Biochimica et Biophysica Acta 1794 (2009) 14–22

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Biochimica et Biophysica Acta

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Bovine Viral Diarrhea Virus infection affects the expression of proteins related toprofessional antigen presentation in bovine monocytes

Sang-Ryul Lee 1, Bindu Nanduri 1, G. Todd Pharr, John V. Stokes, Lesya M. Pinchuk ⁎Department of Basic Science, College of Veterinary Medicine, Mississippi State University, P. O. Box 6100, Mississippi State, MS, 39762-6100, USA

Abbreviations: 2D, 2 dimensional; ANOVA, analyspresenting cells; BT, bovine turbinate; BVDV, Bovine ViraDB, database; DC, dendritic cells; DDF, differentialdeoxycholate; GO, gene ontology; HSP, heat shockchromatography electrospray ionization tandem mass scharide; MHC, major histocompatibility complex; MOImannose receptor; MudPIT, multidimensional proteinnon-cytopathic; PAGE, polyacrylamide gel electrophormononuclear cells; RP, reverse phase; SCO, subcommissexchange; SDS, sodium dodecyl sulfate; TAP, transpoprocessing; TLR, toll-like receptor; Xcorr, cross correlati⁎ Corresponding author. Tel.: +1 662 325 1436; fax: +

E-mail address: pinchuk@cvm.msstate.edu (L.M. Pin1 These two authors contributed equally to this work

1570-9639/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.bbapap.2008.09.005

a b s t r a c t

a r t i c l e i n f o

Article history:

The complete annotation Received 21 February 2008Received in revised form 8 September 2008Accepted 9 September 2008Available online 30 September 2008

Keywords:APCMonocyteBVDVDDF–MudPIT2D-LC ESI MS2

XcorrIn silico

of the cattle genome allows reliable protein identification by tandem massspectrometry (MS2) and greatly facilitates proteomics. Previously, we reported that differential detergentfractionation (DDF) analysis of bovine monocytes reveals proteins related to antigen pattern recognition,uptake and presentation to immunocompetent lymphocytes. Here we have identified 47 bovine proteins,involved in immune function of professional antigen-presenting cells (APC) that have been significantlyaltered after cytopathic (cp) Bovine Viral Diarrhea Virus (BVDV) infection. In particular, proteins related toimmune responses such as cell adhesion, apoptosis, antigen uptake, processing and presentation, acute phaseresponse proteins, MHC class I- and II-related proteins and other molecules involved in immune function ofprofessional antigen presentation have been significantly altered after BVDV infection. Our data suggest thatcp BVDV, while promoting monocyte activation and differentiation, is inhibiting their antigen presentation toimmunocompetent T cells, thus resulting in the uncontrolled inflammation mediated by activatedmacrophages, enhanced viral spread, and impaired anti-viral defense mechanisms in the host.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Professional antigen-presenting cells (APC), dendritic cells (DC)and their myeloid progenitors, monocytes/macrophages are criticalcontrollers of innate and adaptive immunity [1]. Cells of themonocyte/macrophage lineage are important elements of theimmune defense system because these cells can phagocytose foreignmaterial, present antigens to immunocompetent cells and producecytokines [1,2]. Bovine monocytes, as professional APC, expressrelatively high levels of MHC class I and class II molecules,costimulatory molecules induced upon activation, receptors forendo- and phagocytosis, and adhesion molecules [3]. Recent datasuggest that bovine monocytes are capable of directly inducingimmunoglobulin (Ig) secretion in activated bovine peripheral blood Bcells [3,4] as was previously demonstrated in humans [5,6]. Before

is of variance; APC, antigen-l Diarrhea Virus; cp, cytopathic;detergent fractionation; DOC,protein; LC ESI MS2, liquidpectrometry; LPS, lipopolysac-, multiplicity of infection; MR,identification technology; ncp,esis; PBMC, peripheral bloodural organ; SCX, strong cationrter associated with antigenon1 662 325 1031.chuk)..

l rights reserved.

entering the tissues, under the influence of certain cytokines,monocytes are capable of differentiating into macrophages or DC [7].

Bovine Viral Diarrhea Virus (BVDV), a single-stranded RNA virus,Pestivirus genus, and Flaviviridae family, infects a large proportion ofcattle worldwide and causes a number of clinical forms of the diseaseranging from transiently detectable mild clinical symptoms to a fataldiarrheic condition known as mucosal disease [8,9]. The BVDV areactuallymultiple viruseswith antigenic and genotypic differences, alongwith different growth patterns [8,9]. Initially the virus binds to CD46, acomplement receptor expressed on monocytes and lymphoid cells, andserves as a “magnet” for several viral and bacterial pathogens [10]. Uponentry, the virus replicates and spreads in the lymphatic system,impairing the immunity of the infected animal, particularly the functionof antigen-presenting cells (APC) and the production of interferons (IFN)[8,9,11]. Monocytes, infected with cp BVDV in vitro are compromisedin their antigen uptake ability and to stimulate allogeneic and memoryCD4+ T cell responses [12,13]. Taking into consideration that the TLR-mediated signaling triggered by different viruses during innate antigenrecognition that is followed by antigen uptake, processing andpresentation is crucial in the development of viral infection, includingBVDV, we assessed selective and non-selective antigen uptake mechan-isms in cp BVDV-infected monocytes [12]. Following the differences inthe antigen uptake function of infected monocytes and using the sameinfection protocols we determined the differences in toll-like receptors(TLR), cytokine and costimulatory molecules gene expression in the cpBVDV-infected cells [11]. Franchini et al. using higher doses of BVDV invitro did not detect significant differences in the TLR gene expressionlevels in bovine macrophages [14].

15S.-R. Lee et al. / Biochimica et Biophysica Acta 1794 (2009) 14–22

Recently we reported that differential detergent fractionation(DDF) of bovine monocytes yielded four electrophoretically distinctfractions enriched in cytosolic, membrane-organelle, nuclear mem-brane and cytoskeletal-matrix markers, respectively [15]. Further-more, multidimensional protein identification technology (MudPIT)couples 2 dimensional (2D) chromatography of peptides with tandemmass spectrometry (MS2), allowing for the identification of proteinsfrom highly complex mixtures of bovine monocytes and thecombination of DDF–MudPIT was used to increase the proteomecoverage in bovine monocytes [15]. Protein quantitation can be doneby calculating a cross correlation (Xcorr) of experimental tandemmassspectra to in silico generated tandem mass spectra from sequencedatabase [16]. This label-free method allows relative quantitation ofproteins without compromising proteome coverage [16].

Recently, the bovine genome was sequenced and annotated(http://www.hgsc.bcm.tmc.edu/projects/bovine/). The completeannotation of the bovine genome allows reliable protein identificationby MS2 and greatly facilitates proteomics. Here we used DDF–MudPITanalysis and relative quantitation of proteins expressed in bovinemonocytes infected with cp BVDV strain NADL.

2. Materials and methods

2.1. Animals

Nine conventionally reared, healthy BVDV-free cows from aHolstein herd at the Mississippi State University Dairy Facility wereused. The animals have been subjected to a comprehensive vaccina-tion program, including Frontier 4 Plus Vaccine (IBR, BVD, PI3, RSV,Diamond Animal H, Inc). All animal use was approved by TheMississippi State University Institutional Animal Care and UseCommittee. Peripheral blood mononuclear cells (PBMC) separatedfrom the animals used in our study were tested for the expression ofBVDV E2 transcripts with E2 BVDV specific primers by RT-PCR [12]. Aswe expected, all animals were BVDV mRNA-free (data not shown).

2.2. Cell preparation

Blood samples (150 ml) were collected into Blood Collection Tubes(16×100mm, Tyco Healthcare) by jugular venipuncture. Bovine PBMCwere separated as described elsewhere [3,11,12,17]. Briefly, PBMCwereisolated using Histopaque gradients (1.077 g/ml, Amersham Bios-ciences) and resuspended in RPMI-1640 supplemented with 10% FBS,1% Glutamax-1 (Invitrogen), 5×10−5 M 2-mercaptoethanol and100 IU/ml Gentamicin (Invitrogen). Monocytes were separated fromPBMC as described elsewhere [3]. Briefly, 40 ml of PBMC suspension(5×108 cells) was added to Petri-dish (150×25 mm, BD sciences) for2 h at 37 °C. Non-adherent cells were removed and the adherent cellswerewashed twice in PBS (Invitrogen). The yield of adherent cells was20–30% of total PBMC number. After removing non-adherent popula-tions (mostly T and B cells), adherent cells were incubated with mAbsto CD14 (MM61A, VMRD) followed by the addition of magnetic beadsconjugated with mouse anti-IgG1 (Miltenyi Biotech, Auburn, CA) [11].CD14+ monocytes were positively selected by using magnetic cellseparation technique according to the manufacturer's instructions(Miltenyi Biotech). The final yield of bovine monocytes was 2–3% oftotal PBMC number.

2.3. BVDV stock and infection

BVDV strain was prepared as described elsewhere [12]. Briefly, theNADL (cp) strain of BVDV was obtained from the American TypeCulture Collection (ATCC) and amplified by growth in the bovineturbinate (BT) cell line (ATCC) according to the manufacturer'shandling procedures. For infection of BT cells, virus dilutions weremade in DMEMwith 4 mM L-glutamine, 4.5 g/l glucose, 1.5 g/l sodium

bicarbonate and 10% horse serum. To measure the infectivity of theNADL strain, the quantal method of Reed and Muench was performedand the tissue culture infectious dose 50 (TCID50) was determined. Toselect the dose of cp BVDV that did not have a cytopathic effect onmonocytes cultured for 48 h we assessed the viability of the infectedcells by using trypan blue and light microscopy. BVDV strain NADL atthe multiplicity of infection (MOI) 0.002 did not affect the viability ofbovine monocytes after 48 h of infection (data not shown). 5×106

monocytes were added to eachwell of a 6 well tissue culture plate andinfected with cp BVDV at the same MOI of 0.002 for 24 h [12]. Afterinfection, at least 107 cells were pooled in one tube. All data weredetermined using triplicate monocyte cultures. For some experimentswe used monocytes infected for 24 h with the New-York non-cytopathic (ncp) strain of BVDV (ATCC) at theMOI of 00.2 as additionalcontrols.

2.4. Protein extraction, trypsin digestion and 2D-LC ESI MS2

Proteins were isolated using DDF as described [15,18]. Briefly, DDFsequentially extracts proteins from cellular compartments using aseries of detergents. Cytosolic proteins were isolated by repeatedwashes in digitonin buffer. After the digitonin washes, the isolation ofmembrane, nuclear and cytoskeletal proteins were performed withtriton X-100 (TX), deoxycholate (DOC), tween 40, and SDS buffers,respectively. To evaluate the quality of isolated proteins, 1% of theprotein samples were compared using 10% SDS-PAGE (data notshown). For each of the detergent fractions, equal amounts of proteinwere precipitated with 25% trichloroacetic acid to remove salts anddetergents. Protein pellets were solubilized and then digested with100 ng of trypsin (50:1 ratio of substrate to enzyme) overnight at37 °C. Peptides were desalted using a peptide microtrap (MichromBioResources, Inc.) and eluted by a 0.1% trifluoroacetic acid, 95%acetonitrile solution. Desalted peptides were dried and resuspendedin 0.1% formic acid.

2D-LC ESI MS2 was done as described elsewhere [15,18]. Briefly, LCanalysis was accomplished by strong cation exchange (SCX) followedby reverse phase (RP) liquid chromatography (LC) coupled directly inline with electrospray (ESI) ion trap MS. Each DDF fraction samplesfrom three different infections were loaded into a LC gradient ionexchange system including a Thermo Separations P4000 quaternarygradient pump (ThermoElectron Corporation) coupled with a0.32×100 mm BioBasic SCX column and run three times. A flow rateof 3 μl/minwas used for both SCX and RP columns. A salt gradient wasapplied in steps of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 57, 64, 71, 79,90, 110, 300, and 700 mM ammonium acetate in 5% acetonitrile, 0.1%formic acid and the resultant peptides were loaded directly into thesample loop of a 0.18×100 mm BioBasic C18 RP LC column of aProteome X workstation (ThermoElectron). The RP gradient used 0.1%formic acid in acetonitrile and increased the acetonitrile concentrationin a linear gradient from 5% to 30% in 30 min and then 30% to 65% in9 min followed by 95% for 5 min and 5% for 15 min.

The spectrum collection time was 59 min for every SCX step. TheLCQ Deca ion trap mass spectrometer (ThermoElectron) was config-ured to optimize the duty cycle lengthwith the quality of data acquiredby alternating between a single full MS scan followed by three tandemMS scans on the three most intense precursor masses from full scan.The collision energy was normalized to 35%. Dynamic mass exclusionwindows were set at 2 min, and all of the spectra were measured withan overall mass/charge (m/z) ration range of 200–2000.

2.5. Protein identification and analysis

Proteins were identified and analyzed as previously described [15].The non-redundant protein database (DB) downloaded from theNational Center for Biotechnology Information (NCBI; May 2006) byTurboSEQUEST (Bioworks Browser 3.2; ThermoElectron) was used to

Table 1Immune function proteins in bovine monocytes with significantly altered expression after 24 h infection with NADL strain

GANa Description Len (aa) Peptide match ΣXcorrb GOc Species

Control cp BVDV p-value Control cp BVDV Molecular function Biological process Cellular component

AAI08182 Serum amyloid A 3 131 bdt 3 0.00 0.00 8.42 Lipid transporter activity Acute phase response Extracellular region BovineCAA34596 Fetuin 359 bdtd 24 0.00 0.00 79.58 Kinase inhibitor activity Acute phase response Extracellular space BovineAAB32744 PA28 = regulators of the 20S

proteasome {peptide 22b}16 3 bdt 0.00 12.86 0.00 Proteasome activator

activityAntigen presentation Cytosol Mouse

AAI11270 TAP binding protein (tapasin) 447 20 7 0.02 73.81 28.11 NAe Antigen processing,endogenous antigenvia MHC class I

Membrane Bovine

XP_612582 Predicted: similar to myosin,heavy polypeptide 9,non-muscle, partial

1621 40 82 0.00 148.72 321.27 Motor activity Cell–cell adhesion Myosin Mouse

CAA41380 Desmoglein 1043 6 bdt 0.00 16.41 0.00 Calcium ion binding Cell adhesion Integral to membrane BovineAAA30512 Factor V 2211 bdt 16 0.00 0.00 55.08 Calcium ion binding Cell adhesion NA Bovine1SDD_B B Chain B, Crystal Structure

Of Bovine Factor Vai647 bdt 4 0.00 0.00 52.66 Calcium ion binding Cell adhesion NA Bovine

AAX46444 Integrin alpha 2b precursor 937 13 20 0.01 45.35 76.07 Receptor activity Cell adhesion Integral to membrane BovineXP_616376 Predicted: similar to integrin

beta chain, beta 3 precursorisoform 1

784 14 21 0.01 42.58 67.75 Protein binding Cell adhesion integral to membrane Rabbit

AAI14108 ITGB1 protein 801 bdt 4 0.00 0.00 14.77 Receptor activity Cell adhesion Integral to membrane BovineXP_584890 Predicted: similar to

Desmoglein-2 precursor(HDGC), partial

1094 4 bdt 0.00 9.77 0.00 calcium ion binding Homophilic cell adhesion Membrane Human

XP_871309 Predicted: similar toLeukosialin precursor(Leucocyte sialoglycoprote

271 6 2 0.04 19.62 7.51 Protein binding Negative regulationof cell adhesion

Integral to plasmamembrane

Mouse

AAB30209 Calreticulin 400 50 35 0.01 149.84 104.43 Calcium ion binding Cortical actin cytoskeletonorganization and biogenesis

Endoplasmic reticulum Bovine

NP_776588 Rho family, small GTPbinding protein Rac1

192 10 5 0.01 32.02 15.49 GTPase activity Endocytosis Cytoplasm Bovine

XP_873226 Predicted: similar to stonedB-like factor

732 3 bdt 0.00 7.14 0.00 Transcription initiationfactor activity

Endocytosis Clathrin vesicle coat Human

BAA12156 Invariant chain 204 6 bdt 0.00 22.17 0.00 NA Immune response Membrane BovineBAA21517 CD14 373 1 8 0.00 2.98 25.75 GPI anchor binding Immune response Membrane BovineNP_776365 Myxovirus (influenza)

resistance 1648 1 4 0.01 2.36 10.93 GTPase activity Immune response NA Bovine

AAI11203 Similar to interleukinenhancer binding factor 2

390 3 bdt 0.00 11.72 0.00 Transferase activity Immune response NA Bovine

XP_613380 Predicted: similar to CD163antigen isoform a

1075 bdt 4 0.00 0.00 10.22 Scavenger receptor activity NA Membrane Human

AAI12488 CD68 molecule 335 bdt 4 0.00 0.00 11.44 NA NA Membrane BovineP02769 Serum albumin precursor

(Allergen Bos d 6) (BSA)607 220 124 0.00 755.67 426.01 Antioxidant activity Negative regulation

of apoptosisExtracellular space Bovine

Q8MIT6 Rho-associated proteinkinase 1 (Rho-associated,coiled-coil-cont

441 3 bdt 0.00 7.21 0.00 Transferase activity Negative regulationof neuron apoptosis

Cytoskeleton Bovine

AAI1608 Similar to ApolipoproteinA-II precursor (Apo-AII)(ApoA-II)

100 bdt 5 0.00 0.00 17.34 Lipid transporter activity Regulation of interleukin-8biosynthetic process

Extracellular region Bovine

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GAN

Description

Len(aa)

Peptidematch

Sum

ofXco

rrGen

eon

tology

Control

NADL

p-va

lue

Control

NADL

Molecular

func

tion

Biolog

ical

proc

ess

Cellu

larco

mpo

nent

Species

P629

35Pe

ptidyl-prolylc

is-trans

isom

eraseA(PPIaseA)

(Rotam

aseA)(Cyc

164

48

0.03

10.79

20.80

Peptidyl-prolylc

is-trans

isom

eraseactivity

Regu

lation

ofviral

geno

mereplication

NA

Bovine

AAI030

84Hea

tsh

ock70

kDaprotein2

641

4bd

t0.00

10.93

0.00

Nuc

leotidebind

ing

resp

onse

toun

folded

protein

NA

Bovine

AAI023

35Hea

tsh

ock70

kDaprotein9B

(mortalin

-2)

679

4128

0.04

143.34

98.16

Unfolde

dproteinbind

ing

Resp

onse

toun

folded

protein

NA

Bovine

XP_

5856

05Pred

icted:

simila

rto

150kD

aox

ygen

-reg

ulated

proteinprecursor

1083

3120

0.03

99.11

70.13

Nuc

leotidebind

ing

Resp

onse

toun

folded

protein

NA

Rat

aGen

bank

accessionnu

mbe

r.b

Sum

ofXco

rrva

lues

forallp

eptide

siden

tified

from

this

proteinin

allthree

biolog

ical

replicates.

cGO:ge

neon

tology

.d

Below

detectab

lethresh

oldat

ourestablishe

dcu

toffsforXco

rr.

eNot

assign

edto

aGO

catego

ry.

17S.-R. Lee et al. / Biochimica et Biophysica Acta 1794 (2009) 14–22

create a bovine subset (bovine DB; search terms: bos taurus and bosindicus). Trypsin digestion was applied in silico to bovine DB and masschanges due to cysteine carbamidomethylation and methionineoxidation were included. The bovine DB was used to search tandemMS using peptide (MS precursor ion) mass tolerance of 1.5 Da and afragment ion (MS2) mass tolerance of 1.0 Da. Peptide matches wereconsidered genuine if they were ≥6 amino acids with Xcorr values of1.5, 2.2 and 3.3 (+1, +2, and +3 ions respectively) andΔCn (≥0.1) values[19]. We used decoy database search strategy to calculate theprobability that a peptide identification is incorrect and estimatedthe probability that a protein identification is incorrect using Xcorrand delta Cn, two SEQUEST parameters (Tables 1 and 2) [20,21]. Eachprotein was classified by using Gene Ontology (GO) annotation. GOwas obtained available GO annotation from UniProt database (http://www.pir.uniprot.org/) [22]. In addition, we used AgBase tool GOanna[23,24] to provide additional GO annotation for bovine gene products.

2.6. Western blot

Western blot analysis was performed as described elsewhere [25].Equal amounts of proteins from each of the detergent fractions werecombined for cp BVDV-infected, control uninfected and ncp BVDV-infectedmonocytes. The concentration of proteins wasmeasured with2-D Quant Kit (Amersham Biosciences) and 5 μg samples were loadedon SDS-PAGE. Ready Gel™ blotting Sandwiches (Bio-Rad) was used forthe transfer of proteins to the nitrocellulose membrane. After primaryantibody labeling, goat anti-mouse Ig (H+L)-AP, human adsorbed(Southern Biotech), goat anti-rabbit IgG (H+L)-AP (CALTAG) and goatanti-mouse IgG (H+L)-AP (Zymed) were used as secondary antibodies.For developing the proteins labeledwith antibodies, BCIP/NBTalkalinephosphatase substrate (Sigma) was used. β-actin (Ambion) as house-keeping protein,MHC class II expressions in BVDV-infectedmonocytesrelative to that of uninfectedmonocyteswereused in this study (Fig. 3).The arrows indicate the position of the molecular weight marker ineach group. Anti-bovine MHC II (HLA-DQ) mAb (BAQ150A, VMRD),anti-bovine MHC II mAb (TH81A5, VMRD), anti-bovine MHC class ImAb (PT85A, VMRD), and anti-β-actin mAb (AC-15, Ambion) wereused to label the proteins.

2.7. Statistical analysis

To determine significant changes in protein expression betweenthe control and BVDV-infected monocytes, proteins were analyzed aspreviously described [16]. Briefly, only proteins identified by at leastthree peptides in any dataset were considered. A custom programwasused to calculate sum of Xcorr and error of all the identified peptidesfrom all three replicates for each protein, and one way analysis ofvariance (ANOVA) (pb0.05) was used to identify statistically sig-nificant differences in protein expression between treatments [25].

3. Results

3.1. Protein identification

A total of 8272 known proteins were identified in bovine mono-cytes. Specifically, with three replicates 6470 proteins in controlmonocytes and 5459 proteins in cp BVDV-infected monocytes wereidentified using non gel-based proteomic 2D-LC ESI MS2 methods.The identified proteins have been distributed as follows: 34% (2813)in control, 21.8% (1802) in BVDV-infected and 44.2% (3672) in bothgroups (Fig. 1). Overall, BVDV infection significantly altered theexpression of 445 proteins in bovine monocytes compared to un-infected controls (Supplementary Table 3 online, Appendix 1). Asexpected 29 proteins identified in the BVDV-infected and controlmonocytes (Table 1) and all 18MHC proteins (Table 2) were related toimmunological function based on the biological process in GO

Table 2Bovine monocyte MHC proteins with significantly altered expression after 24 h infection with NADL strain

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a) Genbank accession number.b) Sum of Xcorr values for all peptides identified from this protein in all three biologicalreplicates.c) GO: gene ontology.d) Below detectable threshold at our established cutoffs for Xcorr.Light gray: expressed in both groups.Dark gray: expressed in cp infected monocytes only.

Table 2 (continued)

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category. We analyzed several proteins that have not been signifi-cantly altered by BVDV as internal standards (Supplementary Table 3online, Appendix 1).

3.2. Comparative analysis of immunological proteins in control andBVDV-infected monocytes by GO categories

Bovine monocyte proteins that have been significantly altered byBVDV (445 proteins) were assigned function using known GOcategories “Molecular Function”, “Biological Process” and “CellularComponent” (Supplementary Table online). Importantly, 29 immu-nological proteins (6.5%) that have been significantly altered byBVDV infection were placed into known GO categories (Table 1).Furthermore, the percentage representation of identified proteinswas determined for each GO biological process category (Fig. 2).Briefly, all 2 proteins related to acute phase response and 6 out of 9proteins associated with cell adhesion were significantly increased incp BVDV-infected bovine monocytes. In addition, CD14, CD68,peptidyl-prolyl cis-trans isomerase A, similar to apolipoprotein A-IIprecursor and myxovirus resistance 1 proteins were significantlyincreased in the cp BVDV-infected cells. On the other hand,desmoglein, predicted: similar to desmoglein-2 precursor (HDGC),and predicted: similar to leukosialin precursor proteins, which are allrelated to cell adhesion, were significantly down-regulated in the cpinfected group. Moreover, all proteins related to the negativeregulation of apoptosis, invariant chain, and antigen uptake,processing and presentation were decreased in the BVDV-treatedgroup. Proteins related to responses to unfolded protein were alsosignificantly down-regulated in the infected group. Finally, 8immune function proteins (27.6%) were identified as below detect-able threshold (bdt) at our established cutoffs for Xcorr in eachgroup (control and cp infected).

3.3. Analysis of MHC proteins in comparison between control andcp-infected monocytes

In this study, 18 MHC proteins (4%) out of 445 were significantlyaltered in cp BVDV-infected bovine monocytes (Table 2). BVDV

Fig. 1. Distribution of total proteins identified from either uninfected or BVDV-infectedbovine monocytes. The MS2 data from each of groupwere analyzed using the bovine DBsubset and the resulting proteins were classified with respect to their group.

infection significantly altered the expression of 11 MHC class I and 7MHC class II molecules in peripheral blood monocytes. Firstly, 9 ofMHC class I proteins were significantly down-regulated in cp BVDV-infected bovine monocytes. Interestingly, two proteins, accessionnumber AAZ73460 and ABA39524 were only detected in cp BVDV-infected group. Secondly, 6 of MHC class II proteins, including the DQisotype, were significantly decreased in the infected monocytes, andone MHC class II DR-beta chain protein was increased in cp BVDV-infected monocytes only. To confirm the significant changes in proteinexpression between the control and BVDV-infected monocytes byusing DDF–MudPIT analysis and relative quantitation approach,Western blotting was performed to evaluate the protein expressionof the MHC class I, class II and HLA-DQ isotype (Fig. 3). In ourpreliminary observation by DDF–MudPIT, ncp BVDV strain New-Yorkdecreased the protein expression levels of the MHC class I, class II andMHC-DQ isotypes to virtually undetectable levels (unpublishedobservation). Therefore, we used monocytes infected with ncp BVDVas positive controls for inhibition of protein expression levels (Fig. 3).The ncp BVDV had the strongest inhibitory effect on the MHC class I,MHC class II and MHC-DQ isotype protein expression levels and didnot affect β-actin expression levels in bovine monocytes (Fig. 3). Asexpected, cp BVDV infection significantly decreased the levels of theMHC proteins and did not affect β-actin expression levels inmonocytes supporting our findings by DDF–MudPIT in cp BVDV-infected cells.

4. Discussion

BVDV has been described as a group of multiple viruses affectingvirtually all organs and systems in the body, including innate and

Fig. 2. Estimated percentage representation of the immunological proteins (exceptMHCclass molecules) altered by cp BVDV infection in each GO biological process category.Biological process descriptions are as follows: actin cytoskeleton organization andbiogenesis (1), acute phase response (2), antigen presentation (3), cell adhesion (4),endocytosis (5), immune response (6), negative regulation of apoptosis (7), regulation ofinterleukin-8 biosynthetic process (8), regulation of viral genome replication (9),response to unfolded protein (10), and function unknown (11). The arrows on the topindicate the up- or down-regulation of the proteins in cp BVDV-infected monocytes.

Fig. 3.Western blot analysis of MHC class I and II proteins in bovine monocytes infectedwith cp BVDV relative to that of uninfectedmonocytes (negative control) and ncp BVDV-infected cells (positive control) with b-actin as housekeeping protein. Bovinemonocytes were infected with cp BVDV strain NADL and ncp BVDV strain New-Yorkat the multiplicity of infection (MOI) 0.002 for 24 h. Proteins were isolated using DDFsequentially extracting proteins from cellular compartments using a series ofdetergents. Equal amounts of proteins from each of the detergent fractions werecombined for BVDV-infected and control monocytes. The concentration of proteins wasmeasuredwith 2-D Quant Kit (Amersham Biosciences) and 5 μg samples were loaded onSDS-PAGE.

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adaptive immune responses [26]. However, the role of professionalAPC, in particular, monocytes/macrophages in BVDV infection is stillunclear. Multiple studies assessed morphological and functionalproperties of the BVDV-infected monocytes/macrophages, includingNO production, antigen uptake, T cell stimulatory capacities, cytokineproduction, cytokine and TLR gene expression [11–14,27–29].

Previously, we reported the identification of proteins involved inprofessional antigen presentation in bovine monocytes by usingdifferential detergent fractionation (DDF) and multidimensionalprotein identification technology (MudPIT) [15]. Sequenced andannotated bovine genomes allow reliable protein identification usingnon-electrophoretic proteomics by 2D-LC ESI MS2 method [15].Compared with electrophoresis-based methods, non-electrophoreticproteomics is more comprehensive and is a high throughput.

However, comparative protein profiling of BVDV-infected APC,including quantitation of the relative immunologically importantprotein expression while maintaining comprehensive proteomecoverage is still missing. Therefore, in this study, we applied DDF–MudPIT analysis and relative quantitation of proteins expressed inbovine monocytes infected with cp BVDV strain NADL. Following ourprevious observations that demonstrated significant differences in themechanisms of antigen uptake, TLR, cytokine, and costimulatorymolecules gene expression in monocytes infected with cp BVDV strain

for 24 h [11,12], we expected to see differences in the proteinexpression levels mediated by BVDV directly and by BVDV-dependentcytokine and other active substances release acting indirectly after24 h infection.

With virus infection, acute phase response protein expressions areincreased in human and porcine models [30–32]. Multiple reportsdemonstrated that acute phase proteins in cattle were also elevatedwith several viral infections [33–35]. Especially, the acute phaseproteins, such as serum amyloid A (SAA), were altered in BVDV-infected calves [33]. Moreover, apolipoprotein A-II is also related toSAA expression [36]. Here we demonstrate that acute phase responseproteins, fetuins, SAA and apolipoprotein A-II were significantly up-regulated in bovine monocytes infected with cp BVDV. This observa-tion supports our previous report that cp BVDV induced theexpression of protein kinases and related proteins involved in cellactivation and differentiation [37].

Multiple studies demonstrated that uptake, processing andpresentation of protein antigens by APC are down-regulated in someviral infections [38–41]. Especially, MHC class I or transporterassociated with antigen processing (TAP) protein were down-regulated with varicella-zoster virus, bovine herpesvirus 1, and bovinepapillomavirus E5 [38,41,42]. Moreover, BVDV-infected bovine mono-cytes decreased their ability to present antigens to the Th cells andtheir phagocytic activity [13,43,44]. In this study we demonstrate thatmost of the MHC-, endocytosis- and TAP-related proteins weresignificantly decreased in BVDV-infected monocytes.

Our data indicate that all proteins related to the negativeregulation of apoptosis were decreased in cp BVDV-infected bovinemonocytes. This evidence correlates with our previous report that thatthe cp BVDV strain decreased the expression of two protein kinase Cinhibitors and proteins with anti-apoptotic effect [37] and withprevious observation that cp BVDV and other viruses induce theapoptotic pathways of the host [43,45].

In order for circulating leukocytes to enter inflammatory tissue orperipheral lymphoid organs, the cells must adhere to and passbetween the endothelial cells lining the walls and blood vessels [46].Some of these surface molecules are expressed continuously, butothers are only expressed in response to localized concentrations ofcytokines produced during an inflammatory response and serve toincrease the strength of the functional interactions between cells ofthe immune system [46]. We report that most of the cell adhesionmolecules expression was significantly increased in BVDV-infectedmonocytes. However, the desmoglein like proteins and the protein ofnegative regulation of cell adhesionwere decreased in this study. Thisevidence supports our previous report that cp BVDV induced theexpression of protein kinase and related proteins promoting mono-cytes to differentiate into macrophages [37]. Finally, the majority ofcell adhesion molecules up-regulated in cp BVDV-infected monocytesare closely related to cell migration to the tissue, while the ones down-regulated are primarily involved in cell–cell adhesion within tissues[46]. Decreased cell–cell adhesion promotes monocyte migration anddifferentiation into macrophages in the tissue.

Two observations suggested that the BVDV group of viruses useintegrin molecules as their receptors in the bovine system [47,48],BVDV use CD46 and low-density-lipoprotein receptor to enter hostcells [49,50]. Although we have identified low-density-lipoproteinreceptors in both control and infected monocytes, BVDV infection didnot significantly alter their expression (data not shown). Interestingly,low-density-lipoprotein receptor-related molecules were significantlyincreased in cp BVDV-infected monocytes in our study.

Immune response proteins play an important role in professionalAPC function [51–53]. In particular, the CD14/TLR4 complex isinvolved in viral G protein-related production of type I IFN [54].Moreover, Muller-Doblies et al. showed that Myxovirus resistance I(Mx) proteins induced by type I IFN were increased in ncp BVDV-infected bovine white blood cells [55]. Finally, our previous report

21S.-R. Lee et al. / Biochimica et Biophysica Acta 1794 (2009) 14–22

demonstrated that the expression of type I IFN was numericallyincreased in bovine monocytes after infection with cp and ncp BVDVstrains [11]. In this study we extend our previous findings anddemonstrate that both proteins, CD14 and Mx were significantlyincreased in BVDV-infected monocytes.

In this study, the expression of invariant chains non-covalentlyassociated with MHC class II complex was decreased unlike thefindings reported on the expression of this molecule in viral infectionpreviously [56–58].

It was shown previously that interleukin enhancer binding factor(ILF2, NF45) and NF90 (ILF3) regulate the IL-2 gene transcription viainteractionwith the antigen receptor response element [59]. Cp BVDVsignificantly down-regulated the expression levels of the proteinsimilar to interleukin enhancer binding factor in our study.

Previous report showed that heat shock proteins (HSP) producedin response to unfolded proteins are highly conserved proteins thatare strongly induced in physical and chemical stress in bothprokaryotic and eukaryotic cells [60]. HSP are involved in tumorimmunity mediated by APC, T cells and NK cells and enhance thedevelopment of innate and adaptive immune responses by interactingwith viral proteins [61,62]. HSP make complexes with viral proteinsthat bind APC that could affect the cytokine expression, induction ofadaptive immunity, and activation of CTL through antigen presenta-tion with MHC molecules [62]. Previous report demonstrated thatrecombinant bovine HSP70 increased the antigen uptake, theexpression of MHC molecules and CD40 in monocytes, monocyte-derived macrophages and DC [63]. In this study, cp BVDV-infectedmonocytes down-regulated the expression of molecules of responseto unfolded proteins as well as MHC proteins suggesting possiblecorrelation between the two groups of proteins in cp BVDV-infectedbovine monocytes.

Calreticulin is a major intracellular calcium-binding proteinidentified in skeletal muscle sarcoplasmic reticulum that modulatescell adhesion, integrin-dependent calcium signaling, steroid-sensitivegene expression and is involved in glycosylation of MHC molecules inthe ER [64,65]. Calreticulin is related to the signaling pathwayinvolving one of receptors for BVDV, lipoprotein receptor-relatedprotein [49,66]. Our observation that calreticulin expression wassignificantly decreased in bovine monocytes infected with cp BVDVstrain confirms the involvement of this protein in BVDV pathogenesisand its down-regulation in cp BVDV infection is consistent with thedecrease in the MHC class I protein expression in cp BVDV-infectedmonocytes.

It was previously demonstrated that CD163, a hemoglobinscavenger receptor exclusively expressed in the monocyte/macro-phage system plays a major role in dampening the inflammatoryresponse and in scavenging components of damaged cells [67]. It wasalso reported that CD163 was repressed in HIV infected humanmacrophages [68], however up-regulated in porcine monocyte/macrophages infected with African swine fever virus [69] and incattle APC infected with tuberculosis [70]. In our study, CD163 andCD68, monocyte/macrophage differentiation marker [71] were sig-nificantly up-regulated in cp BVDV-infected monocytes.

Finally, several reports described peptidyl-prolyl cis-trans iso-merases (PPIases) as ubiquitous foldases that contribute to theconformational changes during protein folding in both eukaryotesand prokaryotes [72]. In this study, BVDV infection significantlyincreased the PPIases expression levels suggesting their involvementin BVDV infectivity.

In conclusion, we hypothesize that by altering expression levels inmultiple proteins related to immune responses such as cell adhesion,apoptosis, antigen uptake, processing and presentation, and otheracute phase response proteins cp BVDV could significantly compro-mise immune defense mechanisms. In particular, up-regulation ofproteins related to the acute phase response and cell adhesion whiledecreasing the expression of proteins involved in antigen uptake,

processing and presentation suggests that cp BVDV infection ispromoting monocyte migration, differentiation and activation whileinhibiting their BVDV antigen presentation to immunocompetentlymphocytes, in particular, Th1 type and regulatory T cells, thusresulting in the uncontrolled inflammation mediated by activatedmacrophages and enhanced viral spread in the host. Our data suggestthat cp BVDV infection induces monocytes to differentiate intomacrophages, or, alternatively, that monocytes that have alreadyembarked on the differentiation into macrophages are more suscep-tible to cp BVDV infection. In addition, the reduced expression ofapoptosis inhibitors would contribute to the cytopathic mechanismsof cp BVDV thus affecting the anti-viral defense mechanisms in theprofessional APC. Although the DDF–MudPIT analysis is an extremelypowerful tool, in order to increase the number of the target proteinsidentified, and confirm the proteome analysis data, additional proteinidentification approaches have to be applied.

Acknowledgements

This work was supported by the College of Veterinary Medicine,CVM-INST proposal “Monocyte-dependent cytokine polarizationprofiles in cytopathic and non-cytopathic Bovine Viral Diarrhea Virusinfection”, Mississippi State University. This paper is MAFES # J-11185.

The authors wish to acknowledge MSU Life Sciences and Bio-technology Institute (LSBI) facility for the protein analysis of bovinemonocyte DDF.

The authors also wish to thank Drs. N. Filipov, M. Lawrence andF.McCarthy for the helpful critique and suggestions on themanuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbapap.2008.09.005.

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