A Val-25-to-Ile substitution in the envelope precursor polyprotein, gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env, and neurovirulence of ts1, a mutant of Moloney murine leukemia virus TB.

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0022-538X/90/020467-09$02.00/0

A

Val-25-to-Ile Substitution

in the

Envelope Precursor

Polyprotein,

gPr80env,

Is

Responsible

for

the Temperature

Sensitivity,

Inefficient

Processing of

gPr80env,

and

Neurovirulence of

tsl,

a

Mutant

of

Moloney Murine

Leukemia

Virus TB

PAUL F. SZUREK,1P. H. YUEN,' J. K. BALL,2 AND P. K. Y.

WONG'*

The University of TexasM.D. Anderson Cancer Center, SciencePark-Research Division, Smithville, Texas 78957,1 and

DepartmentofBiochemistry, Universityof Western Ontario, London, Ontario, Canada H6A SCJ2

Received 9August1989/Accepted25October 1989

tsl is a neurovirulent spontaneous temperature-sensitive mutant of Moloney murine leukemia virus TB

whichcauses hindlimbparalysis in mice. Previously, it had been shown that the temperature-sensitive defect

resided in the env gene. At the restrictive temperature, the envelope precursor polyprotein, gPr80env, is inefficiently processed intracellularly intotwocleavageproducts, gp7O andPrpl5E. This inefficient processing ofgPr8Oe"v is correlated with neurovirulence. Inthisstudy,itwasshownthatasingle amino acid substitution,

Val-25-IleingPr80"v, is responsible for thetemperaturesensitivity,inefficient processing ofgPr8(W'vatthe restrictive temperature, and neurovirulence of tsl. At the restrictive temperature, a steady-state level of

nonprocessed, endoglycosidase H-sensitive gPr8Oenv remained in the endoplasmic reticulum of cells infected by tsl,butnoendoglycosidase H-resistant gPr8Oe"vandonlytrace amountsofgp70weredetected in the infected

cells. Since the host cell-encoded processing protease resides in the cis cisternae of the Golgi apparatus,

inefficient processing ofgPr80e"V attherestrictive temperature is.most likely due toinefficient transportof gPr8env from the endoplasmic reticulum to the cis cisternae of the Golgi apparatus rather than due to

misfolded gPr80rnVbeingapoorsubstrate fortheprocessingprotease at therestrictivetemperature.

tsl is a neurovirulent spontaneous temperature-sensitive

mutant of Moloney murine leukemia virus TB (MoMuLV-TB) (22, 41, 43, 50). MoMuLV-TBcauseslymphoma in mice

after a long latency period of 179 days (48), whereas tsl

causesaprogressive hindlimb paralytic disease in

suscepti-ble strains of mice after a much shorter latency period

ranging from 24 to 80 days (22, 28, 42, 50). The hindlimb paralyticdisease ischaracterized byadegenerative

spongi-form encephalomyelopathy (22, 41, 50), generalized body wasting (42, 50),severethymicatrophy (22, 42),anddrastic

immunodeficiency (42). Asimilar hindlimb paralyticdisease has been described for other strains of ecotropic MuLV isolated from wild mice(10)andexperimentally generatedin the laboratory (3, 18, 31, 43).

The neurovirulence of tsl seems to be related to its

temperaturesensitivity(44). Onlysusceptiblestrainsof adult mice that were infected with tsl as neonates succumb to hindlimb paralysis (22, 44). Newborn mice have a lower

body temperature (-34.0°C) than adult mice (-38.4°C) (5). Thebodytemperatureofnewbornmice ispermissive forthe replicationof tsl, andtsl is able to replicate intargetcells

andspreadtothecentralnervoussystem(CNS) (22, 41, 50). However,signs of hindlimbparalysisdonotappearuntil the infectedmice becomeyoungadults. Inyoungadultmice, the bodytemperatureisrestrictive for thereplicationof tsl, and defective virus replication is correlated with the onset of signs of hindlimb paralysis. The first signs to appear are

tremors in the hindlimbs, which rapidly progress to

para-paresis, which is then followed by hindlimbparalysis. His-topathological studies indicate that spongiform changes oc-curin thebrain stemand inboth thegreyand whitematter ofthe spinalcord (28, 50).

*Correspondingauthor.

Inprevious studies, the close genetic relatedness between tsl and MoMuLV-TB allowed usto explore the molecular basis for the neurovirulence of tsl (38, 46, 47, 49). By constructing hybrid viruses between tsl and both MoMuLV-TBandMoMuLV, itwas shown thattworegions in theenv

geneoftslarerequiredto causehindlimbparalysis (38, 46,

49). One region, in the XbaI-BamHI restriction fragment (771 base pairs [bp], nucleotides [nts] 5767 to 6537), coded for the temperature sensitivity; inefficient processing of

gPr8OenV atthe restrictivetemperature, and neurovirulence oftsl (38, 44, 46, 49). Theotherregion, in the BamHI-ClaI restrictionfragment(1,138 bp, nts 6538 to 7675), coded for theenhancedability of tsltoreplicateintheCNS relativeto MoMuLV-TB (38, 49). A comparison of the amino acid

sequences(deduced from the nucleotide sequences) encoded

by theenvgenesofboth tsl andMoMuLV-TB showedthat

four amino acid substitutions in tsl, i.e., Val-25--Ile in

gPr8oenv, Ser-157-*Ala in gPr8Oenv, Arg-430--+Lys in gp7O, and Ile-23-*Valin p15E, werethe results ofgenuine muta-tionsintsl(38).BoththeIle-25 and the Ala-157substitutions ingPr8Oenvwereencodedby the XbaI-BamHIregion ofenv,

whereasboth theLys-430 substitution in gp7O and the Val-23 substitution in piSE were encoded by the BamHI-ClaI

region ofenv.

In thisstudy, fine-structuregenetic mappingwas doneby

exchanging restriction fragments containing one or moreof thefouraminoacid substitutionsintheenvelopeproteinsof

tslbetweentsl andMoMuLV-TB togeneratea newset of

hybrid virus genomes. These hybrid viruses were assayed

for the phenotypic characteristics oftsl. It wasfound that theVal-25--Ile substitution ingPr8Oenv was responsiblefor the temperaturesensitivity, inefficientprocessingofgPr8Oenv at the restrictive temperature, and neurovirulence of tsl.

Endoglycosidase H (endo H) sensitivity experiments 467

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showed that a steady-state level of the nonprocessed gPr8oenv remains in the endoplasmic reticulum (ER) of infected cells at the restrictive temperature. These results indicatedthat inefficient processingofgPr8oenvatthe restric-tivetemperatureismostlikely duetoinefficient transport of gPr8Oenv from the ER to the cis cisternae of the Golgi apparatus, which containsthe hostcell-encoded processing protease, rather than due tomisfoldedgPr8oenv beingapoor substrate fortheprocessingprotease at therestrictive tem-perature.

MATERIALS AND METHODS

Cells. Viruses were propagated in mouse TB cells, a thymus-bonemarrowcell line derived fromCFW/D mice (2), and titer was determined on 15F cells, a murine sarcoma-positive, leukemia-negative (S+L-) cell line (45). NIH 3T3 cells were used for transfection and immunoprecipitation experiments. All cell lines were maintained in Dulbecco modified Eaglemedium supplemented with 6% fetal bovine serumand4% bovine serum.

Viruses. Thestandard MoMuLV referred toin thisreport wasdescribed previously inYuen etal. (46).Theprogenitor of MoMuLV-TB, a variant ofMoMuLV, was isolated as described by Wong etal. (43) from the tissue extract ofa sarcoma produced in a MoMuLV-murine sarcoma virus-infected BALB/c mouse providedby J. B. Moloney. Since its isolation, ithasbeenpropagated inTBcells and hasbeen designated MoMuLV-TB. MoMuLV-TB has been molecu-larly cloned into Charon 21A, and clonewt-25 was used in these studies (47). tsl has been molecularly cloned into Charon 21A, and clone tsl-19wasused in thesestudies(47). Constructionof hybridvirusgenomes. Details of methods for the construction of hybrid virus genomes have been describedpreviously (46,49). Hybridtslwt-33 consistsofa tslgenomewithasubstituted BstEII-BstXI restriction frag-ment (116 bp, nts 5924 to 6039) ofMoMuLV-TB. Hybrid tslwt-32 is areciprocal hybridgenome oftslwt-33. Hybrid tslwt-26consists ofa tsl genome witha substituted BstXI-BamHI restriction fragment (498 bp, nts 6040 to 6537) of MoMuLV-TB. Hybrid tslwt-31b consists of a tsl genome withasubstitutedBamHI-HpaIrestriction fragment (660 bp, nts 6538 to 7197) of MoMuLV-TB. Hybrid tslwt-30 is a reciprocal hybrid genome of hybrid tslwt-31b. Hybrid tslwt-23 consists of a tsl genome with asubstituted HpaI-ClaI restriction fragment (478 bp, nts 7198 to 7675) of MoMuLV-TB. Hybrid tslwt-41 consists of a tsl genome withtwosubstituted restriction fragments of MoMuLV-TB, i.e.,BstXI-BamHIandHpaI-ClaI.Hybrid tslwt-42 consists ofanMoMuLV-TB genomewithtwosubstitutedrestriction

fragments

oftsl, i.e.,

BstEII-BstXI

and

BamHI-HpaI.

The hybrid genomes were verified by mapping four restriction enzyme sites that were present in MoMuLV-TB but not in tsl. These four restriction site polymorphisms were identi-fiedby analysis ofthe nucleotidesequences of the env genes for bothMoMuLV-TB and tsl (38). In MoMuLV-TB, there is a ScaI site (nts 5870 to 5875) in the HindIII-BstEII fragment(1,029 bp, nts 4895 to5923), a SfiI site (nts 6237 to 6249) in the BstXI-BamHI fragment (498 bp, nts 6040 to 6537), an ApaI site (nts 6992 to6997) in the

BamHI-HpaI

fragment (660bp, nts 6538 to7197), and a

HaeIII

site (nts 7204 to7207)in the

HpaI-ClaI

fragment (478 bp, nts 7198 to 7675). Nucleotides were numbered by the method of Shin-nicketal. (36).

DNA transfection. NIH 3T3 cells were transfected with viral DNA by the calcium-phosphate precipitation method

(13). Details of this procedure have been described previ-ously in Yuen et al. (47).

Metabolic labeling, radioimmunoprecipitation, and SDS-PAGE. Immunoprecipitation of intracellular virus-specific proteins by using fixed Staphylococcus aureus (Pansorbin cells from CalbiochemCorp., La Jolla, Calif.) as an immu-noabsorbent and sodiumdodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) were performed as described previously (44, 45), except that the infected NIH 3T3 cells were double labeled with both L-[35S]cysteine (50

RxCi/ml)

andL-[35S]methionine (50 ,uCi/ml)and all immunoprecipita-tion steps were done at 4°C. Goat antiserum preparedagainst Rauscher MuLVgp69/71 (lot79S000842) was obtained from Microbiological Associates, Inc., Bethesda, Md. Apparent molecular weights (Mr) of proteins were determined by plotting log Mr versus relative distance migrated (34). The

"4C-protein

standards (Dupont, NEN Research Products,

Boston, Mass.) had Mrs of 97,400 (phosphorylase B), 69,000 (bovine serumalbumin),46,000(ovalbumin), 30,000 (carbon-ic anhydrase), and12,300 (cytochrome c).

Endo H treatment. Streptomyces plicatus endo H (EC 3.2.1.96), purified from a recombinant Escherichia coli strain, was purchased fromBoehringer GmbH, Mannheim, FederalRepublic ofGermany. Theendo Hdigestions were optimized by the procedure described by Trimble and Maley (39). The washed Pansorbin cell pellets obtained from im-munoprecipitation were washed two additional times with 10 mMTris(pH

7.4)-1%

(vol/vol)aprotinin-2mM phenylmeth-ylsulfonyl fluoride andonetime with1%(vol/vol) aprotinin-2 mM phenylmethylsulfonyl fluoride, and then they were suspendedin 30,ul ofantigenrelease buffer(10mM Tris[pH 7.4], 0.2 M

2p-mercaptoethanol,

1% SDS). Antigen was releasedfromthe Pansorbincell-antibody complex by boil-ing the mixture for 5 min, and the Pansorbin cells were removed by centrifugation. Reagents were added to the supernatant to give final concentrations of 50 mM sodium citrate (pH 5.3), 0.1 M

2p-mercaptoethanol,

0.02% SDS, 1 mMphenylmethylsulfonyl fluoride, 1%(vol/vol) aprotinin,5 mM EDTA, 0.02% (wt/vol) acetylated bovine serum albu-min,and 40.0 mUofendoHper mlinavolumeof 50,ul. The samples were digested for 16 h at 37°C, and they were prepared for SDS-PAGE asdescribedpreviously(45).

Mousestrain and inoculationprocedure. TheBALB/c mice (28, 42),theinbred CFW/D mice (2), and theprocedurefor intraperitoneal inoculations (47) have been described previ-ously.Theexperimentsinvolved with theCFW/D micewere conducted as a blind study.

RESULTS

Construction of hybrid viruses. The MoMuLV genome contains8,332nts(36) (Fig. 1). The env gene (1,998 bp, nts 5777 to 7774) codesforanenvelope precursor polyprotein, gPr8Oenv (632 amino acids, encoded by nts 5876 to 7771) which is processed intracellularly by a host cell-encoded protease to yield two envelope proteins, gp7O (435 amino acids, encodedby nts 5876 to 7180) andPrp1SE (196 amino acids,encodedbynts7184 to7771) (37).PrplSEis processed byavirus-encodedprotease to yieldplSE(180 amino acids, encoded by nts 7184 to 7723) and p2E (16 amino acids, encodedbynts 7724to 7771)(37). In a previous study (38), fourpoint mutationsin the env geneof tsl which resulted in amino acid substitutions that may be responsible for the phenotype oftsl wereidentified. A

G-+A

change at nt 5948 resulted in a Val-25---Ile substitution in gPr8oenv, a T- G

change

atnt6344resulted ina Ser-157--Alasubstitution in

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A.

B.

C.

gag^9 | p0p gp7O PrpIE LTR

M I I ~~I l

0 1 2 3 4 5 6 7 8

Sm H X Bx B H C Pa

I I II I rI

Bs

ts gPr8O Neurovirulence A

MoMuLV-TB

b=

IA KV

I I l l

tS1 i tslwt-33 i

I +

P _

IP ++

I I I - I - P

tslwt-26 6 I +

tslwt-31b II I I1+

tslwt-23 b I I I

mb

+

tslwt-41 b +

tslwt-32 k I

tslwt-30 i tslwt-42

.

-I+ I+

II

IP ++

IP +

IP ++

FIG. 1. Determination of the amino acid substitution responsible for the temperature sensitivity, inefficient processing ofgPr8Oenv,and neurovirulence of tsl. (A) Genetic map of the MoMuLV genome. (B) Critical restriction sites used to construct hybrid virus genomes. Restriction site abbreviations: B, BamHI; Bs,BstEII;Bx, BstXI; C,ClaI;H,HindIlI;Hp,HpaI;Ps,PstI;Sm,SmaI;X,XbaI.(C)Genomes ofhybrid viruses used to determine the phenotype of tsl. Hybrid genomes were constructed by exchanging homologous restriction fragments between tsl (black) and MoMuLV-TB (white). The locations of the four amino acid substitutions(Val-25--Ile ingPr8Oe"v [I],Ser-157--+Ala

ingPr8Oenv [A], Arg-430--*Lys in gp7O [K], and Ile-23--+Val in pl5E[V])encodedby the env gene of tsl are shown above the tsl genome. Restriction fragments containing amino acid substitutions of tsl areBstEII-BstXI(I),BstXI-BamHI(A),BamHI-HpaI (K), andHpaI-ClaI

(V). The position of the Thr-347--*Ala substitution ingp7O encoded by the MoMuLV-TB env gene is shown above the MoMuLV-TB genome (A). TheThr-347-.Ala substitution in gp7O of MoMuLV-TB is encoded by the BamHI-HpaI restriction fragment. NIH 3T3 cells were transfected with the hybrid virus genomes to obtain the infectious hybrid viruses. The viruses were assayed for temperature sensitivity(ts), inefficient processing ofgPr8Oenv (gPr80),and neurovirulence. For temperaturesensitivity,+indicates that the ratio of foci produced on 15F cells(34.0°C/39.0°C) was greater than 100. ForgPr80,P indicates thatgPr8Oe"vwasefficiently processed togp7O andPrp15Eat 39°C,and IP indicates thatgPr8Oe"v was inefficiently processed at 39.0°C. For neurovirulence, ++ indicates hindlimb paralysis, + indicatesparaparesis (aslight degree of paralysis whichaffectsthe hindlimbs), and -indicates no signs of paraparesis or hindlimb paralysis.

gPr8Oenv, a G--A change at nt 7164 resulted in an

Arg-430-+Lys

substitution in

gp7O,

and an A--G change at nt 7250 resulted in an Ile-23-)Val substitution in pl5E. Both the Ile-25 and the Ala-157 substitutions in gPr80e"' are encodedby theXbaI-BamHI region ofenv(771 bp, nts5767 to6537)which is responsible for thetemperaturesensitivity, thegPr8Oenv processing defectattherestrictivetemperature, and the neurovirulence of tsl (38, 44, 46, 49). Both the Lys-430 substitution in gp7O and the Val-23 substitution in

pi5E

are encodedbythe BamHI-ClaI region ofenv (1,138 bp, nts 6538to 7675) which isresponsible forthe enhanced ability oftsl toreplicate inthe CNS relative to MoMuLV-TB (38, 49).

Inthis presentstudy, individual amino acid substitutions in the envelope proteins of tsl were assigned to specific

phenotypic

characteristics of tsl

by

assaying

a new set of hybrid viruses. A new set of hybrid genomes was con-structed by exchanging restriction fragments between tsl and MoMuLV-TB (Fig. 1). Thecodons forthe four amino acid substitutions inthe

envelope proteins

of tsl are

con-tained within unique restriction

fragments

(36, 38). The codon for the Ile-25 substitution in

gPr80ern

is in a BstEII-BstXI restriction fragment (116 bp, nts 5924 to

6039),

the codon for the Ala-157 substitution in

gPr8Oenv

is in a BstXI-BamHI restriction fragment (498

bp,

nts 6040 to

6537),

the codon forthe Lys-430 substitution in

gp7O

is in a

BamHI-HpaI restrictionfragment (660 bp,nts6538 to7197),and the codon for the Val-23 substitution inpiSE isin aHpaI-ClaI restriction fragment (478 bp, nts 7198 to 7675). In hybrids tslwt-23, -26, -30, -31b, -32, and -33, single amino acid replacements weremade which involved one of the amino acid substitutions encoded by the env gene oftsl and the

corresponding wild-type

amino acid of MoMuLV-TB. In hybridstslwt-41 and-42,twoamino acidreplacementswere

made which involved two ofthe amino acid substitutions encodedbythe envgeneoftsland twoofthe

corresponding

wild-type amino acids ofMoMuLV-TB. (Note that in Mo-MuLV-TBthereisanAlainposition347 of

gp7O

instead of aThr, whichisin both tsl and MoMuLV [38]

[Fig. 1].)

The Thr-347--Ala substitution in

gp7O

probably arose

during

propagation of MoMuLV-TB virus after tsl was selected from MoMuLV-TB butbefore thegenomeof MoMuLV-TB was molecularly cloned (i.e., clone wt-25) (38, 47). Both hybrids tslwt-31b and tslwt-32 contain the

BamHI-HpaI

restrictionfragment (660 bp, nts6538to7197) of MoMuLV-TB which codes for the Thr-347--Ala substitution in the gp7O ofMoMuLV-TB.

All of theexchangedrestriction

fragments

could be iden-tified by restriction site

polymorphisms

between tsl and MoMuLV-TB except for the BstEII-BstXI

fragment.

In

MoMuLV-TB, the BstXI-BamHI

fragment

contains a

Sfil

site (nts 6237to6249), the

BamHI-HpaI

fragment

contains

I I I I

mimmi

U

=Li==f

I

m

I

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H H

Sc

S fBs

Bx

Sf

B

H+Bs

Sm

I=

Sm

H B Ps

Nd

WMMl

_ M

H BsBx B

[image:4.612.62.301.65.373.2]

I II I

tslwt-33

FIG. 2. Schematicdiagram illustratingtheconstruction of hybrid genome tslwt-33. Plasmids p21 and p40 are pUC9 plasmids that

containtheHindIII-BamHI fragment (1,643 bp,nts4895to6537)of tsl(black) andMoMuLV-TB (white), respectively (46).Plasmidp40 containstwomarker restriction sites,ScaI (nts5870to5875)and

Sf1i

(nts 6237 to6249), that arenot present in p21. The BstXI-BamHI fragment (498 bp, nts 6040 to 6537) of p40 was replaced by the BstXI-BamHI fragment ofp21 to yield plasmid pX. Plasmid pX contains the Scal marker but not the Sfi1 marker. The HindlIl-BstEIIfragment (1,029bp,nts4895to 5923) of pXwasreplaced by

theHindIII-BstEII fragmentof p21 toyield plasmidpS. PlasmidpS

does notcontaintheScaImarkerortheSfi1 marker. Byaprocedure described indetailpreviously(46), the HindIII-BamHI fragment of

pSwasligatedtoboththe BamHI-PstI fragment (2,294 bp, nts6538 to 8264 and 1 to 567) of plasmid p22 (46) and the SmaI-HindIII fragment(4,864bp,nts31to4894)of plasmidp17(46)toyieldhybrid tslwt-33(*). Restrictionsite abbreviations: B, BamHI; Bs,BstEII; Bx, BstXI; H,HindIII; Ps,PstI; Sc, ScaI;Sf, SfiI,and Sm, SmaI.

anApaI site (nts 6992to 6997), and theHpaI-ClaIfragment contains aHaeIII site (nts7204 to 7207). Since the BstEII-BstXI fragment is small (116 bp) and does not contain a

restriction site polymorphism, exchanges involving the BstEII-BstXI fragment were accomplished by an indirect

procedure as shown for the construction of hybrid tslwt-33 in Fig. 2. Instead of exchanging the small BstEII-BstXI fragmentofplasmidp40 withthe corresponding fragment in plasmid p21inonestep, twolarger fragmentsthat contained

restriction site polymorphisms (BstXI-BamHI [498 bp, nts

6040 to 6537] and HindIII-BstEII [1,029 bp, nts 4895 to

5923]) wereexchangedbetweenp21 andp40intwo stepsto

yield plasmid pS. The final steps in the construction of

tslwt-33 from plasmid pS are shown in Fig. 2. After the

hybrid genomes were constructed, they were transfected

intoNIH 3T3 cellstoobtaininfectious hybridviruses. Identificationof theaminoacid substitution ingPr8Oe"vthat isresponsiblefor thetemperaturesensitivity oftsl.Previous studies showed that either the Ile-25 substitution or the

Ala-157 substitution in

gPr8Oenv,

encoded

by

the

XbaI-BamHI

region ofenv, was

responsible

for the temperature sensitivity oftsl (38,46,

49).

In ordertodetermine which of the two amino acid substitutions

(Ile-25

or

Ala-157)

was responsible for temperature

sensitivity,

the

panel

ofviruses in Fig. 1 was assayed for temperature

sensitivity.

A virus was considered to be temperaturesensitive if the ratio offoci producedin

15F

cellsatthepermissivetemperature

(34.0°C)

tofociproduced at therestrictive temperature

(39.0°C)

was greaterthan 100. In hybrid tslwt-26, theAla-157 in

gPr8Oenv

of tsl wasreplaced by thewild-type Serof MoMuLV-TB

by

exchanging the

BstXI-BamHI

restriction fragment (498

bp,

nts 6040 to6537) oftslwith the

corresponding

BstXI-BamHI

restriction fragment of MoMuLV-TB. Hybrid tslwt-26

re-mained temperature sensitive even thoughthe Ala-157 sub-stitution in the

gPr8oenv

of tsl had been replaced by the wild-type Ser of MoMuLV-TB. This resultindicated thatthe Ser-157--Ala substitution in

gPr8Oenv

was not

responsible

for the temperature sensitivity oftsl. Inhybrid tslwt-33, the Ile-25 in the

gPr8oenv

of

tsl

was replaced by the wild-type Val of MoMuLV-TB by exchangingthe

BstEII-BstXI

restriction fragment (116 bp, nts 5924 to 6039) of tsl with the corresponding

BstEII-BstXI

restriction fragment of MoMuLV-TB. However, hybrid tslwt-33 was not tempera-ture sensitive, and this resultindicated that the Val-25-->Ile substitution in

gPr8Oenv

wasresponsible for thetemperature sensitivity of tsl. The resultfor hybrid tslwt-32, a

reciprocal

of tslwt-33, demonstrated that MoMuLV-TB could be con-verted into a temperature-sensitive virus by replacing the Val-25 in the

gPr8oenv

of MoMuLV-TB with Ile. Further-more, all of the viruses containing the

Val-25-Ile

substitu-tion in

gPr8oenv,

i.e., tsl and tslwt-23, -26,-31b, -32, -41, and -42, were temperature sensitive, whereas the viruses con-taining the

wild-type

Val-25 of MoMuLV-TB in

gPr8Oenv,

i.e., MoMuLV-TB and tslwt-30 and -33, were not tempera-ture sensitive. From these results, it can be concluded that the

Val-25-Ile

substitution in

gPr8Oenv,

not the Ser-157-*Ala substitution in

gPr8OenV

encoded by the

XbaI-BamHI

region of env, is responsible for the temperature sensitivity of tsl.

Identification

of the amino acid substitution that is respon-sible for inefficient processing of

gPr8Oe"v

at the restrictive temperature. Previous studies showed that either the Ile-25 substitution or the Ala-157 substitution in

gPr8oenv,

encoded by the

XbaI-BamHI

region of env, was responsible for the inefficient processing of

gPr8oenv

at the restrictive tempera-ture (38, 44, 46, 49). In this study, it has been shown that a single amino acid substitution, Val-25->Ile in

gPr80env,

is

responsible for the temperature sensitivity of tsl. In order to determine whether theVal-25-Ilesubstitution in

gPr8Oenv

is

also responsible for the inefficient processing ofgPr8Oenv at the restrictive temperature, only the critical hybrid viruses in Fig. 1, i.e., tslwt-26, -32, -33, and -42, were assayed for processing ofgPr8Oenvat both the permissive

(34.0°C

[data not shown]) and the restrictive

(39.0°C)

temperatures. In these assays, infected NIH 3T3 cells were pulse-radiolabeled for 10

min

with both

L-[35S]cysteine

and

L-[35S]methionine

(there are 24 cysteines and 7 methionines in

gPr8Oenv

and 20 cysteines and 2 methionines in gp7O [36,

38]).

The pulse period was followed by a chase period of 0, 90, or 300

min.

After the chase period, radiolabeled proteins were immuno-precipitated withanti-gp7Oantisera, and they were separated by SDS-PAGE (Fig. 3 [data shown for only tslwt-32 and

-33

at the restrictive temperature]).

With a 0-min chase in both tslwt-32- and -33-infected cells, most of the radiolabel in the immunoprecipitates was

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ts1wt-32 ts1wt-33 Chase(min) 0 90 300 0 90 300

gp8env_ _ _

gPrao

gp7O-1 2 3 4 5 6

FIG. 3. Pulse-chase analysis ofgPr80envprocessing by tslwt-32 and-33atthe restrictivetemperature. NIH 3T3 cellswereinfected

with tslwt-32 and -33 and incubatedat 39.0°C. The infected cells were pulse-radiolabeled for 10 min with both L-[35S]cysteine and

L-[35S]methionineandchasedfor 0, 90,or300min.Theradiolabeled

cellswerelysed, and samples of the lysatecontaining 2 x 10i cpm

weretreated with anti-gp7O antisera. Immunoprecipitated proteins wereseparatedby SDS-PAGEon a9.0%polyacrylamide gel.

incorporated into gPr80env, and radiolabeled gp7O was not

detected (Fig. 3,lanes 1 and 4). With a90-min chase in the

tslwt-33-infected cells, processing of radiolabeled gPr8Oenv intothe cleavage products ofgp7O andPrpl5E was evident

(lane 5), and processing continued throughout the 300-min

chase period (lane 6). At 300 min, very little radiolabeled

gPr8Oenv remained in the tslwt-33-infected cells. The

radio-labeledgPr8Oenv had been processed into the cleavage prod-uctsofgp7OandPrpl5E, and these cleavage products were

thentransportedtothe plasma membrane, incorporatedinto virions, and exportedfrom the cell. However, in the tslwt-32-infected cells, the processing of radiolabeledgPr80e"vwas

veryinefficientat90 and300min(lanes2and3).Only afaint

gp7Obandwaspresent atboth 90and 300min(Fig. 3, lanes 2 and 3)instead of the intense, prominentgp7Obandthatwas

present for tslwt-33 (lanes 5 and 6). In tslwt-32-infected cells there was some decrease in intensity of the gPr80env

band at 300 min (lane 3), and this may be due to protein degradation. In studies ofa Rauscher MuLV

temperature-sensitive mutant(ts26) which is also defective inprocessing

the envelopeprecursorpolyprotein attherestrictive temper-ature,the nonprocessed envelopeprecursorpolyprotein has

been shownto be degraded (32).

Hybrid tslwt-33, which consists ofa tsl genome except

forasingle nucleotide changewhich resultedin the

replace-ment of Ile-25 by Val ingpPr80e"V, processedgPr8Oenv into gp7OandPrp15Eattherestrictivetemperature (Fig. 3,lanes

5 and 6), whereas the reciprocal hybrid, tslwt-32, ineffi-ciently processed gPr8Oe"v at the restrictive temperature (lanes 2 and 3). These results indicate that the Val-25-Ile

substitution in gPr8Oenv is responsible for the inefficient processing ofgPr80env at the restrictive temperature. The results of the assays for both hybrids tslwt-26 and -42

confirm the results for hybrids tslwt-32 and -33. In hybrid tslwt-26, the Ala-157 in the gPr80env of tslwas replaced by the wild-typeSerof MoMuLV-TB byexchanging the BstXI-BamHI restriction fragment (498 bp, nts 6040 to6537) ofts

lwith thecorresponding fragment ofMoMuLV-TB. Hybrid tslwt-26 inefficiently processed gPr8Oenv at the restrictive

temperature eventhough the Ala-157 substitution in

gPr8Oenv

had been replaced by the wild-type Ser of MoMuLV-TB. This result indicated that the Ser-157--Ala substitution in

gPr8oenv

was notresponsiblefor the inefficient processing of gPr8oenvattherestrictive temperature. On the basis of these

gPr8Oenv

processing results and the temperature sensitivity results, itcanbeconcluded that theVal-25-dle substitution in

gPr8Oenv,

nottheSer-157--Ala substitution ingPr8Oenv,is responsible for both the temperature sensitivity and the inefficiency inprocessinggPr8Oenvat the restrictive temper-atureoftsl.

Intracellular location of the nonprocessed gPr8Oe"v at the restrictive temperature. The MoMuLV gPr8oenv precursor polyprotein is vectorially transported into the lumen of the ERduring synthesis (37). In the ER, gPr8Oenv is cotransla-tionally modified with endo H-sensitive mannose-rich aspar-agine-linkedoligosaccharides (27, 37, 40), and monomersof gPr8oenv are assembled into oligomers (25). The oligomers are transported to the cis cisternae of the Golgi apparatus, where subunits of gPr8Oenv in the oligomers are rapidly cleaved by a host cell-encoded protease to yield subunits consisting of heterodimers of gp7O and Prpl5E (9). The processed oligomers are then transported to the medial cisternae oftheGolgiapparatus,where the endoH-sensitive mannose-rich asparagine-linked oligosaccharides are con-verted into endo H-resistantcomplex-type asparagine-linked oligosaccharides (37, 40). In subsequent steps, the endo H-resistant oligomers, consistingofsubunits ofgp70-Prpl5E heterodimers, are transported to the plasmamembrane (37). However, there are cases in which the nonprocessed enve-lope polyprotein of some dual-tropic MuLVs (8) and of a mutant Rauscher MuLV with a smalldeletion intheenv gene (21) are transported to the cell surface. Since tsl inefficiently processes gPr80env at the restrictive temperature, it was of interest to determine the intracellular location of the

non-processed gPr8oenv.

NIH 3T3 cells were infected with tslwt-32 and -33 atthe restrictive temperature (39.0°C), pulse-radiolabeled with both L-[35S]cysteine and L-[35S]methionine for 10 min, and chased for 0, 90, or 300 min. After the chase period, the radiolabeled cells were lysed, and gp7O and gPr8oenv were immunoprecipitated with anti-gp7O antisera. The radiola-beled proteins, treated or not treated with endo H, were separated by SDS-PAGE(Fig. 4). Endo Hhydrolyzes

man-nose-rich asparagine-linked oligosaccharides from the poly-peptide backbone of glycoproteins to yield apoproteins with increasedelectrophoreticmobilities (39). In all chase periods for both tslwt-32 and-33, the glycosylated gPr8Oenv (Fig. 4, odd-numbered lanes) was hydrolyzed by endo H to an unglycosylated core polypeptide (Fig. 4, even-numbered lanes) with an increased electrophoretic mobility and anMr of 63,000 (7), andendo H-resistant gPr8OenVwasnotdetected (Fig. 4, even-numbered lanes). These results indicated that at the restrictive temperature gPr8Oenv is not transported beyond the cis cisternae of the Golgi apparatus in both tslwt-32 and -33-infected cells (19). However, the intensity of the gp7O bands in lanes 9 and 11 indicated that the endo H-sensitive gPr80env of tslwt-33 is processed by the host cell-encoded processing protease in the cis cisternae of the Golgi apparatus, whereas there is very little processing of the endo H-sensitive gPr8Oenv of tslwt-32(lanes 3 and 5).

Analysis of the amino acid substitution in gPr8Oe"v that is responsible for the neurovirulence of tsl. In this report, neurovirulence is defined as the ability of a virus to cause eitherparaparesis orhindlimbparalysis in mice.Paraparesis is defined as a partial or slight degree of paralysis of the

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ts tsiwit:33

Genorne , ._

Chase(m,n) 0 90 300 0 90 300

EndoH

gPr8oenv

[image:6.612.67.299.83.284.2]

63kD--1 2 3 4 5 6 7 8 9 10 11 12

FIG. 4. Endo HsensitivityofgPr80envat the restrictive temper-aturein tslwt-32and -33-infected cells. NIH 3T3 cellswereinfected with tslwt-32 and -33 and incubated at39.0°C. The infected cells

were pulse-radiolabeled for 10 minwith both L-[35S]cysteine and

L-[35S]methionineand chased for0, 90,or300min.The radiolabeled cellswerelysed,andsamplesof thelysate containing2 x 105cpm

weretreated with anti-gp 70 antisera. Radiolabeled proteinswere

released from theimmunoprecipitates. The releasedproteinswere

treated with endo H (+) or untreated (-), and then they were

separated bySDS-PAGEon a9.0%polyacrylamide gel.The

arrow-headdesignatesanunidentifiedprotein (lanes 3, 4, 5,and6)which

coprecipitatedwithgp7OandgPr8oenv;it hasanMrof77,000,which isthesame asthat of the BiPprotein (11).

hindlimbs. A useful diagnostic sign of paraparesis is the retraction of the hindlimbs when the micearesuspended by

their tails. In addition to retraction and weakness of the hindlimbs, frequent tremors ofthe hindlimbs are also ob-served. Hindlimbparalysisis defined aspartial orcomplete

loss offunction and voluntary movement of the hindlimbs. tsl is a neurovirulent retrovirus, and it causes hindlimb paralysis in mice (22, 41, 50). In previous studies, it was

shown by constructing hybrid viruses between tsland both MoMuLV-TB and MoMuLV that two regions of the env

geneoftsl, i.e.,XbaI-BamHI(771 bp,nts5767 to6537)and BamHI-ClaI (1,138 bp, nts 6538 to 7675), are required to

causehindlimbparalysis. Itwasalsofoundthat the

BamHI-ClaIrestrictionfragment onlyenhanced theability of tsl to replicatein the CNS relative to MoMuLV-TB andthat the BamHI-ClaI restrictionfragmentdid notcode for

neurovir-ulence,asdid theXbaI-BamHIrestrictionfragment (38, 49). TheXbaI-BamHI restriction fragmentof tslencodes for two amino acid substitutions in gPr8Oenv, i.e., Val-25-Ile andSer-157---Ala (38). In this study, in ordertodetermine which one of the two amino acid substitutions (Ile-25 or

Ala-157)wasresponsiblefor theneurovirulence oftsl, mice

were intraperitoneally inoculated with the panel of viruses

shown inFig. 1.The infectedmice wereobservedfor signs ofhindlimb paralysis, and theresultsare shown inTable 1

and Fig. 1. During a postinoculation period of5 months,

mice that were infected with MoMuLV-TB and hybrids tslwt-30 and-33 showedno signsof hindlimbparalysis (-), micethatwere infected with hybrids tslwt-31b and -32had paraparesis (+), and mice that were infected with tsl and

hybrids tslwt-23, -26, -41, and -42 had hindlimb paralysis (+ +). Onlyviruses which contained theVal-25-dle

substi-TABLE 1.

Neurovirulence

oftsl,MoMuLV-TB, and the

hybrid

viruses inBALB/candCFW/Dmice'

No.of No. of miceexhibiting

Virus Mouse mice

strain inocu- Para- Para- No Neurovir-lated lysis paresis signs ulenceb

MoMuLV-TB BALB/c 9 0 0 9

-MoMuLV-TB CFW/D 9 0 0 9

-tsl BALB/c 9 6 3 0 ++

tsl CFW/D 10 9 1 0 ++

tslwt-23 BALB/c 9 6 3 0 ++

tslwt-26 BALB/c 9 6 3 0 ++

tslwt-30 BALB/c 7 0 0 7

-tslwt-30 CFW/D 7 0 0 7

-tslwt-31b BALB/c 9 0 9 0 +

tslwt-31b CFW/D 10 1 7 2 +

tslwt-32 BALB/c 9 0 6 3 +

tslwt-32 CFW/D 10 0 7 3 +

tslwt-33 BALB/c 9 0 0 9

-tslwt-41 BALB/c 7 5 2 0 ++

tslwt-41 CFW/D 12 7 5 0 ++

tslwt-42 BALB/c 9 6 3 0 ++

tslwt-42 CFW/D 11 7 4 0 ++

a Within 48hafterbirth, micewereinoculatedintraperitoneallywith101to 106infectious unitsof virus in 0.1 mlofgrowth medium and observed for signs ofhindlimbparalysis for5months. TheCFW/Dmiceweretreatedas ablind study.

bDegree of neurovirulence: ++, hindlimb paralysis; +, paraparesis (a slight degree of paralysis which affects the hindlimbs); -, no signs of paraparesisorhindlimbparalysis.

tution in gPr8Oenv were neurovirulent (+ or ++) (tsl and hybrids tslwt-23, -26, -31b, -32, -41, and -42), whereas the viruses which contained the Val-25 in gPr80e"v were not neurovirulent (-) (MoMuLV-TB andhybrids tslwt-30 and -33). These results indicate that the Val-25-Ile substitution ingPr80envisresponsible for the neurovirulenceof tsl, and this is the same amino acid substitution thatisresponsible for the temperature sensitivity and inefficient processing of gPr8oe.v attherestrictive temperatureoftsl.

Analysisof the amino acidsubstitutions encoded by the env gene of tslthat areresponsiblefor hindlimbparalysis. There were two classesof neurovirulent virusesin Fig. 1 and Table 1.Thevirusesin one class (+ +) caused hindlimbparalysis in mice, andthey weremoreneurovirulentthan the viruses in the other class (+), whichcausedparaparesis. Onlyviruses which contained both the Val-25-Ile substitution in gPr80e"vand theBamHI-HpaI restriction fragment (660 bp, nts6538 to 7197) oftsl caused hindlimb paralysis (tsl and hybrids tslwt-23, -26, -41, and -42), whereas viruses which contained both the Val-25->Ile substitution in gPr8oenv and the BamHI-HpaI restriction fragment of MoMuLV-TB caused paraparesis (hybrids tslwt-31b and -32). This result confirms ourearlierfindingthatonly one region of the env gene of tsl, i.e., XbaI-BamHI, is required for neuroviru-lence, whereas two regions of the env gene of tsl are required for hindlimb paralysis, i.e., XbaI-BamHI and BamHI-ClaI (38, 46, 49).

Inthisstudy, the size of the latter region has been reduced from a 1,138-bpBamHI-ClaIrestriction fragment to a 660-bp BamHI-HpaIrestriction fragment. TheBamHI-ClaI restric-tionfragment oftslcoded for twoamino acid substitutions,

i.e.,

Arg-430--Lysingp7Oand

Ile-23-*Val

inp1SE,whereas the BamHI-HpaI restriction fragment coded for only the Arg-430->Lys substitution in gp7O. As a result, the Ile-23--Val substitution in

pi5E

was eliminated from having a role in causing hindlimb paralysis. In hybrid tslwt-30, the

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BamHI-HpaI

restriction fragment of tslwas introduced into the genome of MoMuLV-TB, but this hybrid virus was not neurovirulent. However, when both the

BamHI-HpaI

re-striction fragment of tsl and the

BstEII-BstXI

restric-tion fragment of tslwere introduced into the genome of MoMuLV-TB, the resulting hybrid virus, tslwt-42, caused hindlimb paralysis. Since the

BstEII-BstXI

restriction frag-ment coded for the Val-25-*Ile substitution in the

gPr80e"v

of tsl and the BamHI-HpaI restriction fragment coded for the Arg-430--*Lys substitution in

gp70,

this result suggested that both the Val-25--*Ile substitution in the

gPr80env

of tsl and the Arg-430--Lys substitution in the

gp7O

of tsl are required for hindlimb paralysis.

However, there is an additional amino acid difference between tsl and MoMuLV-TB in

gp7O

which must be

con-sidered

when analyzing these hybrid viruses. In

MoMuLV-TB there is an Ala

in

position 347 of

gp7O

instead of the Thr which is in both tsl and MoMuLV-TB (Fig. 1). The Thr-347-)Ala substitution in the

gp7O

of MoMuLV-TB probably arose during propagation of MoMuLV-TB after tslwas se-lected from MoMuLV-TB but before the genome of MoMuLV-TB was molecularly cloned (i.e., clone wt-25) (38, 47). At this time it is not known whether the Ala-347 in the gp7O of hybrids tslwt-31b and -32 has an effect on their neurovirulence. Nevertheless, it can be concluded that the Val-25->Ile substitution in

gPr8oenv

is responsible for the neurovirulence of tsl and that both theVz1-25-d)le substitu-tion in

gPr8Oenv

and the region of

gp7O

encoded by the BamHI-HpaI restriction fragment are required for hindlimb paralysis.

DISCUSSION

tsl is a mutant retrovirus which causes hindlimb paralysis

in

susceptible strains of mice (22, 41, 43, 50). In order to pinpoint individual tsl amino acid substitutions encoded by the env gene to specific phenotypic characteristics of tsl, a new set of hybrid viruses was constructed, and they were assayed for phenotypic characteristics of tsl (Fig. 1). The assays of the new hybrid viruses showed that the Val-25-*Ile substitution in

gPr8Oenv

is responsible for the temperature sensitivity, inefficient processing ofgPr80env at the restric-tive temperature, and neurovirulence of tsl. However, hy-brid viruses tslwt-31b and -32 showed that the Val-25--*Ile substitution in

gPr80env

alone was not sufficient to cause hindlimb paralysis, but only paraparesis. An additional amino acid substitution,

most

likely the Arg-430--,Lys sub-stitution in

gp7O

encoded by the

BamHI-HpaI

restriction fragment (660 bp, nts 6538 to 7197), in combination with the Val-25--*Ile substitution

in

gPr8Oenv

was required for hind-limb paralysis. These results are consistent with our previ-ous observation that two functionally distinct regions in the env gene of tsl are required to induce hindlimb paralysis (49).

The results presented in this report for hybrid viruses tslwt-31b and -32 demonstrate that the Val-25-+Ile substi-tution in

gPr8Oenv

is responsible for the neurovirulence of tsl. This finding appears to contradict the results from a previous report (49) that hybrid viruses

tslwt-11,

-12, and -13, which also contain the Val-25-*Ile substitution in

gPr8Oenv,

were not neurovirulent (although hybrid tslwt-13 did cause a temporary paraparesis in 34% of the infected mice). This seeming contradiction may be due tonucleotide sequence polymorphisms in the BamHI-PstI fragments (2,294 bp, nts 6538 to 8264 and 1 to 567) that were used to construct hybrids tslwt-11, -12, -13, and -32. The

BamHI-PstI fragment of hybrid tslwt-32 was derived from Mo-MuLV-TB, whereas the

BamHI-PstI

fragments of

hybrids

tslwt-11, -12, and -13 werederived fromMoMuLV. Nucle-otidesequence analysis ofa1,872-ntregion

(nts

6538to8264 and 1 to 145,which encodes forthe62% oftheenvgeneand theentirelongterminal repeat) in the

BamHI-PstI fragments

of bothMoMuLV-TB andMoMuLVhas identified 35 differ-encesinnucleotide sequence (38,48). Ithasbeen shown that nucleotidedifferences in the U3 region of the

long

terminal repeats of MoMuLV and MoMuLV-TB have resulted in differencesin thediseaseprofiles induced

by

MoMuLVand MoMuLV-TB (48). Possibly the BamHI-PstI

fragment

of MoMuLV in hybrids

tslwt-11,

-12, and -13 attenuates the potential neurovirulence of these hybrid

viruses,

which is determined bythe Val-25--+Ile

substitution

in

gPr8Oenv.

Although

atpresentthemolecular mechanism

responsible

for theneurovirulence oftslisnot

known,

it is clearfromthe studiesreportedhere that itcorrelates withthe temperature sensitivity and the defect in

processing

gPr8Oenv

at the restrictive temperature oftsl (38, 44,

46, 49).

In newborn mice, the body temperature ispermissive forthe

replication

oftsl, and tslis abletoreplicate in

target

cells and

spread

to

the CNS (5, 41, 50). Inadult

mice,

the

body

temperature is restrictive for the replication of tsl, and tsl induces the degeneration of neural cells in the CNS.

Possibly

the gPr8oenv processing defect of tsl is

responsible

for the degeneration ofneural cells in theCNS. Inorderto investi-gate the mechanism of induced neural cell

degeneration

by

tsl, we soughtto determine the intracellular location of the nonprocessed gPr8oenv at the restrictive temperature.

The

gPr8Oenv ofhybrid tslwt-33 wasprocessed at the restrictive temperature, whereas the

gPr8Oenv

of tslwt-32 was ineffi-ciently processed (Fig. 3). Unlike the

gPr8oenv

of

tslwt-33,

thegPr8Oenv oftslwt-32 is

temperature-sensitive,

andatthe restrictive temperature the

gPr8Oenv

of tslwt-32is

probably

misfolded

(12).

There are two

possible

reasons for the inefficient processing of the misfolded

gPr8Oenv

of tslwt-32. The first possibility is that the misfolded

gPr8oenv

is not

transported from the ER to the cis cisternae of the

Golgi

apparatus (4, 6, 11, 20, 30), which contains the host cell-encoded processing protease (9), and as aresult a

pool

of nonprocessed

gPr8Oenv

remains in the ER. The second possibility is that themisfolded

gPr8Oenv

is

efficiently

trans-ported to the cis cisternae of the

Golgi

apparatus, but the misfolded polyprotein is apoor substrate forthe host cell-encoded processing protease, and as a result a pool of

nonprocessed

gPr8Oenv remains in the cis cisternae of the Golgiapparatus. Experiments have notbeen done to distin-guish between these two

possibilities,

but most

likely

the pool ofmisfolded

gPr8oenv

remainsin the ERandnot in the cis cisternae of the

Golgi

apparatus, because endo H-resistantgPr8oenv is not detected

(8,

19,

21,

30).

Ifinefficient processing of

gPr8Oenv

induces the

degenera-tion of neural cells,it mustbe taken intoconsideration that there are

different

molecular forms of

gPr8Oenv

in the in-fected cells,

and possibly

athreshold

steady-state

level ofa

specific molecularform of

gPr8Oenv

inducesthe

degeneration

ofneural

cells

in the CNS. At the

permissive

temperature,

there are two major molecularforms of

gPr8Oenv

in theER.

There are both newly

synthesized

monomers of endo H-sensitive

gPr8Oenv

and

oligomers,

consisting

of subunits of endo H-sensitive

gPr8Oenv,

which were assembled from the monomers. The steady-state

pool

of

gPr8Oenv

monomers is

maintained by the influx of

newly

synthesized

monomers

into the pool of

gPr8Oenv

monomers and theefflux of

mono-mers from the pool of

gPr8Oenv

monomers to formsubunits

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of

oligomers.

The steady-state pool of oligomers is main-tainedby the influx oftheoligomer subunits into thepoolof oligomers and the efflux ofoligomersfrom the ER to thecis cisternae of the Golgi apparatus, where processing of the gPr8oenv subunits occurs. As a result, at the permissive temperature there is a constant ratio between steady-state levels of endo H-sensitive gPr8oenvin monomers and endo H-sensitive gPr8Oenv in subunits ofoligomers in the ERs of infected cells.

However, Fig. 4 shows that in cellsthat were infected at therestrictive temperature by hybrid tslwt-32 (which con-tains theVal-25-*Ile substitutioningPr8oenv), asteady-state level ofendo H-sensitive gPr8OefV is maintained in the ER,

probably

asaresultof inefficienttransportof gPr8Oenvto the ciscisternae of the Golgiapparatus. SincethegPr8Oenv oftsl isatemperature-sensitive protein,at therestrictive

temper-aturethe

protein

isprobably misfolded (12). Instudies ofthe vesicular stomatitis virus G protein (6, 20) and the influenza

hemagglutinin

protein (4, 11), it has been shown thatproper

folding

of the monomerforms of both the G protein and the

hemagglutinin

protein in theERis required for oligomeriza-tion andtransportfrom the ERtotheGolgiapparatus. If this

finding

alsoappliestotsl, thenattherestrictivetemperature the

steady-state

levelof misfolded gPr8Oenvin monomers in the ER is

probably

much higher than that of gPr8Oenv in subunits of

oligomers

inthe ER.Possiblyahigh steady-state level of misfolded monomers of gPr8oenv in the ERinduces

degeneration

of neural cells, and this is the reason for the

degenerative

effect oftsl onneural cells in the CNS. Another aspect ofthe replication of tsl is its enhanced

ability

to

replicate

in theCNS relativetoMoMuLV-TB (41);

previously

we have shown that the BamHI-ClaI restriction

fragment

(1,138 bp,nts6538to7675) inthe env geneoftsl is

responsible

for this ability (38, 49). In this report, it was shown

(Table

1 and

Fig.

1)that the Arg-430--Lys substitu-tion in

gp7O

which is encoded bythe BamHI-HpaI restric-tion

fragment

(660 bp, nts 6538 to 7197) was most likely responsible for this property of tsl. The

Arg-430-+Lys

substitution is located in the carboxy-terminal portion of

gp7O

(36, 38). The putative ecotropicreceptorbindingregion for MuLVs has been localized in the carboxy-terminal

portion

of

gp7O

(16, 26). Possibly, the Arg-430-(Lys substi-tution in

gp7O

alters the

affinity

of thegp7O surface proteinof tslfor the

ecotropic

receptorprotein (1), thereby increasing the

infectivity

of tsl in neural cells ofthe CNS.

Alterna-tively,

the alteration in gp7O caused by the Arg-430->Lys substitutionmayenabletsl tobindto a receptor molecule on neural cells that is different from the ecotropic receptor protein. It has been proposed that the neurovirulent wild mouse ecotropic MuLVs WM1504-E and Cas-Br-E infect neuralcells by usingareceptorprotein that is not the same

as the ecotropic receptor protein (23, 24). The ecotropic WM1504-E MuLV has a greater in vitro binding affinity for neural cells of the spinal cord than the lymphomagenic amphotropicWM1504-A virus(23).Furthermore,it has also beenreported thathumanimmunodeficiencyvirus type 1 is able to enter neural cells other than through the CD4 receptor, which is thereceptor for humanimmunodeficiency virus type 1 inlymphoid cells (15).

It is not clear why tsl is neurovirulent whereas other

ecotropic

MuLVs with phenotypic properties similar to those oftsl are notneurovirulent. Atemperature-sensitive

mutantof RauscherMuLV hasbeen isolated (ts26), and like tsl it is inefficientin processing gPr8Oenv at the restrictive temperature(32), yet ts26 doesnot cause hindlimb paralysis inmice (14).The merereplicationofMuLVs intheCNS is

notsufficient for neurovirulence either. MoMuLV-TB

repli-cates in neural cells of the CNS to some extent and causes

slight

histological

lesions

(41),

but it is not neurovirulent (50).

Furthermore,

it has been

reported

that mice which express

large

amounts of MoMuLV in the CNS do not

develop

neurological

disease

(35).

An

understanding

of the molecular mechanism of the neurovirulence of tsl may be importantfor studyingthe growing list ofhuman

neurologi-cal diseaseswhich have been

implicated

as

being

caused

by

retroviruses, which include human

immunodeficiency

virus type1-associatedneurological diseases(17),multiple sclero-sis (29), and tropical spastic paraparesis (33).

ACKNOWLEDGMENTS

We thank D. Hollowell and I. Beauchamp for excellenttechnical assistance. We alsothank Joyce Mayhugh forassistance in prepa-ration of the manuscript.

This investigationwas supported by Public Health Service grant CA 45124 from the National Cancer Institute to P.K.Y.W. andbya grant from The National Cancer Institute ofCanada (NCIC) to J.K.B. J.K.B. is a Terry Fox Career Research Scientist of the NCIC.

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