Quantitative Label-Free Phosphoproteomics Reveals Differentially

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Quantitative Label-Free Phosphoproteomics Reveals Differentially Regulated Protein Phosphorylation Involved in West Nile Virus–Induced Host Inflammatory Response Hao Zhang, Jun Sun, Jing Ye, Usama Ashraf, Zheng Chen, Bibo Zhu, Wen He, Qiuping Xu, Yanming Wei, Huanchun Chen, Rong Liu, Zhen F. Fu, and Shengbo Cao J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00424 • Publication Date (Web): 20 Oct 2015 Downloaded from http://pubs.acs.org on October 21, 2015

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Journal of Proteome Research

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Quantitative

Label-Free Phosphoproteomics Reveals

Differentially

RegulatedProtein

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Phosphorylation Involved in West Nile Virus–Induced Host Inflammatory Response

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Hao Zhang 1,2, Jun Sun 3,Jing Ye 1,2, Usama Ashraf 1,2, Zheng Chen 1,2,Bibo Zhu1,2,Wen He 1,2,

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Qiuping Xu1,2, Yanming Wei1,2,Huanchun Chen 1,2, Zhen F. Fu1,2,4,Rong Liu 3* and Shengbo Cao

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1,2,*

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1

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Wuhan, Hubei, 430070, PR China

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University,

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2

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University, Wuhan, Hubei, 430070, PR China

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3

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Huazhong Agricultural University, Wuhan, Hubei, 430070, PR China

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Laboratory of Animal Virology, College of Veterinary Medicine, Huazhong Agricultural

Agricultural Bioinformatics Key Laboratory of Hubei Province, College of Informatics,

Department of Pathology, University of Georgia, Athens, GA30602, USA

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*Corresponding author

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Email: [email protected] (Rong Liu)

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[email protected] (Shengbo Cao) Tel: +86-27-87618028; Fax: +86-27-87282608

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ACS Paragon Plus Environment


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Abstract

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West Nile virus (WNV) can cause neuro-invasive and febrile illness that may be fatal to humans.

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The production of inflammatory cytokines is key to mediating WNV-induced immunopathology

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in the central nervous system. Elucidating the host factors utilized by WNV for productive

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infection would provide valuable insights into the evasion strategies used by this virus.

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Although, attempts have been made to determine these host factors, proteomic data depicting

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WNV–host-protein interactions are limited. We applied liquid chromatography-tandem mass

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spectrometry for label-free, quantitative phosphoproteomics to systematically investigate the

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global phosphorylation events induced by WNV infection. Quantifiable changes to 1,657

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phosphoproteins were found; of these, 626 were significantly upregulated and 227 were

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downregulated at 12 h post-infection. The phosphoproteomic data were subjected to gene

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ontology–enrichment analysis, which returned the inflammation-related spliceosome, ErbB,

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mitogen-activated protein kinase, nuclear factor kappa B, and mechanistic target of rapamycin

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signaling pathways. We used short interfering RNAs to decrease the levels of glycogen synthase

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kinase-3 beta, bifunctional polynucleotide phosphatase/kinase, and retinoblastoma 1 and found

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that the activity of nuclear factor kappa B (p65) is significantly decreased in WNV-infected

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U251 cells, which in turn led to markedly reduced inflammatory cytokine production. Our results

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provide a better understanding of the host response to WNV infection and highlight multiple

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targets for the development of antiviral and anti-inflammatory therapies.

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INTRODUCTION

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Mosquito-borne West Nile viral disease can affect humans, birds, and horses1. The increase in

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the number of outbreaks of this severe and often fatal neurological disease is a major concern

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globally2. The etiological agent, West Nile virus (WNV), is a small, spherical, enveloped virus

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belonging to the genus Flavivirus of the family Flaviviridae 3. WNV has a positive-sense, single-

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stranded RNA genome of ~11 kb, which contains a single open-reading frame encoding a

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polyprotein precursor of ~3000 amino acid residues. This precursor undergoes proteolysis by

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cellular and viral proteases to yield three structural and seven non-structural proteins3. The

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structural proteins contribute to virion formation, whereas the non-structural proteins are

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important for viral genome replication and pathogenesis by facilitating formation of viral

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replication complexes and by interacting with host immune responses4,5. WNV pathogenesis has

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been divided into three distinct stages: initial infection and spread (early phase), peripheral viral

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replication (viremic phase), and neuroinvasion (central nervous system phase) 6. During the first

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two phases, macrophages and dendritic cells are targeted by the virus7,8. In the neuroinvasion

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phase, the virus breaches the blood brain barrier and subsequently targets brain cells 9.During the

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earliest stage of infection, macrophage migration inhibitory factor is upregulated, which

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increases the permeability of the blood brain barrier via peripherally expressed inflammatory

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cytokines and by inducing matrix metalloproteinase synthesis, so as to facilitate WNV entry into

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brain 10. WNV can also replicate in brain microvascular endothelial cells and can enter the brain

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without compromising the integrity of the blood brain barrier9. Thereafter, WNV causes neuronal

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damage accompanied by activation of microglia and astrocytes. Over-activated glial cells induce

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the production of inflammatory cytokines, which increase the possibility of extensive neuronal

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damage 11–13.



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The mechanisms of WNV pathogenesis involve interactions between host and viral proteins. The

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interplay of host-viral interactions determines the outcomes of viral infection and disease

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progression14,15. A better understanding of these host-virus interactions may allow the

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identification of potential druggable targets, and consequently, design of new antiviral therapies.

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Because of the highly complex nature of host-virus interactions, recent studies have shifted from

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simpleor one-pathway approaches, to large-scale screening approaches, e.g., metabolomics,

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transcriptomics, and proteomics 16. Such large-scale screens are quite useful for the identification

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of unexpected protein-protein interactions.

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Of the large-scale screening strategies, quantitative proteomics has provided new insights into

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the host cellular responses against viral infections, including those of human immunodeficiency

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virus 17, pseudorabies virus 18, human respiratory syncytial virus 19, influenza virus 20, infectious

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bronchitis virus 21, foot-and-mouth disease virus , and porcine circovirus 22, thereby providing a

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better understanding of the molecular mechanisms involved in viral pathogenesis . Multiple

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quantitative proteomics approaches have been used to investigate cellular gene expression

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profiles induced by flavivirus infection. The use of stable isotope labeling by amino acids in cell

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culture has led to the identification of 158 host proteins that show remarkable changes in their

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expression levels in HeLa cells infected with Japanese encephalitis virus

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previously uncharacterized proteins were identified. Moreover, two-dimensional fluorescence

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difference gel electrophoresis followed by mass spectrometry (MS) has been used to investigate

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differentially expressed proteins in WNV-infected Vero cells. Interestingly, 127 proteins that

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were differentially expressed were identified, 68 of which were upregulated and 59

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downregulated. Among these 127 proteins, the differential regulation of 93 was confirmed by

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RNA interference depletion studies 24.


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. Of these, 35

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Label-free quantitative proteomic analysis can provide a valuable platform for functional

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analysis of the proteome

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electrophoresis and no-label quantitative method, the label-free quantitative method is more

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efficient and suffices to discover and study protein-protein interactions.

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Phosphorylation is a common post-translational modification that involves the transfer of a

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phosphate group by a protein kinase to a targeted tyrosine(s), threonine(s), and/or serine(s) in a

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protein. Phosphorylation acts as a molecular switch to regulate the activities of targeted proteins

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and signaling molecules involved in several cellular processes such as differentiation,

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proliferation, apoptosis, and signal transduction

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. Compared with two-dimensional fluorescence difference gel

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. Similar to other viruses, WNV modulates

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several cellular pathways by regulating the phosphorylation and dephosphorylation of host

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proteins, thereby enhancing its replication

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production by blocking the phosphorylation of the proteins signal transducer and activator of

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transcription 1 and eukaryotic initiation factor-2 α via activation of transcription factor 6 and

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inositol-requiring enzyme 1 signaling, thereby facilitating its replication 28.

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For the study reported herein, quantitative phosphoproteomic analysis was used to identify

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cellular phosphoproteins that are involved in the host response to WNV infection. We utilized

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the label-free approach in conjunction with liquid chromatography-tandem MS (LC-MS/MS) to

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explore changes in the global cellular phosphoproteome that occur during WNV infection. Using

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bioinformatics tools to examine the functions of the differentially regulated phosphoproteins, we

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identified several inflammatory signaling–related pathways that are triggered in response to the

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altered levels of these phosphoproteins. In addition, the types of motifs and kinases that mediate

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phosphorylation as well as the interacting networks of phosphoproteins were also determined.

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Our results suggest that the interplay between WNV and host proteins results in many more

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. For example, WNV inhibits type-I interferon



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signaling events than had previously been considered. Ours is the first study to apply quantitative

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phosphoproteomic to uncover global phosphorylation events upon WNV infection in host cells

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and may, therefore, provide a better understanding of the molecular mechanisms of WNV

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pathogenesis in addition to aiding in the development of anti-inflammatory and antiviral drugs

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against WNV.

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MATERIALS AND METHODS

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Cell Cultivation and Viral Infection for Characterization of the Global Phosphoproteome

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12-h Post Infection

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WNV strain NY99 was propagated in BHK-21 cells. Human glial cell line (U251)was cultured

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and maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) heat-

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inactivated fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin sulfate at 37 °C in

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a 5% CO2 atmosphere. Cells were seeded in six-well plates (6 × 105 cells/well) and grown to

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80% confluency. Non-adherent cells were removed by washing with medium before virus

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inoculation. After washing, the cells were infected with WNV NY99 at a multiplicity of

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infection(MOI) of 5 for 1.5 h and harvested 12-h post-infection (hpi). A mock-infected cell

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preparation was also prepared using the same procedures but omitting the viral infection.

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Immunofluorescence Analysis

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U251 cells were infected with WNV NY99 strain at MOI of 1, 2, and 5. At 36 h, cells were fixed

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with ice-cold methanol for 10 min and then washed with phosphate-buffered saline (PBS) and

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incubated with primary antibodies against WNV E protein for 1 h at room temperature. After

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washing, cells were incubated with a 1:500 dilution of fluorescence-conjugated secondary

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antibodies for 30 min, and then stained with 4′, 6-diamidino-2-phenylindole dihydrochloride

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(DAPI; Invitrogen) for another 10 min. The cells were finally washed and observed using a

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fluorescence microscope (Zeiss) with 20× magnification.

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Protein Hydrolysis, Digestion, and Phosphopeptide Enrichment



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Harvested cells were centrifuged at 3,000 × g for 5 min and washed thrice with cold PBS

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containing 1 mM NaF and 1 mM pervanadate. The cells were lysed in 1 mM

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phenylmethylsulfonyl fluoride, 1% (w/v) SDS, 2 mM EDTA, 5 mM β-glycerophosphate, 10 mM

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NaF, 10 mM DTT, 100 mM sodium pyrophosphate decahydrate. Lysis debris was removed by

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centrifugation at 25,000 × g for 20 min. Sample reduction and alkylation were performed with an

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additional amount of 10 mM DTT and 55 mM iodoacetamide, respectively, at room temperature.

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After centrifugation, the precipitate was washed twice with 80% (v/v) ice-cold acetone, dried

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under vacuum, and then suspended in 0.5 M triethylammonium bicarbonate. A Bradford assay

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was performed to quantify the supernatant protein concentration, and those proteins were then

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digested with sequencing-grade modified trypsin (Promega; 10 µg trypsin/mg protein) for 16 h at

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37 °C. Peptides were desalted on a C18 Sep-Pak cartridge (Waters) and dried under vacuum.

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Peptide elution was performed with 3.5% (v/v) TFA, 65% (v/v) acetonitrile solution saturated

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with glutamic acid.

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Phosphopeptides were enriched using TiO2, as described Ref. Briefly, 500 µg of TiO2 (GL

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Sciences) and 1 mg of the phosphopeptides were mixed and then incubated for 20 min with end-

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over-end rotation. The mixture was washed first with 0.5% (v/v) TFA/65% (v/v) acetonitrile and

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then with 0.1% (v/v) TFA/65% (v/v) acetonitrile. The phosphopeptides were eluted with 0.3 M

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NH4OH/50% (v/v) acetonitrile and dried under vacuum.

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LC-MS/MS

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TiO2-enriched phosphopeptides were dissolved in buffer A 0.1% (v/v) formic acid/2% (v/v)

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acetonitrile at a concentration of 0.5 µg/µL and then subjected to LC-MS/MS. Samples (10 µL)

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were individually resolved through a 10-cm analytical C18 column after being eluted from a 2-


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cm C18 trap column, controlled by a Shimadzu LC-20AD nano HPLC system (Shimadzu)

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coupled to an autosampler. After loading each sample, the column was first washed with 2%

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(v/v) buffer B 0.1% (v/v) formic acid/98% (v/v) acetonitrile at 15 µL/min for 12 min and then

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subjected to a gradient of 2 to 35% buffer B over 91 min, followed by a 5-min wash of 80% (v/v)

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buffer B, and, finally, a 10-min re-equilibration with 2% (v/v) buffer B.

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A precursor MS scan of each intact phosphopeptide was recorded using a LTQ Orbitrap Velos

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spectrometer (Thermo Scientific) from 350 to 2000 m/z. The eight most intense multiply charged

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precursor ions were subjected to collision-induced dissociation for 30 ms at a normalized

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collision energy of 35.0. The automaticgain-control targets were 10,000 ions for the MS/MS

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scans and 100,000 for the Orbitrap scans. A dynamic exclusion time for 30 s was applied to

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avoid repeated analysis of the same component.

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Data Processing and Analysis

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The raw MS data were analyzed using Progenesis LC-MS software (version 4.1, Nonlinear

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Dynamics, Newcastle, U.K.). Quantification was performed for each condition after automatic

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retention time calibration, peak detection, and normalization in Progenesis LC−MS. The peak list

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was created by Progenesis LC-MS and searched in Mascot (version 2.4.1, Matrixscience,

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London, U.K.). The Uniprot Sus Scrofa database (release Mar. 2015) was used, and the decoy

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option was turned on in Mascot. The following search parameters were included: a fragment

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mass tolerance of 0.6 Da, a peptide mass tolerance of 10 ppm, and a maximum of two missed

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trypsin cleavages. The program also searched for carboxyamidomethylated Cys, which was a

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required modified residue, and deamidated Asn and Gln, N-terminal acetyl groups, oxidized Met,

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N-terminal pyro-Glu, and phosphorylated Ser, Thr, and Tyr, which were allowed modified



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residues. Results were analyzed with Protein Pilot software (version 4.0 SCIEX USA) with a

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peptide probability value set to 95%. These results in a peptide FDR are 0.95 for 12 h samples

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data set. All highly confident phosphopeptides were exported and quantified with Progenesis

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LC−MS. For comparison between samples, label-free quantification was performed with a

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minimum of 2 fold change to determine the differentially expressed phosphopeptides.

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Furthermore, p value

Quantitative Label-Free Phosphoproteomics Reveals Differentially

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