Journal of Medical Virology 70:470–474 (2003)

Manganese Superoxide Dismutase InductionDuring Measles Virus Infection

Michael Wang,1 Joseph M. Howell,2 Jane E. Libbey,2 John A. Tainer,3 and Robert S. Fujinami2*1Department of Pediatrics, School of Medicine, University of Utah, Salt Lake City, Utah2Department of Neurology, School of Medicine, University of Utah, Salt Lake City, Utah3Department of Molecular Biology, The Scripps Research Institute, La Jolla, California

Measles virus infection of B-cells results inmarked alterations in proliferation and immuno-globulin production. Very little is known aboutthe changes of gene expression, if any, duringacute measles virus infection. To elucidate cel-lular genes that are induced during measlesvirus infection, we carried out a subtractiontechnique, representational differential analysis.The mitochondrial protein, manganese super-oxide dismutase (MnSOD), was upregulated inB-cells during measles virus infection. Althoughmeasles virus-infected B-cells did not secreteMnSOD into the environment, itwas found, usinganMnSODmimetic, that intracellularMnSODdidinhibit proliferation of the B-cells. MnSOD alsodecreases the titer of virus produced from infect-ed cells. Therefore, MnSOD seems to play a rolein the alteration of immune function seen uponinfection of B-cells with measles virus. J. Med.Virol. 70:470–474, 2003. � 2003Wiley-Liss, Inc.

KEY WORDS: paramyxovirus; morbillivirus;host gene expression

INTRODUCTION

Measles virus infection of B-cells can modulateimmune function. After infection, B-cells secrete asoluble factor that inhibits proliferation of cells oflymphoid lineage [Fujinami et al., 1998]. The immuno-suppressive soluble factor was neither interleukin-10,transforming growth factor b, nor a/b interferon. Theimmunosuppressive soluble factor has a size greaterthan 50 kDa and is heat labile at 558C. B-cells treatedwith the immunosuppressive soluble factor were unableto present antigen to T-cells. B-cells, when infected bymeasles virus, also cannot present antigen and have adiminished capacity to secrete immunoglobulin (Ig) orproliferate [McChesney et al., 1986, 1989; Fujinamiet al., 1998]; however, the mechanism(s) by whichmeasles virus alters proliferation is not known.

To address this issue, we determined on a limitedbasis what cellular genes are induced or repressed

during virus infection. The subtraction technique,representational differential analysis (RDA), usingmRNAs from measles virus infected versus uninfectedB-cells, was used to identify candidate genes that couldrepresent host genes that regulate the immunosup-pression causedbydirectmeasles virus infection of cells.We found that one of the genes, which is upregulatedduring measles virus infection, is manganese super-oxide dismutase (MnSOD).

The family of superoxide dismutases (SOD) can bedivided into two groups of proteins. The first group iscomprised of copper/zinc SODs that are present in thecytoplasm of both eukaryotes and bacteria [Fridovich,1974a,b, 1975; Beyer et al., 1991]. The second group ismanganese SODs and present normally within mito-chondria [Bannister et al., 1987; Ho and Crapo, 1988;Beyer et al., 1991]. SODs protect cells from oxida-tive damage and regulate superoxide concentrations.MnSOD functions as an antioxidant molecule thatprotectsmitochondria fromdamage by superoxides gen-erated during respiration and in response to cellularstress [Chance et al., 1979; Jones et al., 1997; Li andOberley, 1997; Manna et al., 1998; Takahashi et al.,1998; Epperly et al., 1999].

Various factors can initiate MnSOD induction. Heartmuscle has been shown to increase MnSOD activityafter ischemia and reperfusion [Das et al., 1993], and

M. Wang and J.M. Howell contributed equally to thissubmission.

Grant sponsor: National Multiple Sclerosis Society; Grantnumber: RG 29258; Grant sponsor: American Academy ofPediatrics; Grant sponsor: The Primary Children’s MedicalCenter.

M.Wang’s present address is Division of Hematology/Oncology/Bone Marrow Transplantation, The Children’s Hospital, Denver,1056 E 19th Avenue, B115, Denver, CO 80218.

*Correspondence to: Robert S. Fujinami, PhD, Department ofNeurology, School of Medicine, University of Utah, Salt Lake City,UT 84132. E-mail: Robert.Fujinami@hsc.utah.edu

Accepted 15 October 2002

DOI 10.1002/jmv.10419

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2003 WILEY-LISS, INC.

treatment with interleukin (IL)-1 [Nogae et al., 1995].Cytokines have been shown to induce MnSOD expres-sion via protein kinase C pathways [Meier et al., 1989;Wong et al., 1989]. Although the mechanism(s) of in-duction of MnSOD by stress, such as hyperoxia, andcytokines may be similar, Warner et al. [1996] haveshown that antioxidants blocked the induction ofMnSOD by cytokines, but not by hydrogen peroxide. Inaddition, the temporal induction by these two pathwayswas different.

Increased amounts of MnSOD also are detectable inthe serumof patientswith acute viral infections, such asinfluenza virus, human immunodeficiency virus (HIV),Epstein Barr virus (EBV) and cytomegalovirus (CMV)infections [Raoul et al., 1994, 1998; Knobil et al., 1998;Semrau et al., 1998]. In local or systemic inflammationboth increased oxidative metabolism and cytokinesinduce the expression of MnSOD [Janssen et al., 1993].This may protect the host against the cytokine-inducedoxidative burst caused by infectious pathogens.

As noted above, MnSOD mRNA was upregulatedupon infection. To investigate measles virus interac-tion with B-cells, the supernatant from measles virusinfected B-cells was tested for SOD activity with acolorimetric assay and a polyacrylamide gel electro-phoresis (PAGE) assay to determine whether cellssecreted MnSOD into the environment. The super-natant was also examined for the presence of a proteinof the appropriate size for MnSOD.

To study the effect of intracellular MnSOD, we usedits mimetic, manganese (III) tetrakis (4-benzoic acid)porphyrin (MnTBAP) that diffuses through cell mem-branes and is able to detoxify reactive oxygen specieswithin cells. MnTBAP has been shown recently toprevent apoptosis of activated T-cells caused by reactiveoxygen species [Hildeman et al., 1999].

We found that MnSOD was not secreted into theenvironment bymeasles virus infectedB-cells; however,intracellular MnSOD was capable of inhibiting prolif-eration of B-cells while also decreasing the amount ofvirus produced from infected cells. Thus,MnSOD seemsto be involved in the alteration of immune functionapparent upon infection of B-cells with measles virus.

MATERIALS AND METHODS

Cell Lines and Infection

An EBV-transformed B-cell line (JBB) was estab-lished from whole peripheral blood mononuclear cells(PBMCs) harvested from a healthy volunteer as des-cribed previously [Fujinami et al., 1998]. The cells weremaintained in RPMI 1640 medium (Invitrogen, Carls-bad, CA) supplemented with 10% fetal calf serum(Invitrogen) and antibiotics (Mediatech, Herndon, VA).

The Edmonston strain of measles virus (AmericanType Culture Collection, Rockville, MD) was used to in-fect or mock-infect, as described previously [Fujinamiet al., 1998], 106 JBB-cells per ml at a multiplicity ofinfection (MOI) of 1 as determined by viral plaque assay([Zurbriggen and Fujinami, 1989], data not shown). The

cells were cultured for 24 hr at 378C in 5% CO2. Theharvesting of supernatants containing the immuno-suppressive soluble factor from measles virus-infectedcells was carried out as described previously [Fujinamiet al., 1998].

Representational Differential Analysis (RDA)

After measles virus infection, the JBB-cells wereharvested and total RNA was isolated using TRIZOL1

Reagent (Invitrogen). Poly(A) mRNA was isolatedfrom total RNA using an oligo (dT)-cellulose column(Amersham Pharmacia Biotech, Piscataway, NJ);cDNA libraries were generated from mRNA isolatedfrom measles virus infected (Tester) and non-infected(Driver) JBB-cells by reverse transcription (Invitrogen).Residual mRNA was degraded with RNase H (Invitro-gen) and gaps were filled in using an enzyme cocktailconsisting of E. coli DNA polymerase I (Promega,Madison, WI) and E. coli DNA ligase (Invitrogen),followed by incubation with T4 DNA polymerase(New England Biolabs, Beverly, MA).

The subtractive hybridization process was carried outusing the PCR-Select cDNA Subtraction Kit (Clontech,Palo Alto, CA) as described previously [Lisitsyn et al.,1993].NewEnglandBiolabs supplied the frequent, four-base pair restriction enzyme RsaI used to digest thetester and driver cDNAs.

PCR amplified, subtracted tester and driver cDNAswere ligated into the plasmid vector pT-Adv using theAdvanTAge PCR Cloning Kit (Clontech) and trans-formed into TOP10F0 E. coli competent cells (Clontech).Bacterial transformants were spread onto Luria agarplates supplemented with kanamycin (50 mg/ml;Sigma, St. Louis, MO), 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-gal, 40 ml of 40 mg/ml stock; Invitrogen)and isopropylthio-b-galactoside (IPTG, 40 ml of 100 mMstock; Invitrogen). Plates were incubated at 378C over-night and 120 colonies were selected based on blue/white screening. New streak plates were generated forcandidate colonies.

Liquid cultures (100 ml) of each of the selected colonieswere prepared and used to PCR-amplify the putativeinserts via the T7 and M13 reverse promoter sequencespresent in thevector.Aliquots of eachPCRreactionwereelectrophoresed on 1% agarose gels and visualized withethidium bromide. Of the 120 starting colonies, 95 werefound to have a verifiable insert.

Dot blots were prepared in the manner described bythe PCR-Select Differential Screening Kit (Clontech).Probes were prepared, incorporating [a-32P]dCTP, fromforward- and reverse-subtracted cDNAs by the randomprimer method [Sambrook et al., 1989]. Blots werehybridizedwith probes overnight at 728Cand visualizedvia exposure to X-ray film for varying lengths of time.Autoradiographswere analyzed visually and 47 positivesamples were selected based on the presence of a spot onthe blot probed with the forward-subtracted cDNA andthe absence of the corresponding spot on the blot probedwith the reverse-subtracted cDNA.

MnSOD and Measles Virus 471

The 47 plasmids selected as bearing a differentiallyexpressed mRNA fragment were subjected to unidirec-tional sequencing at the DNA Sequencing Core Facilityat the University of Utah using the T7 promotersequence present in the vector. Sequence data wasanalyzed using BLAST (NCBI). The 19 plasmids carry-ing an insert for a measles virus gene were eliminatedfrom further consideration, as was the one plasmid thatcoded for vector only. The remaining 27 plasmids wereused to prepare slot blots produced and probed in amanner similar to that described above. Slot blots wereprobed with radiolabeled cDNA produced directly fromtester and driver mRNA by reverse transcription andrandom primer incorporation. Samples were run induplicate. Twelve differentially displayed plasmidswere identified by autoradiography and subjected tobidirectional sequencing and BLAST analysis.

MnSOD Activity Assays

Supernatants from B-cells treated with either theimmunosuppressive soluble factor, measles virus, ormock-infected were subjected to a colorimetric assay,the BIOXYTECH1 SOD-525TM SpectrophotometricAssay for Superoxide Dismutase Activity (OXIS HealthProducts Inc., Portland, OR), as described previously[Nebot et al., 1993]. A portion of those samples tested bythe colorimetric assay was tested via a PAGE analysismethod for activity and size [Beauchamp and Fridovich,1971;Sambrooket al., 1989]. To test for activity, aliquotsof the samples, approximately 25 mg of protein each,were electrophoresed into native polyacrylamide gels.The resultant gels were first stained with nitrobluetetrazolium (NBT, 2.43 mM; Sigma) and then withriboflavin (28mM;Sigma) andTEMED (28mM;Sigma).The gels were then exposed to UV light. To test for size,aliquots, of approximately equal amounts of protein,were electrophoresed into denaturing polyacrylamidegels and then stained with Coomassie (Sigma-Aldrich).

Proliferation and Plaque Assays

Proliferation assays, utilizing the JBB-cells, werecarried out as described previously [Fujinami et al.,1998], with some modification. JBB-cells were testedin proliferation assays in the presence of MnTBAP (100and 300 mM; Alexis Biochemicals, San Diego, CA), N-acetyl-L-cysteine (25 mM; Sigma), S-methylisothioureahemisulfate salt (500 mM; Sigma) and recombinanthuman MnSOD (100 and 500 mM; kindly provided byJohnA. Tainer). Plaque assays were carried out on Verocell monolayers as described previously [Fujinami andOldstone, 1981]. The virus titer produced from measlesvirus infected JBB-cells with or without MnTBAP(100 2 mM) was compared.

RESULTS

Representational Differential Analysis (RDA)

To better understand the molecular events caused bydirect measles virus infection, measles virus-infected

and uninfected cells were compared by RDA, conductedas described in the Methods. We found 47 clones/genes were upregulated during measles virus infection.As expected, 19 of 47 clones were found by partialsequencing to bemeasles virus-specific, representing allmeasles virus genes except the L (large or polymerase)gene. One clone coded for the vector. The remaining27 clones were analyzed by slot blotting. Twelve cloneswere further selected based on increased expression ininfected cells; the entire insert of these 12 clones wassequenced.Twocloneswere found to carrymeasles virusgenes and the remaining 10 clones represented hostcellular genes. Six clones, found to have homology orsimilarity to known cellular genes, were designatedIVI (inducible by virus infection) 1 through 6 (Table I).One of the genes, IVI-3, has 100% homology to humanMnSOD.

MnSOD Activity Assays

Todetermine ifB-cells releasedMnSODuponmeaslesvirus infection, supernatants were tested for MnSODactivity after mock-infection, treatment with the immu-nosuppressive factor secreted by infected B or T-cells[Fujinami et al., 1998; Sun et al., 1998] or measles virusinfection. They were tested by two different methods forsuperoxide dismutase activity. In a colorimetric assay,no superoxide dismutase activity was detectable (datanot shown). A portion of these samples, including thesupernatant containing the soluble factor, was assayedfor SOD activity on nitroblue tetrazolium-stained poly-acrylamide gels; no SOD activity was detected (data notshown). Further, denaturing polyacrylamide gel elec-trophoresis for size did not reveal a band at theappropriate size for MnSOD (data not shown). Largeprotein bands were present at approximately 23 and70 kDa, however, roughly the same size as the proteinwith antiproliferative properties identified in our pre-vious report on the characterization of the solubleimmunosuppressive factor [Fujinami et al., 1998].

TABLE I. Tentative Designation of IVI-1 to 6

Clone Tentative designation

IVI-1 KIAA09764 Protein of unknown function fromhuman brain mRNA

IVI-2 High homology to Rattus norvegicusUDP-glucoxe:ceremideglycosytranfersase MRAN (no humanhomologue reported).

IVI-3 Human mRNA for MnSODIVI-4 50% homology with mouse T-complex

proteins (novel protein fragment)IVI-5 Bears passing similarity to human

histone protein, a replication factorand may have an EGF-domain (novelprotein fragment)

IVI-6 Putative ATP-binding protein (novelprotein fragment)

472 Wang et al.

Proliferation and Plaque Assays

B-cells were tested in proliferation assays withMnTBAP to study the effect of intracellular MnSOD.The addition of MnTBAP caused a decrease in B-cellproliferation, but never to the extent of the soluble factor[Fujinami et al., 1998] or measles virus infection. Thisdecrease in proliferation was not due to cell death (datanot shown). The addition of MnTBAP to infected B-cellsdid not ameliorate the inhibition of proliferation caus-ed by measles virus infection (Table II) and did notprevent the majority of the infected cells from dying.Other anti-oxidants such as N-acetyl-L-cysteine andS-methylisothiourea hemisulfate salt had no effect onthe proliferation of our B-cell line (data not shown).Interestingly, the addition of recombinant humanMnSOD to B-cells in culture did not have an inhibitoryeffect on proliferation; in fact, the proliferation of thesecells seemed to increase. Plaque assays comparingmeasles virus titers of B-cells infected with measlesviruswithandwithoutMnTBAPshowed thatMnTBAP-treatedB-cells produced less infectious virus (Table III).

DISCUSSION

MnSOD is known to be induced by infection withseveral viruses [Raoul et al., 1994, 1998; Knobil et al.,1998; Semrau et al., 1998]. MnSOD is also induced inB-cells upon infection with measles virus. MnSOD doesnot seem to be the antiproliferative factor secreted bymeasles virus-infected lymphocytes we described pre-viously [Fujinami et al., 1998], however, but may con-tribute to the immunosuppression seen inmeasles virus

infection. MnSOD is upregulated quickly after measlesvirus infection, and may play a role in the limitedproliferation of infected lymphocytes. B-cells incubatedwith MnTBAP had a decreased rate of proliferation;however, toxicity caused by MnTBAP itself cannot beoverlooked. This decrease in proliferation may be animportantmechanism to limit viral burden presented tothe host. Treatment of measles virus infected B-cellswith MnTBAP resulted in a decreased production ofinfectious virus. Therefore, one consequence of increas-ed amounts of MnSOD may be to reduce the prolifera-tion of infected cells, thereby decreasing the amountof virus produced by infected lymphocytes and the viralburden during a time when the host is relatively immu-nosuppressed. Increased MnSOD production may alsorepresent a state in the cell where multiple oxidativesystems are not compensating in a coordinated fashionthereby sensitizing the cell to oxidative damage ulti-mately leading to cell death by apoptosis.

It is interesting to speculate about the complexities ofhost-virus interaction during infection. Measles virusinfection of B-cells increases the production of MnSOD;this response may be secondary to the increase inreactive oxygen species generated during infection.Reactive oxygen species have been shown to be involvedin the apoptosis of superantigen-activated T-cells[Hildeman et al., 1999]. Survival of these T-cells occur-red only after incubation with MnTBAP. A possibleexplanation as to why the proliferation of our measlesvirus infected B-cells was not rescued by the addition ofMnTBAP, is that the process of infection was ongoing inour systemand probably involves dysregulation of other

TABLE II. Intracellular Manganese Superoxide Dismutase Inhibits Proliferation But DoesNot Rescue Proliferation of Measles Virus Infected B Cells

3H-Thymidine cpm (�SEM)

JBB 81,480 1,448JBBþSupernatant 1:2 3,627 227JBBþMnTBAP 100 mM 71,240 4,207JBBþMnTBAP 300 mM 46,006 2,641JBBþ rhMnSOD 100 mM 92,468 2,645JBBþ rhMnSOD 500 mM 97,661 2,167JBBþMV MOI 1.0 19,929 77JBBþMVMOI 1.0þMnTBAP 100 mM 26,205 353JBBþMVMOI 1.0þMnTBAP 300 mM 21,105 1,698

TABLE III. Intracellular Manganese Superoxide Dismutase Inhibits Viral Production ofMeasles Virus From Infected B Cells

Measles virus titers (pfu/ml)

24 hr incubation 48 hr incubation 72 hr incubation

Mock-infected 0 0 0MOI 0.05 6.0� 102 4.2� 102 4.36� 104

MOI 0.5 2.4� 104 4.4� 104 1.78� 105

MOI 5.0 2.0� 105 2.0� 105 3.36� 105

Mock-infectedþMnTBAP 100 mM 0 0 0MOI 0.05þMnTBAP 100 mM 3.0� 102 — 7.0� 103

MOI 0.5þMnTBAP 100 mM 2.0� 103 1.28� 103 3.44� 104

MOI 5.0þMnTBAP 100 mM 2.92� 104 2.72� 104 4.12� 104

MnSOD and Measles Virus 473

free radical systems, which lead to the death of cellsinfected with measles virus.

The overexpression of MnSOD has been associatedwith the inhibition of proliferation, and contributes tocell death by oxidative agents, depending on levels ofperoxide enzymes. A large proportion of measles infect-ed B-cells die [Wang et al., 2002]. A potential cause ofdeath involves the dysregulation of free radical genera-tion and antioxidant systems. Thus, the inhibition ofproliferation seen in directly infected B-cells is, in part,due to the death of measles virus-infected cells by freeradical species.

The induction of MnSOD does not explain the globalimmunosuppression associated with measles. We haveshown previously that infection of lymphocytes withmeasles virus causes them to secrete a soluble factorthat inhibits proliferation of uninfected cells [Fujinamiet al., 1998; Sun et al., 1998]; we have also shown thatthis factor does not cause cell death. Therefore, its dif-ferences with MnSOD are broad. Although the identityof this factor remains unknown, this report emphasizesthe complexity of measles virus immunosuppressionand argues that the mechanisms that cause immuno-suppression of infected versus uninfected B-cells arenot the same.

ACKNOWLEDGMENTS

We thank J. Blackett, T. Alexander, and M. Stroupefor their excellent technical assistance, andK.Borick forthe preparation of the article. We also thank the DNASequencing Core Facility at the University of Utah.

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