. There was good correlation for some genes and proteins. For example, OAS1 and PML mRNA and protein levels were highly up-regulated, 9 other proteins we identified as up-regulated also had up-regulated mRNA levels, although the degree of up-regulation differed, and some proteins and genes (i.e. UBC and POLD1) were similarly non-regulated or only slightly down-regulated. Analysis of the 30 proteins after assigning proteins and genes to highly-up, slightly-up, non-regulated, slightly-down and highly-down regulated classes resulted in an r2 correlation of 0.62 (data not shown), slightly higher than what has been found in other studies that correlate mRNA levels to protein levels [3, 4]. Unfortunately, most mRNAs and proteins could not be compared because the other ~70 interferon- and NF-κB-responsive genes reported by O’Donnell et al.  were not found by us.
We previously determined HEK293 responses to reovirus T1L infection . That study identified and measured 2992 proteins at 24 hpi, 104 of which were up-regulated and 49 of which were down-regulated. Only 194 (~ 6.5%) of these same proteins were identified in the current study (Additional file 1: Figure S3). A small number of proteins were similarly regulated by both viruses in both cell types. Most proteins (159-187, depending upon how cutoffs were set) were non-regulated in both cell types, and no proteins were up-regulated in one cell but down-regulated in the other (Additional file 1: Figure S3C). Although there was only ~6.5% overlap between the 2 protein datasets, which could represent differences in the virus used and/or in the type of cell analyzed, many of the highly regulated pathways and processes were similar between the two experimental conditions. For example, regulation of interferon signaling, immunomodulation and responses to virus were highly up-regulated in both studies. Only 5 of the 194 host proteins identified and measured in both the T1L-infected HEK-293 and T3D-infected HeLa cells were significantly regulated. Of these, only 1 (STAT1), discussed in more detail earlier, was significantly regulated by both cell/virus conditions, being up-regulated 2.1-fold in T1L-infected HEK293 cells and up-regulated 3.2-fold in T3D-infected HeLa cells. One other protein (SCO1) was up-regulated 8.4-fold in T1L-infected HEK-293 cells but only moderately up-regulated (1.4-fold, Z-score = 1.088σ) in T3D-infected HeLa cells. SCO1 is a metallochaperone involved in copper homeostasis and found in the mitochondrial intermembrane space . The SCO genes appear evolutionarily conserved  and have not yet been reported to have roles in virus replication. Two proteins were significantly up-regulated in one cell type but not regulated in the other. PRKCDBP, a potential tumor suppressor gene , and COMT, a degradative methyltransferase  that has been associated with cognitive deficits in herpes simplex type 1 virus infections , were up-regulated 2.3-, and 3.1-fold, respectively, in T1L-infected HEK-293 cells. However, the COMT ratio is based upon a single measured peptide so less reliable. One protein was significantly down-regulated in one cell type but not regulated in the other. PLCG1, involved in immune regulated signal transduction , was down-regulated 3-fold (I:M ratio of 0.33) in T3D-infected HeLa cells. Twenty six other proteins were moderately regulated (Z score 1.000 – 1.959σ, corresponding to ~ 0.9- – 2.2-fold up-regulation; or -1.000 – -1.959σ, corresponding to ~ 0.9- – 1.6-fold down-regulation [I:M ratios of 0.63 – 1.13) (Additional file 3: Table S2). Although measured values were not significant, one additional protein (C2orf43), a gene that has been associated with prostate cancer , was moderately down-regulated in both the T1L and T3D infections, with infected:mock values of .66 and 0.61, respectively. A few of these determined values are based upon 1 or 2 peptides. Therefore, additional work is needed to determine whether these apparent similarities and differences are real and virus- and/or cell-specific. We are currently analyzing T1L- and T3D-infected HeLa cells labeled by the iTRAQ reagent to simultaneously compare multiple virus types to mock and to use a complementary approach that may identify additional regulated proteins.
In summary, this non-biased global analysis has identified numerous host proteins significantly affected by reovirus T3D infection. These proteins complement others determined in other studies, fit within numerous inflammatory and innate immune pathways, and will provide the starting point for more detailed kinetic studies, such as initiated herein, as well as studies aimed at delineation of virus-specific pathways.
This work was supported by grant MT-11630 from the Canadian Institutes of Health Research to KMC and is MIAPE-compliant. The author thanks Kolawole Opanubi for cell maintenance, Peyman Ezatti for mass spectrometry, John Cortens for database assistance, Matthew Stuart-Edwards for developing scripts for some bioinformatics analysis, and members of the laboratory for reviewing the manuscript.
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