Jpn. J. Infect. Dis., 52, 228‑233, 1999

Review

Theiler's Murine Encephalomyelitis Virus (TMEV): the Role of a Small
0ut‑of‑Frame Protein in Viral Persistence and Demyelination
Masatsugu Obuchi and Yoshiro Ohara*
Department of MicT10biology, Kanazawa Medical University
Uchinadb一machi, Kahoku‑gun, Ishikawa 920‑0293, Japan(Received December 6, 1999. Accepted December 14, 1999)

SUMMARY: Theiler's murine encephalomyelitis virus (TMEV) belongs tothe genus CaTdiovirus of the family
Picornaviridaeand is divided into two subgroups on the basis of different biologiCalactivities. GDVII subgroup
strains produce acute and fatalpolioencephalomyelitis in mice with no viruSpersistence. In contrast, DA or TO
subgroup strains causeanearly nonfatalpolioencephalomyelitis. TMEV is thought to beanexcellent amimalmodel forthe humandemyelinating disease, multiple sclerosis･ Data suggest that macrophages are a major
reservoir harboring the virus. A small out‑of‑frame protein designated L* is synthesized in DA subgroup strainsfrom an altemative, out‑of‑frame, initiation site. Studies of a DA mutant virus, having an ACG ratherthanan
AUG and therefore does not synthesize L* protein, demonstrate that this protein is important for virus growth inparticular cell types and is critical for DA‑induced demyelinating disease and virus persistence･ In addition,

TMEV canbe used as a vector for delivenng forelgn Sequences into the centralnervous system.

Table 1. Ch打aCterisdc in vivo and in vitro acdvityofthe TMEV subgroup

Introductiom
Subgroup

Theiler's murine encephalomyelitis virus (TMEV) belongs

DAOt))

to the genus Cardiovirus of the family Picornaviridae.

GDVII

Representad ve

Strains of TMEV are divided into two subgroups on thes也ns :

basis of different biological activities ( I ‑3). GDVII subgroup

DA,TO,WW, BeAnetaL. GDVII, FA

in vivo acdvi也es:

strains produce acute and fatal polioencephalomyelitis with

Netmvirulenceno viruSpersistema inmice (1‑4). In contrast, DA orTO subgroup

+±++

{+‑一

Acute FKdioencephalomyelids

strains induce an early nonfatalPolioencephalomyelitis ofhnic dcmyelinadon

weanling mice fわllowed by chronic demyelination. Virus

Persistence

persists in the demyelinated splnal cord in the face of ain vitro acdvides:

vlgOrOushumoral immune response. PathOloglCal features of

PIaque size in BHK‑21 cells (mm)demyelination with inflammatory cell infiltration are remi‑

Ultrastructurc in BHK‑2 1 cells

niscent of those found in the human demyelinating disease,multiple sclerosis (MS) (I‑3). Therefore, TMEV‑induced

Growthin macrophages

demyelinating disease is thought to be an excellent animalmodel for MS. In the TMEV animal model, however, themechanisms of vims persistence and demyelination remain

unclear. In this article, we will focus on a small altematively

in vitro biological activities Crable 1)･

initiated viral pmtein (5,6) that plays a key role in the late

GDVII subgroup strains,typified by the GDVII strain, aredemyelinating disease. The protein is only synthesized in DA

highly virulentand cause an acute fatalpolioencephalomyelitis

subgroup strainsand is therefore thought to be a key determi‑

in weanling mice after intracerebral andperipheral routes ofnant of the different biological activities observed between
the two subgroup strains of TMEV.

inoculation (10). After an incubation period of usually lessthan 2 weeks, infected mice show circling, cachexia, andruffled fur, in addition to flaccid paralysis. Almost all theinfected mice die. Neither virus persistence nor demyelina‑

In yivo and in vitro biological actiyities of TMEV

tion is obseⅣed in the few suⅣiving mice (4). Histologicalsubgroups

examination reveals a severe necrosis of neurons of the

EnEel10Virus, it is now classified in the genusCaTdiovirus based

hippocampus, cortex, and splnal cord anterior hom,with
microgliosis, neuronophagia, and innammatory cell infiltra‑

on sequence homology (7‑9). All strainsofTMEV are groupedtion (1‑3). Tsunoda et al. recently reported that the apoptosis

together tmusethey are of one serotypeand cantx neutralizedof neurons may be responsible for the fataloutcome in GDVII

by the same polyclonal antiserpm ( 1 )･ TMEV strains produce

infection (l I).

enteriCand neurologic diseases ln mice,andthey canbe divided
into two subgroups on the basis of their different in vivo andIn contrast to GDVII subgroup strains, DA subgroup strainscause a biphasic disease after intracerebralinoculation (1‑*Corresponding author: Tel: +81‑76‑286‑221 I ext. 3011, Fax:

inoculation, has clinical and pathological features that are

Although TMEV was onglnally classified in the genus

3,12,13). The early disease, which appears 1‑3 weeks after

similar to those seen in GDVII subgroup strains, but milder･+8 I ‑761286‑396 1 , E‑mai1･. ohara@kanazawa‑ned.acJP

228

Perivascular inflammation and necrosis are observed inthe(7 to 12 h p.i.) in BHK‑21 cells, the GDVII strain foms welレ

gray matter, pnmarily in the spinalcordanterior hom, cerebral

developed crystalline arrays in the cytoplasm of infected cells

cortex, and the hippocampus･ Mice recover from the early

and is readily released upon cell lysis (15). In contrast, thediseaseand then develop a chronic, progressive white matter‑

DA strain foms membranous structures consisting of two一demyelinating disease beginnlng about 1 ‑month postinocu‑

membrane units enclosing a slngle layer of viral particles andlation (p･i･)･ ClimiCalsigns include spastic paralysis, inactivity,
uQnary incontinence, and priapism ( 1 ,3)･ The demyelination

This obseⅣation is of interest because such an amngement

pnmarily affects the spinalcord, With the white matter of themay prohibit or hinder the exposure of virus particles to thebrain being spared. The pathology lS remimiscent of MS, withimmune system. Subgroup‑Specific host cell‑restricted infection

inflammatory cell infiltration and a loss of myelin occumng

is another in vitro feature distinguishing the two subgroups

with a relative preservation of axons･ Mononuclearand plasma
adjacent white matter (Fig･ I)･ Electron micrPscopic study(16). In BHK‑21 cells, both DAand GDVII strains produc‑tively grow, although the titers of the DA strain are about 1
log unit lower than those of the GDVII strain. On the otherdemonstrated the stripplng Of myelin lamellae ln response tO

hand, the growth kinetics is remarkably different between

does not appear to be released freely upon cell lysis (15).

cells infiltrate the leptomenlngeS, Perivascular areas, andan invasion of mononuclear cells similar to that seen in

the two strains in J774‑1 cells, a macrophage‑like cell line

experimentalallerg!c encephalitis (14)･ This chronic demyeli‑

derived from a mouse tumor. The GDVII strain does notnating disease in mlCe is thought to be an appropriate animal

actively replicate inthese cells,althoughthere is a slgmificant

model fわr MS (1‑3).

inhibition of cellular protein synthesis. In contrast, the DA

Asshown in Table I, TMEValso shows subgroup‑Specificstmin productively infects these cells with little if any cellularin vitro activities･ In BHK‑21 cells, DA subgroup strains
produce small plaques (less than0･3 mm in diameter), where㌘

protein synthesis shutoff(Fig. 2) (16). A subgroupISPeCific
growth difference is also observed in other macrophage cell

GDVII subgroup strains show a plaque size of 2.5 mm lnlines (Table 2) (17).

diameter (4). Electron microscopy demonstrated anotherfeature differentiating TMEV subgroups･ During late infection

Target ceu for virus persistence

As described above, both subgroup strains of TMEV
pnmarily infect neurons during acute polioencephalomyelitis(1‑3). It is interest that DA viral antigen and RNA that arepresent in neurons in the acute stage are not observed inTable 2･ TMEV growthin neuronaland non‑neuronal cell lines
Virus growth

Cell line Celltype
C‑ I 3 00

DA GDVII DAL* ‑ 1

Neurobl astoma

OS3

+ +

+ +

+++++++ + lll +01i godendrocyte/

type12 astr∝yte

Fig･ 1 ･ TMEV‑induced demyelination. Spinal cord from an SJIJJ mouseapproximately 8 Weeks postinoculation with a DA strain of TMEV.

C6Gl

G26‑ 20

Gl i oma

J774‑ 1

Macrophage

RAW264･ 7

Demyelination withinnammatory cell infiltradon is found in the

円3

anterior funiculusofthe spinalcord. XIBver‑Barrera stain, × 50.

L‑929

DA

8D

i

oma

+ + lIl +Macrophage1

Mac10phage
Fi brobl ast

GDVIl(一EJnLd)SJal!tSnJ .^1111

7 8 Lb 一 3 N 10000000

10

20

30

40

50

0

10

20

30

40

50

0

10

20

30

Hours posトinfection

Fig･ 2･Kinetics of DA, GDVII, and DAL*‑1 virus growthin BHK‑2l and J774‑I cells. The cultLlre SupematantS (solid line,

closed symbol)and cell lysates (broken line, open symbol) of infected BHK‑21 (circle) or J774‑1 (square) cells were
harvested atthe indicated dmesand subjected toaninfectivity assay by a standard plaque dtration on BHK‑21 cells･ Data

are expressed asthe meantiter from three independent expenments.

229

40

50

are the major Cell containing the persistent viral genome,

りeuronsduringthe chronic stage (18,19)I The actuallocaliza‑

t10n Of DA subgroup strainsin the chromic demyelinating stage

although the virus can also be found in astrocytes and oligo‑

is somewhat controversial･ R∝lriguez et all( 18) used immun㌣

dendrocyte s.

electron microscopy to detect the presence of viral antlgen ln

A small out･of･frame protein synthesi2Cd only inneurons and astrocytes in the acute stage of infection as well

DA subgroup strains

as in macrophages, astrocytes, and olig∝lendrocytes at 28

days p･l･, and reportedthat most infected cells at 45 days p･l･
and later were oligodendr∝ytes. An in situ hybridization study

TMEV belongs to the family Picornaviridae and infects
the gastrointestinal tract and the CNS (I‑3). The virion has

demonstratedthat a small amount of viral RNA was presentanicosahedron shapeabout 28 nm in diameter without a lipid

within glial cells in the white matter of the splnal cord during

envelope. A single‑stranded RNA is enclosed by the shell

the chronic demyelinating stage. It was, however, impossible toconsisting of four capsid proteins, referred to as VPl , VP2,

identify the lab:led glial cells as oligcKlendr∝ytes or astruytes

on purely morphological criteria (19). Ohara et al. reported

VP3 , and VP4. The three‑dimensionalstructure was resolved
by means of X‑ray crystalIography in the early 1 990S (27,28)I

that a pnmary culture of oligodendr∝ytes can be infected

The nucleotide and predicted amino acid sequences werewith both subgroup strains (20)･ Using a simultaneous
immunoperoxidase‑ln Situ hybridization assay, Aubert etal.(21 ) quantitativelyanalyzed the population of DA‑infected
cells in the chronic stage, and reportedthat 25140% were
oligodendrocytes, 5‑ 1 0% astrocytes, 1 0% microglia/macro‑

determined in late 1 980S (7, 8, 29); neutralizing epitopes have
TMEV strains were constmcted (33,34).

phages,and the rest unknown.

the 5'and 3'noncoding reglOnS is translated into a long

In contrast, other investigators claimedthat the virus resided
in macrophages in the chronic demyelinated stage. Using

polyprotein, which is then cleaved into L, Pl, P2,and P3･ L

ultrastructural immunohistochemical teclmiques, researchersaphthoviruses. Although the function of L protein is notobserved viral inclusions in macrophages in and around

known, L plays a role in host cell‑restricted infection (35)･

been identified (30132),and fulHength infectious cDNAsof
The genome is of positive sense and is approximately 8 loo

nucleotides long. A long open reading frame (ORF) between

is a small leader protein present only in cardiovimses anddemyelinating lesions (22). It wasalSo reportedthat infectious

A noteworthy feature regarding the L region is that DA

BeAn virus, which is a demyelinating TMEV strain similar

subgroup strains have an altemative translation initiation

to DA, was recovered from infiltrating mononuclear cells
isolated directly from the central nervoussystem (CNS) ofcodon within the L coding region at nucleotide (nt) 1079, inchronically infected mice (23). Two‑Color immunonuorescentaddition tothe authentic initiation site forthe polyprotein attheamino terminus of L (nt 1066) (5, 6) (Fig. 3). This altema‑

staining with conventionaland confocalmicroscopy showed

tive initiation site is out‑of‑frame with the polyprotein and isthat the viral antigen burden was predominantly within

used to translate a small 17 kDa protein, designated L*･ The

macrophages infiltrating demyelinating lesions (24). A small

synthesis of L* protein is subgroup‑specific because thefraction of cultured pnmary brain macrophages could be

altemative initiation site is present in DA, TO, WW, BeAn,

efrlCiently infected with the DA strain without significant

and Yale strains, but not the GDVII or FA strains (wherethe

cytopathic effects (25). Rossi etal. (26)also examined the

L* AUG is substituted byanACG) (36). Therefore,thismiqueprotein wasthought to potentially play a role as a determinant

types of cells infected at different times p.i. Approximately
lO% of infected cells were astrocytes at any time p.i. Theof the different activities observed in both virus subgroups.

number of infected macrophages increased with time andreached a plateau by 21 days p.I. When approximately 50%
of infected cells were macrophages. In addition,the depletion ofRole of L* protein in TMEV growth in nacrophages

Aspreviously noted, DA grows well in J774‑ I cells, whilebl∝山一bome macrophages by dichloromethylene di phosphonate

GDVII does not. The importance of L* protein in thisdifference was demonstrated in studies with a mutant virus,
DAL*‑ 1 , which hasanACG rather thananAUGand therefore

prevented virus persistence in mice infected withthe DA
strain, further emphasizing the importance of macrophages

in the late demyelinating disease (26).

From the above data, it appears likely that macrophages

does not synthesize L* protein (Fig. 3). Takata et al･ foundp呈yprotein+ E･,pl･ P2･ ド L★ protein

DA subgroup

人UG‑‑･‑‑‑‑‑‑‑‑AUG･‑‑‑‑‑‑
1066

1079

1066

1079

5■

VPg

QDVtl subgroup AUG･･･････････････････‑････････ACG････････‑･･･.････
DAL'‑1 virus pまyprotein‑三･3Pl･ P2･ ｣‑毎Fig. 3. Authentic and altemativc initiadon sites in TMEV. An authentic initiation site is located at nucleotide (nt) 1066 in bothstrainsand a mutant virus, DAL*‑I. Fromthis AUG, a long polyprotein is translated and subsequently cleaved into L
protein, Pl, P2, and円･ On the other hand,analtemative, out‑of‑frame, imitiadon site that is only present in DA subgroup

strains is located at nt 1079 and is used to tradlSlate a small 17 kDa protein, L*. GDVII subgroup strains do not synthesize L*

since U at nt logo is substituted with C. The DAL*‑ I viruSalso fails to synthesize L* becauscthe U is substituted withC (asa result of site‑directed mutagcnesis).

230

that the DAL*‑1 vims failed to grow in J774‑1 macrophagecells, whereasthe virus grew well in BHK‑2l cells, suggestingthat L* protein plays a role in the TMEV subgroup‑specificthe lymphotoxin (IJ) gene into the L region, resulting in an

host cell‑restricted infection (Fig. 2). The restriction of virus

The above studies indicate that TMEV may be used as avector for delivering foreign sequences into the CNS (43,44)･ One potentialshortco.ming of this approach is that theexpressi‑on of IJ, which was demonstrated by cytotoxicity
against L‑929 cells (one of the biological activities of I:T).

growth in these cells is regulated at the step of viral RNA
synthesis (37). Obuchi et al. found that the DAL*11 andGDVII viruses, neither of which produce L* protein, failed
to grow in macrophage cell lines, but grew as well as DA inother neuraland non‑neuralcell lines (17) (Table 2). In addi‑tion, a host cell‑specific antiapoptotic effect was recently
demonstrated in a macrophage cell line, P388Dl (38). These

coding sequence of L proteln OVerlapswith that of L* protein,
as a result, insertion of a foreign Sequence into the L coding

reg10n interrupts L* translation. Since L* protein is importantfor viruSpersistence, interruption of L* translation prevents
virus persistence and therefore prevents continuous expresI

features are of interest because macrophages are one of thesion of the tmnsgene in the CNS. Therefore, a more appropnate

major Cell types for TMEV persistence duringthe stage ofchronic demyelination, and because virus growthandsite of insertion must be explored.

minimum host cell damage are critical for maintenance ofstrain as a virus vector is the probability that the vims will

the viral genome and viralpersistence (39).

induce demyelination. Of interest is the recent report by Sato

Another potential problem related to the use of the DA

et al. (32) Conceming a DA mutant vims in which lysinechanges to asparaglne at amino acid 141 in VP2; this muta‑

Role of L* protein in yirus persistence andtionpersists but does not cause demyelination･ There is adenyelination

continulng role for genetic manlpulation of DA vimS in the

There are a number of reports demonstrating that L* protein

future in order to produce non‑pathogenic vectors.

plays a role in vims persistence and demyelination. The

DAL*‑ 1 virus prducesanearly acute polioencephalomyelitis

CoTLChs ion

similar to parentalDA. However, the viral RNA genome wasno longer detected in the spinalCord of mice 6 weeks p.I.

DA subgroup strains of TMEV cause demyelination in micethat is reminiscent of that seen in the humandemyelinating(38)･ In addition, there was minimal if any evidence of
demyelination or innammation in the splnal cord at this timedisease, MS. Althoughthe mechanism(S) of demyelination(40)･ It was also demonstrated that L* protein appears toremains to be clarified, a small out‑of‑frame protein designatedinhibit the generation of H‑2K‑restricted TMEV‑Specific

L* seems to play an important role in virus persistence andcytotoxicity, therefore permitting a persistent infection indisease pathogenesis. L* Protein is synthesized in DA subgroupsusceptible mouse strains (41 ). Yamasaki etal. recently rerx)rted

strains from an alternative, out‑of‑frame, initiation site.

a variation inthe utilization of translation initiation atthe

Studies of a DA mutant vinュs Which has an ACG rather thanpolyprotein'S AUG versusthe L* AUG (42). These investi‑
gators proposedthat L* (ratherthan the polyprotein) is

an AUG and therefore does not synthesize L* protein

preferentially synthesized in cenain CNS cells (e.g., microglial
cells) following infection with DA subgroup strains, leading
to a restricted expression of the virus. The production of onlyin particular celItypeSand is critical for DA‑induced demyeli‑
nating diseaseand viruSpersistence. Recent studies have shownthat TMEV canbe used as a vector for delivenng forelgn

smallamounts of capsid protein would decrease neurovirulencesequences into the CNS. The availability of a murine ex‑in the acute stage andalso foster a persistence of the virus inperimental host susceptible to DA infection providesanat‑

the chronic stage.

tractive model system for an elucidation of the pathogenesis

demonstrated that this protein is important for virus growth

The above data indicate that L* protein is a key determinantof vims‑induced demyelination and/or the development ofof TMEVpersistence, withsubsequent inflammatory demyeli‑
nation in the CNS similar to that in MS. All the data havegene therapies for intractable human neurologlCal diseases･

been obtained from studies uslng the mutant virus, DAL*1 1 ,which does not synthesize L* protein. Additional data using

AcknowledgmeAtS

a recombinant virus which synthesizes L* protein inthe back‑This work was supported by a grant for Project Researchground of the parentalGDVII virus would be valuable in orderfrom Kanazawa Medical University (n9‑7) and a grant forto confirm the role of L* protein.

the Science Research Promotion Fund (1999) from the Pr0‑

motionand Mutual Aid Corporation for Private Schools ofJapan.

TMEV as a rector for delivery of foreign genes
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