Infectious cDNA clones of the DA strain of Theiler's murine encephalomyelitis virus.

Infectious cDNA Clones of the

DA

Strain

of

Theiler's

Murine

Encephalomyelitis

Virus

RAYMOND P.

ROOS,'*

STEVEN STEIN,1 YOSHIRO OHARA,3 JIANLIN FU,1 AND BERT L. SEMLER2 Department ofNeurology, University of Chicago Pritzker School of Medicine, Chicago, Illinois 606371; Department of

Microbiology and Molecular Genetics, College ofMedicine, University ofCalifornia, Irvine, California 927172; and Department ofNeurological Sciences, Tohoku University Schoolof Medicine, 1-1

Seiryo-Machi,

Sendai

980, Japan3

Received 13April1989/Accepted2August1989

TheDAstrainand other membersoftheTO subgroupofTheiler's murineencephalomyelitisvirusescause apersistent demyelinating infection, whereas theGDVII strain and other GDVII subgroupstrainscausean

acutelethalpolioencephalomyelitis. Wegeneratedaninfectious DAcDNAclone insertedintoa transcription

vector.Virus derivedfrom transfectionoftranscripts producedademyelinatingdiseaseindistinguishablefrom

that of wild-type virus. The infectious clone provides a critical reagent for the production of interstrain

recombinant virusestohelp identify genetic loci responsible forthebiologicalactivitiesof thestrains.

Although strains of Theiler's murine encephalomyelitis viruses(TMEV) belongtooneserotype,theycanbe divided

intotwo subgroupson thebasis of their different biological

activities (2, 3)as wellasreactivity withneutralizing

mono-clonal antibodies (4, 6). DA and other TO subgroup strains

cause apersistent demyelinating infection in mice. In

con-trast, GDVII and other GDVII subgroup strains are more

neurovirulent and produce an acute, fatal polioencephalo-myelitis withnopersistenceordemyelination. A major goal

for investigators of this virus group is the delineation of molecular determinants ofthevaried biological activities-neurovirulence, persistence,demyelination-of the strains.

A powerful tool in molecular pathogenesis studies of picornavirus has been theuseof infectiouscDNA clones of viralRNAs. In poliovirus,infectious clones of neurovirulent and attenuated strains have been recombined to produce chimeric cDNAs andrecombinant viruses to delineate ge-nome segments and nucleotides responsible for neuroviru-lence (5). Investigation of the TMEV model system has severalpotential advantagesoverpoliovirus studies. (i) The

mouseisboth the natural and experimental host for TMEV;

for thisreason,the effect of TMEV interstrainrecombinants

or mutated TMEV on disease in the natural host can be easily and effectively studied. (ii) The varied biological activities of strains from the two subgroups represent a

natural comparative system for investigations with TMEV recombinant viruses. (iii) TO subgroup strains manifest

biological activities that are unusual for

picornaviruses-persistenceand demyelination.

Togenerate afull-length clone, previously described DA

subgenomic cDNA clones in pUC9 (9) were digested with

restriction enzymes and then, inanumber ofsteps, ligated together (Fig. 1). pDAFL1 contains the full-length cDNA cloneof DA(including 13 adenylate residuesatthe 3' end of the heteropolymeric sequences) inserted into the PstI and BamHI sites of the transcription vector Bluescript pSK-(Stratagene).

We were concerned thatthe 69 nucleotides of the poly-linker between the T7 promoter and the 5' end of the DA

genome in pDAFL1 might interfere with infectivity. A

previous report demonstrated that the elimination of

nucle-*Correspondingauthor.

otides between the T7 promoter and the 5' end of the viral

genome increasesinfectivity oftranscripts derived froman

infectiouspoliovirus cDNA (13).For thisreason,we

reengi-neered the 5' end of the DA genome in pDAFL1 two nucleotides downstream from the T7 promoterto generate pDAFL3 (Fig. 2). Tobringthe5' end of the DAgenomein pDAFL1 nearer the T7 promoter, we synthesized paired

complementary oligonucleotides to replace the part of DAFL1 that extended fromaBglIsite(N472)inpSKtoN14 in the DA genome. The paired oligonucleotides (Fig. 2B) contained the T7 promoter 2 nucleotides upstream from the first14nucleotides of the DAgenome; oneendof thepaired oligonucleotideswas one-halfof theBglI recognitionsiteat pSKN472followedbyaunique ClaI site(tofacilitate future

manipulations),while the otherendwasone-half of the SmaI

recognition site at N12. The N14-N8093 fragment from SmaI-XbaI-digested pDAFL1 was ligated to the annealed paired oligonucleotides and the two pSKII-fragments to finally yieldpDAFL3 (Fig. 2A).

pDAFL1 was digestedwithXbaI and thentranscribed in

vitrowith T7polymerase by previouslypublishedmethods (14). The products of this transcription wereof the

appro-priate size when analyzed by electrophoresis on a 1%

agarose gel (data not shown). In vitro-derived transcripts

werequantitated by comparing asamplewithdilutionsofa

known control RNA and by using 10 ,uCi of [o-32P]GTP (specificactivity, 3,000Ci/mmol)perreaction.The in vitro-derived transcripts ofXbaI-digested pDAFL1 had a

maxi-mum infectivity of 1.5 x

103

PFU/,ug of RNA when

trans-fected into Lcells(1) (Table 1). In contrast,transcriptionof pDAFL3,which containedonlytwonucleotides between the T7 promoter and the 5' end of the DA genome, produced RNA with an increased infectivity averaging 1.8 x 104 PFU/,ug (Table 1). This infectivity was approximately 20-fold less than theinfectivityoftransfected virion RNA. The infectivitywas aresult of RNA since therewas acomplete

loss ofinfectivityaftertreatmentwith 10,ug ofRNase for 30 min, while there was no significant change in titer after

treatmentwith 5 U of DNasefor 30min (datanot shown). Virusproduced from transfectedpDAFL1 was neutralized by neutralizing monoclonal antibodies directed against TO subgroupstrains(4) (datanotshown).Thesequenceofthe 5'

end of RNA extracted from virusproducedfrom transfected

5492

0022-538X/89/125492-05$02.00/0

Copyright © 1989,AmericanSociety for Microbiology

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Hindl BamHI

T7-P(T3-P

BamH

ori

pSK-HindI/BamHI

digest

Isolate

large

fragment

T4

DNA

Ligase

BspHI(7496)

_

pSKn

BspHI/BamHI dige.

Isolate

DA7496-D

fragment

1I

(8093)

HindM

(7306)

pDAn3

HindIII/BamHI

digest

Isolate DA7306-DA8093 fragment

pDAf42-10

st

BspHI/PstI digest

k8093

Isolate

DA6689-DA74'

fragment

PstI BamHI

(orikd

pSK-PstI/BamHI

digest

96

Isolate

large

fragment

1

(930)

PstI

(1043)

pDAfSKn

pDAk300

PstI/NcoI

digest

Isolate

largefragment

(containing

DA6896-DA8093,

SK

sequences

and T7

promoter)

fragment

1-I

PstI(l)

AmpmHI

(8093)

FIG. 1. Construction ofaninfectious cDNA clone ofDAstrainin atranscription vector.pDAn3,pDAf42-10,pDAf42,andpDAk300are

DAsubgenomiccDNAclones inpUC9thathave beenpreviously described(9). Inthis figureandFig. 2,numbers refertothenucleotide number of theDAgenomeorSK,atranscriptionvector;numbers inparenthesescorrespondtotherestrictionenzymesiteonthe DA genome. The T3 promoter (T3-P) and T7 promoter (T7-P), theampicillin-resistant site(Amp),and theoriginsofreplication(ori)areshown.

pDAFL3

showeda terminus

indistinguishable

from that of DA

wild-type

viral RNA

(data

not

shown).

After transfection of L

cells,

virus was

picked

from a

plaque

and grown

for

oneandtwopassagesonBHK-21cells to produce stock virus for inoculation of nine 3-week-old

SJL/J

mice

(Jackson

Laboratory,

Bar

Harbor,

Maine).

His-topathological

evaluationof the centralnervoussystemof all theinfected mice showed

prominent inflammatory,

demyeli-nating

disease of the

spinal

cord 3 and 4 months after inoculation.

Figure

3showsa

section

ofa

spinal

cord

froma

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[image:2.612.96.526.82.617.2]

(12)

BglI

Clal SmaI

L T7-P

(1) (14)

I

T4DNA Ligase

`pSKH-BglI /XbaI

digest

IsolateBglI

(SK472)

-BglI

(SK2166)

and

BglI

(SK2166)-Xba

fragments

B BglI ClaI SmaI

.~~~~~~~+

I

I-AGGCATCGATAATACGACTCACTATAGGTTGAAAGGGGGCCC AAGTCCGTAGCTATTATGCTGAGTGATATCCAACTTTCCCCCGGG

(1) (14)

T7 promoter

SmaI (1

2)

-(1' ClaI

BaimHI

(8093)

BgI--I

FIG. 2. Construction of pDAFL3,aninfectiouscDNAcloneDAstrainin atranscription vector, inwhichpDAFL1wasreengineeredso

thatthe 5'end of the DA genomewasplaced twonucleotides downstream from theT7promoter. (A)Outline of the construction ofpDAFL3. The brackets representpaired oligonucleotides andaredetailed below. (B) Pairedoligonucleotides used in construction of pDAFL3 which containone-half ofaBglI restriction enzymerecognition site inSKII-,aClaI site,the T7promoter, and thefirst14nucleotides ofthe DA

[image:3.612.63.561.61.494.2]

genome upto oneendofaSmaIsite.

TABLE 1. Resultsof transfection into L-929cellsa

Sample Infectivity(PFU/j,g)

pDAFL1 invitro-transcribed RNA 2.5 x 101 5.0 x

102

1.5 x 103 pDAFL3 in vitro-transcribedRNA 3.6 x 104 1.0 x 104 1.4x 104 2.8 x 103

DAvirionRNA 3.6 x 105

aEachinfectivity resultwasobtainedin a separateexperiment.

mouse

sacrificed

3 months after

inoculation;

thereare

lym-phocytic

infiltrates in the

meninges

and whitematter

perivas-cular spaces with

macrophages

in the

disrupted

white mat-ter. The brain

appeared normal,

aside from a few

perivascular

inflammatory

cuffs in the brain stemand occa-sional calcified foci in the

hippocampus.

Theabove

findings

were characteristic of disease seen after

inoculation

with

wild-type

DAvirus.

Whilethis

manuscript

wasin

preparation,

Tangyetal.

(12)

published

data

concerning

the

preparation

of infectious GDVII cDNA

clones; however, infectivity

titersof in

vitro-derived GDVII

transcripts

were not

presented.

Wehave also

produced

an

infectious GDVII

cDNA

clone

as well as a series of DA-GDVII chimeric cDNA clones

(J.

Fu,

S.

Stein,

L.

Rosenstein,

T.

Bodwell,

B. L.

Semler,

andR. P.

Roos,

manuscript

in

preparation).

Our

preliminary

data

(using

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[image:3.612.62.303.605.712.2]

FIG. 3. Sections ofspinalcord frommouseinoculated 3month, previously with virus derivedfromtransfected pDAFL3. See text for

details. (A)Transversesection of spinalcordshowing inflammatory cells in meninges and perivascular spaces with disruption of white matter

(hematoxylin-eosin-stained section of paraffin-embeddedtissue; x40).(B)High-power viewof whitematterofabove section

(hematoxylin-eosin-stained section ofparaffin-embedded tissue; x300).

recombinant viruses generated from the chimeric cDNAs) have

identified

a segmentfrom the polyprotein

correspond-ing

to the

lB-coding

area to the

2C-coding

area critical in

determining

the enhanced neurovirulence of the GDVII strain. These recombinant virus studies will also provide

information

concerning

genomesegments critical in

produc-ing

demyelination and virus persistence. Site-directed

muta-genesis of

the

infectious

cDNAclones will

provide

adetailed

localization

ofparticular nucleotides key in producing spe-cific

biological

effects. We are

especially

interested in

mu-tating

the carboxyl end of VP1, which contains a

trypsin-sensitive,

immunodominant neutralization site

(8),

because of its apparent role in theDA

demyelinating

disease

pheno-type

(11).

Infectious clones of

positive-strand

viruses haveallowed

genetic manipulations

to delineate the functions of

specific

viral gene

products.

For

example,

invitro translation of in vitro-derived

transcripts

of

wild-type

and mutated

full-length

poliovirus

infectious clones has

provided

information

con-cerning

viral

polyprotein processing

and the viral

protein-asesthatcarryoutthe

processing

(14).

Ina similar

fashion,

we used in vitro-derived

transcripts

from

wild-type

and

Oi-Audg,W 'W- 40, .Pr

wlqw -0v

B

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mutated pDAFL3 for invitrotranslation in rabbit reticulo-cyte lysates (see accompanying manuscript [10]). These studies have provided important information concerning TMEVpolyprotein processing and viral proteinases and, in addition, because of the availability of the mouse as an experimental host, allow testing for the effect of the muta-tionsondemyelinating

disease.

This research was supported by Public Health Service grants 1POlNS21442 (R.P.R.) and A122693 (B.L.S.) from the National Institutes of Health. R.P.R. was a recipient of a senior research fellowship from the National Multiple Sclerosis Society and a national research service award from the Public Health Service. B.L.S. isthe recipient of a research career development award from theNational Institutes of Health (AI00721).

The technical assistance of S. Hwang and S. Cofer and the secretarial help of M. Witt and L. Baksas are gratefully acknowl-edged.

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