Loss of Interferon Regulatory Factor 3 in Cells Infected with Classical Swine Fever Virus Involves

J OURNAL OF V IROLOGY,June2005,p.7239–7247Vol.79,No.11 0022-538X/05/$08.00?0doi:10.1128/JVI.79.11.7239–7247.2005

Copyright?2005,American Society for Microbiology.All Rights Reserved.

Loss of Interferon Regulatory Factor3in Cells Infected with Classical Swine Fever Virus Involves the N-Terminal Protease,N pro

S.Anna La Rocca,1Rebecca J.Herbert,1Helen Crooke,2Trevor W.Drew,2Thomas E.Wileman,1

and Penny P.Powell1*

Department of Immunology,BBSRC Institute for Animal Health,Ash Road,Pirbright,Surrey GU240NF,1and Department of Virology,Veterinary Laboratories Agency,Weybridge,Surrey KT153NB,2United Kingdom

Received22September2004/Accepted5January2005

We show that cells infected with the pestivirus classical swine fever virus(CSFV)fail to produce alpha/beta

interferon not only following treatment with double-stranded RNA but also after superinfection with a

heterologous virus,the alphavirus Sindbis virus,a virus shown to normally induce interferon.We investigated

whether the inhibition of interferon synthesis by CSFV involved a block in interferon regulatory factor3(IRF3)

activity.Cells infected with CSFV exhibited a lack of translocation of green?uorescent protein-IRF3to the

nucleus;however,constitutive shuttling of IRF3was not blocked,since it could still accumulate in the nucleus

in the presence of leptomycin B.Interestingly subcellular fractionation analysis showed that IRF3was lost

from the cytoplasm of infected cells from18h postinfection https://www.360docs.net/doc/729705035.html,ing IRF3promoter-luciferase reporter

constructs,we demonstrate that loss of IRF3was due to an inhibition of transcription of the IRF3gene in

CSFV-infected cells.Further,we investigated which viral protein may be responsible for the inhibition of

interferon and loss of IRF3.We used cell lines expressing the CSFV N-terminal protease(N pro)to show that

this single viral protein,unique to pestiviruses,inhibited interferon production in response to Sindbis virus.

In addition to being lost from CSFV-infected cells,IRF3was lost from N pro-expressing cells.The results

demonstrate a novel viral evasion of innate host defenses,where interferon synthesis is prevented by inhibiting

transcription of IRF3in CSFV-infected cells.

Classical swine fever virus(CSFV)belongs to the Pestivirus genus in the Flaviviridae family,together with bovine viral diarrhea virus(BVDV)and border disease virus(26).The CSFV genome is a single positive-stranded RNA about12.5kb in length,with a single large open reading frame encoding a polyprotein that is processed into12known proteins(38). CSFV causes a severe disease of pigs characterized by fever, leukopenia,and hemorrhage(40),and there is widespread apoptosis of uninfected lymphocytes(35,36).The disease causes signi?cant economic loss worldwide and is an Of?ce International des Epizooties list A pathogen(25).Pestiviruses can also cross the placenta,resulting in the birth of persistently infected animals due to failure of the dam to raise an innate immune response capable of preventing infection of the fetus (8).This interesting property of pestiviruses may partly be due to their ability to inhibit interferon production in cells they infect.CSFV causes no visible cytopathic effect in cells in culture,it induces the production of proin?ammatory cyto-kines,yet does not stimulate interferon secretion(5).More-over,both CSFV and BVDV can inhibit the induction of in-terferon and apoptosis induced by double-stranded RNA (dsRNA)(5,29).Production of interferon during a viral infec-tion is dependent on the activation of several transcription factors,including interferon regulatory factor3(IRF3),NF-?B,and ATF2(reviewed in references3and13).IRF3is central for induction of antiviral genes,such as alpha interferon,RANTES,ISG-15,ISG-54,ISG-56,and inducible nitric oxide(14).IRF3is expressed constitutively as two forms, one of which is phosphorylated at its N terminus(27).In unstimulated cells,IRF3shuttles between the nucleus and the cytoplasm,with cytoplasmic localization predominating(18). During viral infection,dsRNA produced during viral replica-tion activates the latent IRF3via phosphorylation on C-termi-nal serine residues(20,30,32).It is thought that there are multiple pathways leading to activation of IRF3following virus infection(31),with a number of kinases involved,including among others,DNA-dependent protein kinase(16),IKK ep-silon,and TBK1(10,33).Recent work has described an im-portant pathway distinct from the dsRNA-dependent protein kinase R in the activation of IRF3by viral RNA(34).The activated IRF3then dimerizes and translocates to the nucleus, where it can bind one of the histone acetylases,CREB binding protein(CBP)or p300(44).This causes IRF3localization to become predominantly nuclear.IRF3and CBP/p300form a virally activated factor as part of the enhanceosome which binds to the beta interferon promoter and stimulates inter-feron production(41).

A number of small RNA viruses encode proteins that block interferon induction through inhibition of IRF3activity;for the?aviviruses,several studies have looked for a mechanism of blocking IRF3.The serine protease complex NS3/4A of hep-atitis C virus expressed from subgenomic replicons inhibits IRF3phosphorylation and translocation to the nucleus(11). Also,cells infected with both noncytopathic and cytopathic strains of the pestivirus BVDV prevent IRF3binding to DNA, although the transcription factor does translocate to the nu-cleus in response to infection with a heterologous virus(1,2).

*Corresponding author.Mailing address:Department of Immunol-ogy,Institute for Animal Health,Ash Road,Pirbright,Surrey GU24

0NF,United Kingdom.Phone:441483231090.Fax:441483232448.

E-mail:penny.powell@https://www.360docs.net/doc/729705035.html,.

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A variety of other virus families have the ability to block in-terferon through IRF3,including the paramyxoviruses(7), Ebola virus(4),and Bunyamwera virus(17).

In the present study,we show that the pestivirus CSFV inhibits interferon induced both by a heterologous virus,the alphavirus Sindbis virus,and by dsRNA.In contrast to?ndings for BVDV,CSFV infection did not induce IRF3translocation to the nucleus.However,there was no inhibition of the con-stitutive shuttling of IRF3between the cytoplasm and nucleus, shown by its accumulation in the nucleus following blockage of nuclear export by leptomycin B treatment.Interestingly,the cytoplasmic form of IRF3was lost from cells during CSFV infection,becoming undetectable after24h postinfection (hpi).The loss of IRF3was not affected by proteolysis inhib-itors and was concomitant with viral protein https://www.360docs.net/doc/729705035.html,ing constructs containing the IRF3promoter upstream of a lucif-erase reporter gene,we present evidence that the loss of IRF3 is due to inhibition of transcription in CSFV-infected cells. Recently,the importance of the N-terminal protease(N pro) of CSFV in virulence and in the inhibition of interferon pro-duction has been demonstrated(23,27).N pro is a cysteine autoprotease which cleaves itself from the core protein(28);it is also nonessential,as it is not required for virus replication in culture(23).In animals,viruses with a deletion in N pro were attenuated and protected the animal from lethal doses of highly virulent CSFV(23).Importantly,mutant virus with a deletion in the N-terminal protease N pro abrogated the inhi-bition of interferon production observed in wild-type virus (27).In our study,we have extended these?ndings by express-ing CSFV N pro in cells,and we have found that this single viral protein not only inhibits interferon production induced by Sindbis virus but N pro expression also results in a loss of IRF3 protein which is similar to that observed in cells infected with virus.

MATERIALS AND METHODS

Reagents.The rabbit anti-IRF3antibody was a gift from Michael David (UCSD,California)and was raised against amino acids107to208of human IRF3fused to glutathione S-transferase.The antibody recognizing N pro was raised in rabbits by immunizing them with peptides with the sequence KTNKQ KPMGVEEPVYDATGKPLFGDPS,which corresponds to amino acids11to37 of the Brescia strain of CSFV.Alpha and gamma tubulin antibodies were from Sigma.CSFV was stained with WH303anti-E2monoclonal antibody(9).Sindbis virus was stained with a rabbit anti-E2polyclonal antibody provided by Sondra Schessinger(Washington University Medical School,St.Louis,Missouri).The plasmid encoding green?uorescent protein(GFP)-IRF3was a kind gift from John Hiscott(McGill University,Montreal,Canada).The IRF3promoter plas-mids pIRF3-79Luc and pIRF3-80Luc,containing fragments of the5?upstream region of the IRF3gene(from KpnI to HindIII and BamHI to HindIII,respec-tively)cloned upstream of the luciferase cDNA in the pGL3basic vector(21) were a gift from Paula Pitha-Rowe(Johns Hopkins School of Medicine,Balti-more,Maryland).

Cell culture and viruses.Cells were maintained at37°C in5%CO2.PK15 cells,derived from a pig kidney,were grown in Dulbecco’s modi?ed Eagle’s medium,10%fetal calf serum(demonstrated to be BVDV free),100units/ml glutamine,and penicillin-streptomycin.The Max cell line is from an inbred NIH minipig major histocompatibility complex d/d haplotype,kindly provided by A. Saalmuller,and was grown in Iscove’s modi?ed Dulbecco’s medium,10% BVDV-free fetal calf serum,100units/ml glutamine,and penicillin-streptomycin. MDBK cells,stably transfected with a construct in which the human interferon-induced MxA promoter drives transcription of a chloramphenicol acetyltrans-ferase(CAT)cDNA(MDBK T2cells)were provided by Bryan Charleston(12). The virulent isolate of CSFV Brescia,used for all infections,was kindly provided by the Institute for Animal Science and Health,Lelystad,The Netherlands(43),and adapted for cell culture by serial passage on Max cells and PK15cells as described by Van Gennip et al.(39).Virus was isolated by freeze-thaw lysis, titrated by immunostaining with anti-E2antibody WH303(5),and used in ex-periments at a multiplicity of infection(MOI)of250%tissue culture infective doses(TCID50)per cell.Sindbis virus was generated from an infectious cDNA clone provided by Sondra Schessinger(SinTOTO1101).Brie?y,cDNA encoding the entire Sindbis virus genome was linearized with XbaI.RNA(?10kb)was transcribed in vitro with SP6RNA polymerase using the Message Machine kit (Ambion).RNA was electroporated into BHK cells,and cells were grown for 24h.Virions were harvested from the supernatant and titrated by enzyme-linked immunosorbent assay using an anti-Sindbis virus antibody(a gift from S.Schess-inger).Sindbis virus was adapted to PK15cells and Max cells by serial passages and used to infect cells at an MOI of2TCID50per cell.In some experiments, Sindbis virus strain Edgar339obtained from the national collection of patho-genic viruses(CAMR)was used to stimulate interferon from PK15cells.

IRF3promoter transfections and luciferase assay.PK15cells were infected with CSFV for24h and then transfected with IRF3promoter plasmids,either pIRF3-79luc or pIRF3-80luc(21),using Fugene6(Roche).Cell extracts were collected48h after transfection,and the luciferase assay was performed by following the manufacturer’s instructions(Promega).

Interferon bioassay.Interferon bioactivity was measured using a sensitive reporter gene assay consisting of MDBK cells stably transfected with Mx-CAT (MDBK-t2cells)(12).Interferon was induced in PK15cells by infection with Sindbis virus or by transfection with pIpC(100?g/ml;Sigma)using Fugene6 (Roche).Cells were washed thoroughly1hour after treatment,and media were removed at the time point speci?c for each experiment.Supernatants were heated to56°C for1hour to inactivate viruses or treated with RNase to remove residual pIpC.MDBK-t2cells,maintained in10?g/ml blasticidin,were seeded into six-well plates.Cells were incubated with sample supernatants overnight. Cell extracts were prepared by repeated freezing-thawing,and the protein con-centration was measured by bicinchoninic acid(Pierce).Lysates(30?g)were assayed for CAT using an enzyme-linked immunosorbent assay(Roche). Western blots.Total cell extracts were prepared by lysis of cells in boiling sodium dodecyl sulfate(SDS)sample buffer.Protein extracts were quanti?ed with the bicinchoninic acid kit(Pierce).Cell lysates,containing equal amounts of protein,were separated on SDS-polyacrylamide gel electrophoresis gels and transferred to a Hybond-C membrane(Amersham).Blocking and subsequent incubation with primary and secondary antibodies were performed in5%dry skim milk dissolved in phosphate-buffered saline(PBS)and0.02%Tween20. Filters were probed overnight at4°C with the primary antibodies.After extensive washing,the immune complexes were detected with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antiserum as appropriate (Bio-Rad Laboratories)followed by an enhanced chemiluminescence reaction (Pierce).

Subcellular fractionation.PK15cells,either uninfected or infected with CSFV,were washed in PBS,scraped,and pelleted.Nuclear and cytoplasmic extracts were prepared using the ReadyPrep protein extraction kit according to the manufacturer’s instructions.Brie?y,the cells were resuspended in CPEB buffer,incubated30min on ice,and then passed through a syringe needle.The lysates were centrifuged,and the supernatant was kept as a cytoplasmic fraction. The nuclear pellet was resuspended in PSB buffer and then centrifuged at maximum speed.This last step was repeated twice.The last supernatant is used as a nuclear fraction extract.Equivalent amounts of cytoplasmic and nuclear protein were separated by10%SDS-polyacrylamide gel electrophoresis.Proteins were transferred to a Hybond-C membrane(Amersham)and probed with the relevant antibodies.

Cloning and expression of the N-terminal protease,N pro.PK15cells were infected with CSFV at an MOI of2.0TCID50/cell for0,8,18,and24hpi.The cells were washed and lysed in Trizol(Invitrogen).Total RNA was extracted, resuspended in water,and reverse transcribed using avian myeloblastosis virus reverse transcriptase and random hexanucleotide primers at42°C for1hour. Ampli?cation of the N pro open reading frame with Taq polymerase(Promega) was carried out by PCR with15cycles of94°C for1min,55°C for1min,and72°C for1min using forward primer5?-ATGGAGTTGAATCATTTTGAACTTTTA TAC-3?and reverse primer5?-GCAACTGGTAACCCACAATGGACA-3?.The PCR product was cloned into pcDNA3.1and transfected into PK15cells using Fugene6(Roche).For stable expression in all cells,either cultures were grown in G418(1mg/ml;Invitrogen)for several weeks or,where indicated,cells were cotransfected with pBabe-puro(0.5?g/ml;obtained from Silvia Soddu,Regina Elena Cancer Institute)and selected for3days in puromycin(1.5?g/ml;Sigma). N pro expression was enhanced by infecting cells with vaccinia virus modi?ed

7240LA ROCCA ET AL.J.V IROL.

vaccinia Ankara (MVA)T7carrying the T7RNA polymerase cDNA for 24h before cell lysis.

Immunohistochemistry and ?uorescence microscopy.Porcine kidney cells (Max)were grow on 13-mm glass coverslips to 50%con?uence and infected with CSFV for 24h.Cells were then transiently transfected using Lipofectamine (Invitrogen)with a plasmid containing IRF3-GFP and left overnight before ?xing in 100%methanol,permeabilizing in 0.1%Triton X-100,and blocking in PBS containing 30%normal goat serum and 0.2%gelatin.CSFV-infected cells were stained with anti-CSFV E2antibody (WH303).Sindbis virus was stained using an anti-Sindbis virus E2antibody.In some experiments,leptomycin B was added at 10nM for 2hours before ?xing.Cells were incubated with Alexa Fluor 488-or Alexa 594-conjugated secondary antibody for 60min before being stained for 5min with 4?,6?-diamidino-2-phenylindole (DAPI)(Sigma).Cells were examined using a Nikon E800microscope or with a Leica TCS NT confocal microscope.

RESULTS

CSFV inhibits interferon production by a heterologous virus and by dsRNA.Interferon secreted from PK15cells was mea-sured using a sensitive bioassay in which supernatants were assayed on cells expressing the Mx-CAT reporter gene,and CAT expression was measured by enzyme-linked immunosor-bent assay.We induced interferon from PK15cells using a heterologous virus,the alphavirus Sindbis virus.The charac-teristics of Sindbis virus infection of PK15cells are shown in Fig.1a.Although there were lower viral titers and longer cell survival seen in these cells than in BHK cells,there was a substantial amount of interferon produced even by 2hpi

(Fig.

FIG.1.(a)Sindbis virus replication in PK15cells and kinetics of interferon production.SV induced similar levels of interferon secre-tion from PK15cells after 2,4,6,and 48hpi (left graph).The optimum virus titer was determined by decreasing dilutions from 16-fold to 1-fold (MOI of 2TCID 50/cell).PK15cells treated with heat-inacti-vated (HI)Sindbis virus did not induce interferon (right graph).(A to C)Sindbis virus replicates in CSFV-infected cells.Cells were ?xed in methanol and stained for viral proteins.(A)Cells stained with rabbit anti-E2glycoprotein from Sindbis virus and counterstained with goat anti-rabbit conjugated to Alexa 594(red);(B)cell stained for E2glycoprotein of CSFV and counterstained with goat anti-mouse Alexa 488(green);(C)merge to show both viruses replicating in same cell.(b)CSFV inhibits interferon induction by the heterologous virus,Sind-bis virus and by dsRNA.Supernatants from noninfected PK15cells (control)or PK15cells induced to produce interferon by SV or syn-thetic dsRNA (pIpC)were tested on Mx-CAT reporter cells.PK15cells were infected with CSFV for 48h,and interferon was measured in supernatants from unstimulated cells (CSFV)or after induction with the heterologous virus SV (CSFV ?SV)or with synthetic dsRNA (CSFV ?pIC).

V OL .79,2005LOSS OF IRF3IN CELLS INFECTED WITH CSFV 7241

1a,left graph).Increasing incubation times or titer of Sindbis virus did not increase interferon production.For the interferon assay,supernatants from Sindbis virus-infected PK15cells were heat treated before addition to MDBK cells to prevent the virus from inducing interferon from the reporter cell line.Heat treatment of Sindbis virus completely prevented its ability to induce interferon (Fig.1a,right graph).To determine whether Sindbis virus could superinfect and replicate in PK15cells infected with CSFV,cells were infected with CSFV for 24h and then infected with Sindbis virus overnight.Infection of both PK15cells and Max cells with CSFV at an MOI of 2TCID 50/cell resulted in 100%of cells infected with CSFV (staining for CSFV E2is shown in Fig.2D and G).For double labeling cells infected with the two viruses,cells were stained for both the E2antigen of Sindbis virus and the E2glycopro-tein of CSFV (Fig.1a,panels A to C).Both viral antigens were detected in the same cell,indicating that Sindbis virus could infect and replicate in the presence of CSFV.Sindbis virus

E2

FIG.2.CSFV does not stimulate IRF3translocation to the nucleus.(A to C)In control experiments,Sindbis virus promoted IRF3translocation to the nucleus.Max cells were transfected with GFP-IRF3(B,green)and infected with Sindbis virus for 24h (A,stained for Sindbis virus E2in red).(C)IRF3is located in the cytoplasm of uninfected cells but is seen as nuclear speckles in Sindbis virus-infected cells.(D to F)In CSFV-infected cells,IRF3is located in the cytoplasm.CSFV-infected cells were detected with an anti-CSFV E2antibody (D).Cells were transfected with a GFP-IRF3plasmid (E),and IRF3was located to the cytoplasm of CSFV-infected cells (F).(G to I)Constitutive shuttling of IRF3to the nucleus is maintained in CSFV-infected cells.Max cells infected with CSFV for 48h (G)and transfected with GFP-IRF3(H)were treated with leptomycin B to block the nuclear export pathway.IRF3accumulated in the nucleus,demonstrating no effect of CSFV on nuclear traf?cking of IRF3(I).

7242LA ROCCA ET AL.J.V IROL .

levels and the titer of secreted virus were similar to those of cells infected with Sindbis virus alone.Interestingly,there was no change in the rate of cell death in cells infected with both viruses,with both CSFV-infected and uninfected PK15cells dying between24and48hpi with Sindbis virus,showing that the inhibition of interferon by CSFV had no effect on Sindbis virus-induced cell death.These results are in agreement with work on BVDV and Semliki Forest virus,an alphavirus similar to Sindbis virus,where coinfection did not block Semliki Forest virus-induced apoptosis and also enhanced plaque size(1). Sindbis virus or double-stranded RNA was then used to induce interferon from CSFV-infected cells.Both control PK15cells and those infected with CSFV did not secrete in-terferon(Fig.1b;5).Infection with Sindbis virus(SV)for2h or transfection with pIpC for2h induced interferon,measured as pg/ml of CAT from the Mx-CAT reporter cells(Fig.1b). Preinfection with CSFV inhibited interferon production in-duced by both the heterologous virus(SV)and dsRNA treat-ment(pIpC).We went on to characterize the mechanism of inhibition of interferon synthesis.

IRF3does not translocate to the nucleus in CSFV-infected cells.It has been shown that in cells infected with the related pestivirus BVDV,IRF3translocates to the nucleus but does not bind to DNA and induce interferon(1).We investigated the subcellular distribution of IRF3in cells infected with

CSFV.The localization of IRF3was monitored using a plas-mid encoding an IRF3-GFP fusion protein(20).In control experiments,the IRF3plasmid was transfected into cells over-night,and these cells were then infected for24h with Sindbis virus(Fig.2A to C).In uninfected cells,IRF3localization was predominantly in the cytoplasm.In each cell infected with Sindbis virus,IRF3was translocated from the cytoplasm to the nucleus.In contrast,in CSFV-infected cells(Fig.2D to F), IRF3remained predominantly in the cytoplasm,although oc-casional cells show some nuclear localization.This suggests that there is no change in the localization of IRF3in CSFV-infected cells compared to unstimulated cells,where IRF3 constitutively cycles between the cytoplasm and the nucleus, with cytoplasmic localization predominating.In the next ex-periment,we treated CSFV-infected cells with leptomycin B to prevent the nuclear export of IRF3.IRF3accumulated in the nucleus of CSFV-infected cells(Fig.2G to I).Uninfected PK15cells treated with leptomycin B showed an accumulation of GFP-IRF3in the nucleus identical to that of CSFV-infected cells(data not shown).These results show that IRF3was able to shuttle into the nucleus in CSFV-infected cells,demonstrat-ing that traf?cking of this factor is not blocked during infection. Taken together,the results indicate that infection with CSFV does not provide an effective signal for IRF3nuclear translo-cation and retention and that IRF3distribution in CSFV-infected cells is similar to that seen in uninfected cells.

IRF3is lost from PK15cells infected with CSFV.We showed above that IRF3is not translocated to the nucleus following CSFV infection.In the next experiments,IRF3pro-tein was analyzed biochemically by Western blot analysis in the nuclear and cytoplasmic fractions.As with the human protein, porcine IRF3was detected by Western analysis of PK15cell lysates as a doublet of53to55kDa(Fig.3).In uninfected cells, IRF3was seen in the cytoplasm and not in the nuclear fraction (Fig.3,top panel).The same samples were probed with an anti-alpha tubulin antibody to con?rm the purity of the cyto-plasmic fractions.In CSFV-infected cells,IRF3was also seen in the cytoplasm and not the nucleus,and moreover,there was a decrease in IRF3protein seen in the cytoplasm compared to uninfected cells.We did not detect multiple forms of IRF3 migrating faster than the two basal forms on denaturing or native gels in either the nuclear or cytoplasmic fraction follow-ing CSFV infection.The biochemical fractionation data con-?rm the results with GFP-IRF3(Fig.2),showing that infection with CSFV does not stimulate translocation of IRF3to the nucleus.In addition,it showed that IRF3is lost directly from the cytoplasm of CSFV-infected cells without prior activation through phosphorylation and translocation to the nucleus The expression of the two forms of IRF3at53to55kDa was monitored over a time course of infection with CSFV(Fig.4). The higher-molecular-weight protein was always more abun-dant than the smaller protein in these cells.In HeLa cells,two protein forms have been identi?ed,the lower-molecular-weight form(form I)represented nonphosphorylated IRF3, while the higher-molecular-weight form(form II)may repre-sent a form phosphorylated on the N terminus,which does not appear to activate the factor(30).Interestingly,by20hpi,there was signi?cantly less of the IRF3protein doublet.By22hpi, the upper form had completely disappeared,with the lower form remaining but at a reduced level(Fig.4,top).Expression of viral protein in these same samples was then checked using an antipeptide antibody to N pro.N pro is cleaved from the CSFV polyprotein to give a?nal protein product of19kDa.A 19-kDa protein appeared at8hpi and increased up to24hpi. Hence,IRF3protein levels decreased at a time when viral protein expression was increasing.

After activation by Sendai virus and double-stranded RNA, IRF3has been shown to be degraded by the proteasome(20, 32).Therefore,we added the proteasome inhibitor MG132 and inhibitors of cellular proteases,including the cysteine

pro-FIG.3.Subcellular fractionation of CSFV-infected cell lysates shows a loss of IRF3from cytoplasm.Lysates from PK15cells either uninfected(?)or24hpi with CSFV(?)were separated into nuclear and cytoplasmic fractions and analyzed by immunoblotting.(Upper panels)IRF3was detected as a doublet at53to55kDa.There was a decrease in the cytoplasmic form of IRF3following CSFV infection. (Middle panels)Subcellular fractionation was assessed using an anti-?-tubulin antibody to show separation of the nuclear and cytoplasmic proteins.(Lower panels)?-Tubulin shows equal lane loading of cyto-plasmic and nuclear extracts.

V OL.79,2005LOSS OF IRF3IN CELLS INFECTED WITH CSFV7243

tease inhibitor E64D and serine protease inhibitors,at 18hpi with CSFV.None of these protease inhibitors was able to block loss of IRF3(data not shown).We investigated whether the loss of IRF3was due to transcriptional down-regulation of the IRF3gene.The promoter region of the IRF3gene,either pIRF3-79from KpnI-HindIII or pIRF3-80from BamHI-Hin-dIII cloned upstream of luciferase,was transfected into PK15cells (Fig.4,bottom).The promoter was found to have basal expression in these cells similar to that seen in human cells (21).When transfected into cells infected with CSFV for 48hpi,basal activity was inhibited to 20%of control values in CSFV-infected cells.The loss of IRF3protein from CSFV-infected cells was therefore due to down-regulation of tran-scription from the IRF3gene.

Expression of the N-terminal protease N pro inhibits inter-feron production and promotes loss of IRF3.Previous work (27)using a recombinant virus with a deletion of the open reading frame of the N-terminal protein N pro showed that this virus no longer inhibited interferon production and dsRNA-induced apoptosis.These workers suggested that N pro plays an important part in innate immune evasion.We investigated whether expression of N pro alone could inhibit interferon in-duction by a heterologous virus (Fig.5).Two types of cell lines were developed.In the ?rst experiment,PK15cells were co-transfected with a plasmid encoding N pro and with pBabe-puro (experiment 1).In this experiment,PK15cells expressing N pro were selected in puromycin for 3days [PK15(N pro )].In exper-iments 2to 4,cells were transfected with N pro in pcDNA3and stable transfectants were selected for by treatment with G418for several weeks.The experiments differed in the length of Sindbis virus infection (see the legend to Fig.5).The cloned cell lines expressing N pro were analyzed for constitutive secre-tion of interferon by testing supernatants on the MDBKt2Mx-CAT reporter cell line.Expression of N pro without stimu-lation of PK15(N pro )cells did not induce interferon (Fig.5).

Infection of PK15cells with Sindbis virus stimulated interferon production,as shown in Fig. 1.Signi?cantly,however,PK15(N pro )cells infected with Sindbis virus for 3.5h (experi-ments 1,2,and 4)or 48h (experiment 3)showed very little induction of interferon.Individual experiments are shown be-cause of the variation on Sindbis virus-induced interferon from control PK15cells.

We checked that N pro was being expressed in the cloned cell lines by analyzing RNA and protein production (Fig.6).RNA levels of N pro determined by reverse transcription-PCR were high in PK15(N pro )cells,expressing more N pro RNA than

an

FIG.4.IRF3loss from CSFV-infected cells is through inhibition of gene transcription.(Top)Time course of infection of PK15cells with CSFV.(Upper panels)IRF3protein,seen as a doublet at 53to 55kDa,is lost from cells from 18hpi with CSFV;(Middle panels)CSFV infection of cells was monitored using an antipeptide antibody to N pro and shows an increase in viral gene expression from 8hpi;(Lower panels)equal lane loading is shown by probing the same samples for ?-tubulin using mouse anti-?-tubulin antibody.(Bottom)Transcrip-tional inhibition of the IRF3gene.IRF3promoter plasmids (pIRF3-79Luc and pIRF3-80Luc)were transfected into uninfected PK15cells or PK15cells 24hpi with CSFV.CSFV infection inhibited IRF3gene transcription to 20%of the basal

level.

FIG.5.N pro -expressing cells [PK(N pro )]show decrease in Sindbis virus-induced interferon production.The graph shows secretion of interferon from control cells (PK15)or after induction with Sindbis virus (PK15?SV)and from cell stably expressing N pro without inducer [PK15(Npro)]and following induction with Sindbis virus [PK(Npro)?SV].In experiment 1,cells were selected for 3days in 0.5?g/ml puromycin and challenged with Sindbis virus for 3.5h.In ex-periments 2to 4,cells were selected in G418and challenged with Sindbis virus for 3.5h (experiments 2and 4)or 48h (experiment 3).Interferon produced from supernatants of PK15cells expressing N pro were measured on Mx A-CAT reporter cells measured as pg/ml.

7244LA ROCCA ET AL.J.V IROL .

equivalent amount of RNA from CSFV-infected cells (Fig.6A).However N pro protein levels were low in PK15(N pro )cells.Protein levels were only detectable when cells were infected with vaccinia virus MVA T7,enabling the expression of T7RNA polymerase to enhance the level of N pro being produced by the N pro pcDNA3plasmid (Fig.6B).Lysates from PK15(N pro )cells were probed for IRF3.Interestingly,very little IRF3was detected compared to control cells transfected with vector alone (Fig.6C).In PK15(N pro )cells,the lower-molecular-weight form has completely disappeared,leaving reduced amounts of the higher-molecular-weight form of IRF3.These results correspond to the effect seen in CSFV-infected cells.However,at 24hpi with CSFV,the higher-molecular-weight form of IRF3disappears,leaving reduced amounts of the lower-molecular-weight form of IRF3.

DISCUSSION

In this study,we have identi?ed a novel mechanism for CSFV evasion of the host innate immune response to infec-tion.CSFV infection can potently block the synthesis of inter-feron induced by two pathways,a heterologous virus,Sindbis virus,and by double-stranded RNA.Sindbis virus stimulates

the translocation of IRF3into the nucleus,where it plays a crucial role in the production of interferon by forming part of the enhanceosome complex that binds upstream of the beta interferon promoter.IRF3is also a crucial regulator of other proin?ammatory genes with ISRE sites,including RANTES,ISG15,ISG56,and interleukin-15.An IRF3-GFP fusion pro-tein was translocated into the nucleus following Sindbis virus infection and was found as discrete dots which may represent a distinct nuclear subcompartment.Although large amounts of interferon were induced by Sindbis virus,higher-molecular-weight phosphorylated forms of IRF3were not detected even on native gels.Phosphorylation on C-terminal serine residues of IRF3is a characteristic of activation by negative-stranded RNA viruses such as measles virus and Sendai virus (3).Sind-bis virus,a positive-stranded RNA virus,may activate different signaling pathways to IRF3than paramyxoviruses.In contrast to Sindbis virus,CSFV did not change the cellular distribution of IRF3,which was seen predominantly in the cytoplasm of infected cells.However,IRF3was able to translocate to the nucleus,since leptomycin B treatment led to nuclear accumu-lation of IRF3,thus indicating that CSFV infection did not impede nuclear traf?cking.Signi?cantly,not only did infection with CSFV fail to activate IRF3but we also showed that this factor,essential for the transcription of interferon,was lost from the cytoplasm of infected cells by 24hpi.Proteolytic inhibitors,including proteasome inhibitors,did not affect the rate of loss of IRF3,suggesting that IRF3was not being de-graded.We show a novel transcriptional down-regulation of the IRF3gene following CSFV infection.The transcriptional down-regulation of IRF3is a late event in infection,occurring after 18hpi,at a time when viral proteins are synthesized.Importantly,this mechanism does not explain how the imme-diate induction of interferon on CSFV entry into cells is in-hibited.We have shown for Sindbis virus that interferon can be detected in supernatants as rapidly as 2hpi.One would expect CSFV infection to have other mechanisms to inhibit this early increase in interferon secretion.Several viruses have been re-ported to have more than one mechanism to block host inter-ferons (22,24).Since IRF3is also important for mediating virus-induced apoptosis (15),loss of IRF3may play an impor-tant part in the establishment of the persistence of CSFV in vitro.Persistently infected cells are resistant to dsRNA-in-duced apoptosis (5),and whether this is due to lack of IRF3remains to be determined.Cells chronically infected with CSFV survive and divide normally for many weeks and have the same growth properties as uninfected cells (5).This is also the case for dominant-negative IRF3cell lines,which survive as normal,except that they do not produce interferon in re-sponse to virus (17,44).The mechanism of loss of transcription during CSFV infection is unknown.As well as effects on the IRF3gene,there may be general effect on host transcription,as yet unde?ned.In the case of Rift Valley fever virus,the transcription factor TFIIH is targeted,leading to suppression of host cellular RNA synthesis (6,19).

In contrast to other ?aviviruses,members of the Pestivirus genus encode an extra protein at the N terminus,N pro ,and we show here for CSFV that this protein is crucial to suppressing interferon production.N pro is an important virulence factor,since deletion leads to attenuation of the virus (23).Deletion of the N pro gene from the virus-induced interferon in

macro-

FIG.6.IRF3is lost from cells stably expressing the CSFV N pro gene.(A)Reverse transcription-PCR showing RNA levels of N pro in PK15cells (?),PK15cells infected with CSFV (csfv),and PK15stably transfected with N pro (Npro).(Upper panels)N pro -transfected cells express high levels of N pro RNA;(Lower panels)actin RNA shows equal lane loading.(B)Western blot showing N pro protein expression in CSFV-infected cells (csfv),control cells transfected with vector alone (vector),and PK15(N pro )cells.N pro protein is highly expressed as a 19-kDa protein in CSFV-infected cells (csfv),not detectable in control cells transfected with vector alone (lower vector band is a nonspeci?c band seen with this antibody).Expression of N pro was enhanced in PK15(N pro )cells from the T7promoter using vaccinia virus MVA T7(MVA).(C)Western blot showing disappearance of IRF3from PK15(N pro )cells.Control PK15cells (?)or cells infected with CSFV 24hpi (csfv)or transfected with empty vector (vector)or N pro (Npro)were lysed,and equal amounts of protein were blotted with anti-IRF3antibody.IRF3protein is detected as a doublet at 53to 55kDa.The higher-molecular-mass form of IRF3disappears from CSFV-infected cells,while most of the higher form and all of the lower form disappear from PK15cells expressing N pro but not from cells expressing vector alone.

V OL .79,2005LOSS OF IRF3IN CELLS INFECTED WITH CSFV 7245

phages and PK15cells in the absence of dsRNA(27).Here we demonstrate that expression of N pro alone was suf?cient to cause inhibition of Sindbis virus and pIpC-induced interferon synthesis and loss of IRF3protein.N pro is a cysteine-like au-toprotease with subtilisin-like activity(28).There is no known inhibitor,and the only known site of cleavage is between itself and the core protein.Although it is possible that N pro proteo-lytic activity degrades IRF3directly in cells,the time course of expression suggests that N pro has an indirect effect on the loss of IRF3,possibly targeting a factor involved in its transcrip-tion.

The list of viral proteins which inhibit IRF3is growing and includes the nonstructural proteins NS1and NS2of bovine respiratory syncytial virus,which block the activity of an IRF3-driven plasmid and phosphorylation of IRF3(7)and the NS1 protein of in?uenza virus(37).The Ebola virus VP35protein also inhibits activation of IRF3through inhibition of IRF-3 phosphorylation and subsequent dimerization(4).In the Bu-nyaviridae family,the NSs proteins of Bunyamwera virus and Rift Valley hemorrhagic fever virus were shown to block IRF-3-dependent promoter activity(6,17).This is the?rst demon-stration of transcriptional inhibition of IRF3by a virus involv-ing a speci?c viral protein,N pro.It remains to be determined which cellular factors are inactivated by this interesting pesti-virus protein.

ACKNOWLEDGMENTS

We thank the following people for reagents:Sondra Schlesinger (Washington University Medical School,St.Louis,Missouri)for Sind-bis virus reagents,John Hiscott(McGill University,Montreal,Canada) for GFP-IRF3,Paula Pitha Rowe(Johns Hopkins School of Medicine) for IRF3reporter constructs,Bryan Charleston(IAH)for Mx-CAT T2 cells,Michael David(UCD,California)for the anti-IRF3antibody, Silvia Soddu(Regina Elena Institute)for pBabe puro,and Armin Saalmuller(Tubingen,Germany)for the Max cell line.

This work was partly funded by the BBSRC and by DEFRA grant SE0773.

REFERENCES

1.Baigent,S.J.,G.Zhang,M.D.Fray,H.Flick-Smith,S.Goodbourn,and

J.W.McCauley.2002.Inhibition of beta interferon transcription by noncy-topathogenic bovine viral diarrhea virus is through an interferon regulatory factor3-dependent mechanism.J.Virol.76:8979–8988.

2.Baigent,S.J.,S.Goodbourn,and J.W.McCauley.2004.Differential acti-

vation of interferon regulatory factors-3and-7by non-cytopathogenic and cytopathogenic bovine viral diarrhoea virus.Vet.Immunol.Immunopathol.

100:135–144.

3.Basler,C.F.,and A.Garcia-Sastre.2002.Viruses and the type I interferon

antiviral system:induction and evasion.Int.Rev.Immunol.21:305–337. 4.Basler,C.F.,A.Mikulasova,L.Martinez-Sobrido,J.Paragas,E.Muhl-

berger,M.Bray,H.-D.Klenk,P.Palsae,and A.Garcia-Sastre.2003.The Ebola virus vp35protein inhibits activation of interferon regulatory factor3.

J.Virol.77:7945–7956.

5.Bensaude,E.,J.L.Turner,P.R.Wakeley,D.A.Sweetman,C.Pardieu,T.W.

Drew,T.Wileman,and P.P.Powell.2004.Classical swine fever virus induces proin?ammatory cytokines and tissue factor expression and inhibits apopto-sis and interferon synthesis during the establishment of long-term infection of porcine vascular endothelial cells.J.Gen.Virol.85:1029–1037.

6.Billecocq,A.,M.Spiegel,P.Vialat,A.Kohl,F.Weber,M.Bouloy,and O.

Haller.2004.NSs protein of Rift Valley fever virus blocks interferon pro-duction by inhibiting host gene transcription.J.Virol.78:9798–9806.

7.Bossert,B.,S.Marozin,and K.-K.Conzelmann.2003.Nonstructural protein

NS1and NS2of bovine respiratory syncytial virus blocks activation of inter-feron regulatory factor3.J.Virol.77:8661–8668.

8.Charleston,B.,M.D.Fray,S.Baigent,B.V.Carr,and W.I.Morrison.2001.

Establishment of persistent infection with non-cytopathic bovine viral diar-rhoea virus in cattle is associated with a failure to induce type I interferon.

J.Gen.Virol.82:1893–1897.

9.Edwards,S.,J.J.Sands,and J.W.Harkness.1988.The application of

monoclonal antibody panels to characterize pestivirus isolates from rumi-nants in Great Britain.Arch.Virol.102:197–206.10.Fitzgerald,K.A.,S.M.McWhirter,K.L.Faia,D.C.Rowe,https://www.360docs.net/doc/729705035.html,tz,D.T.

Golenbock,A.J.Coyle,S.-M.Liao,and T.Maniatis.2003.IKKεand TBK1 are essential components of the IRF3signaling pathway.Nat.Immunol.

4:491–496.

11.Foy,E.,K.Li,C.Wang,R.Sumpter,Jr.,M.Ikeda,S.M.Lemon,and M.

Gale,Jr.2003.Regulation of interferon regulatory factor3by the hepatitis

C virus serine protease.Science300:1145–1148.

12.Fray,M.D.,G.E.Mann,and B.Charleston.2001.Validation of an Mx/CAT

reporter gene assay for the quanti?cation of bovine type-I interferon.J.Im-munol.Methods249:235–244.

13.Goodbourn,S.,L.Didcock,and R.E.Randall.2000.Interferons:cell sig-

nalling,immune modulation,antiviral response and virus countermeasures.

J.Gen.Virol.81:2341–2364.

14.Grandvaux,N.,B.R.ten Oever,M.J.Servant,and J.Hiscott.2002.The

interferon antiviral response:from viral invasion to evasion.Curr.Opin.

Infect.Dis.15:259–267.

15.Heylbroeck,C.,S.Balachandran,M.J.Servant,C.DeLuca,G.N.Barber,R.

Lin,and J.Hiscott.2000.The IRF-3transcription factor mediates Sendai Virus-induced apoptosis.J.Virol.74:3781–3792.

16.Karpova,A.Y.,M.Trost,J.M.Murray,L.C.Cantley,and P.M.Howley.

2002.Interferon regulatory factor-3is an in vivo target of DNA-PK.Proc.

https://www.360docs.net/doc/729705035.html,A99:2818–2823.

17.Kohl,A.,R.F.Clayton,F.Weber,A.Bridgen,R.Randall,and R.Elliott.

2003.Bunymwera virus nonstructural protein NSs counteracts interferon regulatory factor3-mediated induction of early cell death.J.Virol.77:7999–8008.

18.Kumar,K.P.,K.M.McBride,B.K.Weaver,C.Dingwall,and N.C.Reich.

2000.Regulated nuclear-cytoplasmic localization of interferon regulatory factor3,a subunit of double-stranded RNA-activated factor1.Mol.Cell.

Biol.20:4159–4168.

19.Le May,N.,S.Dubaele,L.Proietti De Santis,A.Billecocq,M.Bouloy,and

J.-M Egly.2004.TFIIH transcription factor,a target for the Rift Valley hemorrhagic fever virus.Cell116:541–550.

20.Lin,R.,C.Heylbroeck,P.Pitha,and J.Hiscott.1998.Virus-dependent

phosphorylation of the IRF3transcription factor regulated nuclear translo-cation,transactivation potential,and proteasome-mediated degradation.

Mol.Cell.Biol.18:2986–2996.

21.Lowther,W.J.,P.A.Moore,K.C.Carter,and P.M.Pitha.1999.Cloning

and functional analysis of the human IRF-3promoter.DNA Cell Biol.

18:685–692.

22.Macdonald,A.,and M.Harris.2004.Hepatitis C virus NS5A:tales of a

promiscuous protein.J.Gen.Virol.85:2485–2502.

23.Mayer,D.,M.A.Hofmann,and J.-D.Tratschin.2004.Attenuation of clas-

sical swine fever virus by deletion of the viral N pro gene.Vaccine22:317–328.

24.Munoz-Jordan,J.L.,G.G.Sanchez-Burgos,https://www.360docs.net/doc/729705035.html,urent-Rolle,and A.Gar-

cia-Sastre.2003.Inhibition of interferon signalling by Dengue virus.Proc.

https://www.360docs.net/doc/729705035.html,A100:14333–14338.

25.Paton,D.J.,and I.Greiser-Wilke.2003.Classical swine fever–an update.

Res.Vet.Sci.75:169–178.

26.Paton,D.J.,J.J.Sands,J.P.Lowings,J.E.Smith,G.Ibata,and S.

Edwards.1995.A proposed division of the pestivirus genus using monoclo-nal antibodies,supported by cross-neutralisation assays and genetic sequenc-ing.Vet.Res.26:92–109.

27.Ruggli,N.,J.-D.Tratschin,M.Schweizer,K.McCullough,M.A.Hofmann,

and A.Summer?eld.2003.Classical wine fever virus interferes with cellular antiviral defenses:evidence for a novel function of N pro.J.Virol.77:7645–7654.

28.Rumenapf,T.,R.Stark,M.Heiman,and H.-J.Theil.1998.N-terminal

protease of pestiviruses:identi?cation of putative catalytic residues by site-directed mutagenesis.J.Virol.72:2544–2547.

29.Schweizer,M.,and E.Peterhans.2001.Noncytopathic bovine viral diarrhea

virus inhibits double-stranded RNA-induced apoptosis and interferon syn-thesis.J.Virol.75:4692–4698.

30.Servant,M.J.,B.ten Oever,C.LePage,L.Conti,S.Gessani,I.Julunen,R.

Lin,and J.Hiscott.2001.Identi?cation of distinct signaling pathways leading to the phosphorylation of interferon regulatory factor3.J.Biol.Chem.

276:355–363.

31.Servant,M.J.,N.Grandvaux,and J.Hiscott.2002.Multiple signaling

pathways leading to the activation of interferon regulatory factor3.Biochem.

Pharmacol.64:985–992.

32.Servant,M.J.,N.Grandvaux,B.R.ten Oever,D.Duguay,R.Lin,and J.

Hiscott.2003.Identi?cation of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor3in response to virus and double-stranded RNA.J.Biol.Chem.278:9441–9447.

33.Sharma,S.,B.R.ten Over,N.Grandvaux,G.-P.Zhou,R.Lin,and J.

Hiscott.2003.Triggering the interferon antiviral response through an IKK-related pathway.Science300:1148–1151.

34.Smith,E.J.,I.Marie,A.Prakash,A.Garcia-Sastre,and D.E.Levy.2001.

IRF3and IRF7phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or Ikappa B kinase but is blocked by Vaccinia virus E3L protein.J.Biol.Chem.276:8951–8957. 35.Summer?eld,A.,S.M.Knoetig,and K.C.McCullough.1998.Lymphocyte

7246LA ROCCA ET AL.J.V IROL.

apoptosis during classical swine fever:implication of activation-induced cell death.J.Virol.72:1853–1861.

36.Summer?eld,A.,S.M.Knoetig,R.Tschudin,and K.C.McCullough.2000.

Pathogenesis of granulocytopenia and bone marrow atrophy during classical swine fever involves apoptosis and necrosis of uninfected cells.Virology 272:50–56.

37.Talon,J.,C.M.Horvath,R.Polley,C.F.Basler,T.Muster,P.Palese,and

A.Garcia-Sastre.2000.Activation of interferon regulatory factor3is inhib-

ited by the in?uenza A virus NS1protein.J.Virol.74:7989–7996.

38.Thiel,H.J.,P.G.W.Plagemann,and V.Moennig.1996.Pestiviruses,p.

1059–1073.In B.N.Fields,D.M.Knipe,and P.M.Howley(ed.),Fields virology,3rd ed.Lippincott-Raven,Philadelphia,Pa.

39.Van Gennip,H.G.,A.C.Vlot,M.M.Hulst,A.J.De Smit,and R.J.

Moormann.2004.Determinants of virulence of classical swine fever virus strain Brescia.J.Virol.78:8812–8823.

40.Van Oirschot,J.T.1988.Description of the virus infection,p.1–25.In B.

Liess(ed.),Classical swine fever and related viral infections.Martinus Nij-hoff,Boston,Mass.

41.Wathelet,M.G.,C.H.Lin,L.Parekh,L.V.Ronco,P.M.Howley,and T.

Maniatis.1998.Virus infection induces the assembly of coordinately acti-vated transcription factors on the IFN-beta enhancer in vivo.Mol.Cell 1:507–518.

42.Weidman,M.K.,R.Sharma,S.Raychaudhuri,P.Kunda,W.Tsai,and A.

Dasgupta.2003.The interaction of cytoplasmic viruses with the nucleus.

Virus Res.95:75–85.

43.Wensvoort,G.,C.Terpstra,E.P.de Kluijver,C.Kragten,and J.C.War-

naar.1989.Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus.Vet.Microbiol.21:9–20.

44.Yoneyama,M.,W.Suhara,Y.Fukuhara,M.Fukada,K.Nishida,and T.

Fujita.1998.Direct triggering of the type I interferon system by virus infec-tion:activation of a transcription factor complex containing IRF3and CBP/ p300.EMBO J.17:1087–1095.

V OL.79,2005LOSS OF IRF3IN CELLS INFECTED WITH CSFV7247