A 1.6-kilobase-pair fragment in the genome of the ts1 mutant of Moloney murine leukemia virus TB that is associated with temperature sensitivity, nonprocessing of Pr80env, and paralytogenesis.

Copyright ©D 1985,American Society for Microbiology

A

1.6-Kilobase-Pair

Fragment

in the Genome

of the tsl Mutant of

Moloney Murine Leukemia Virus

TB

That

Is

Associated

with

Temperature

Sensitivity,

Nonprocessing

of Pr80env,

and

Paralytogenesis

P. H. YUEN, D. MALEHORN, C. KNUPP, ANDP. K. Y. WONG*

Department of Microbiology and SchoolofBasicMedical Sciences, University of Illinois, Urbana, Illinois 61801

Received 13 November1984/Accepted 21 January 1985

tslandtS7, twotemperature-sensitive mutants of Moloney murine leukemiavirus strain TBinducehind-limb

paralysis in 100% ofCFW/D mice injected. These two paralytogenic mutants also share a defect in their

inability to process the env precursor protein, Pr8Oe"v, at the restrictive temperature. To identify the

mutation(s) in the genomes of the paralytogenic mutants whichcausetheinabilitytoprocessPr8Oe"v efficiently

and confer the ability to cause hind-limb paralysis instead of lymphoma, we constructed chimeric genomes

between tsl and Moloney murine leukemiavirus or the TB strainofthevirus.Weidentifieda3.9-kilobase-pair

HindIII-PstI sequence from nucleotides 4895 through 8264 and 1through567oftsl, comprising the3' end of

thepol and all of the env genes, the long terminal repeat, and the 5' noncoding sequence, as being responsible

for the temperature sensitivity, the inefficiency in processing Pr8Oe"v, and the induction of paralysis. We

extendedthese findings by demonstrating that the 1.6-kilobase-pairpol-gp7O HindIII-BamHI DNAsequence

fromnucleotides4895 through 6537 oftsl within the3.9-kilobase-pairHindIII-PstI fragment is necessary for tsl toinduceparalysis.Inaddition,we showed thatthis1.6-kilobase-pair fragment also controls theprocessing

of Pr8Oe"v and thetemperaturesensitivity oftsl.

Murineleukemia viruses(MuLVs)are acomplex groupof viruses with the ability to induce a variety of diseases in

mice. For example, Moloney MuLV (MoMuLV) induces T-cell lymphomas (9), and members of the Friend virus

complex induce erythroleukemias. On three separate

occa-siuns we have isolated from MoMuLV-TB, a strain of

MoMuLV, a group of temperature-sensitive mutants, tsl,

ts7, and tsll,

which

induce hind-limb paralysis in 100% of

CFW/D mice injected with them. This is in contrast to the

induction of lymphoma byMoMuLV-TBin the same mouse

strain. Inductionof paralysis has also been reported for the

Cas-Br-E strain of MuLV, whichwasinitially isolated from

the brain of a paralyzed wild mouse (for a review, see

reference 3), and for four clones of MuLV isolated from a

paralyzed Fischerrat whichhad beeninfected with

rat-pas-saged Friend leukemia virus (5). The mechanism by which

theseneurotropic viruses induce paralysis is still unknown.

The tsl group ofmutant viruses is especially suited for

investigations intothe molecularbasis of this

retrovirus-in-duced neurological disease because (i) these paralytogenic

mutants were derived from a nonparalytogenic parent, so

that thechange (ormutation) in the gene responsible for the

induction ofparalysis

could

be identified by comparing the

genome ofthe mutants with that ofthe parent, (ii) the tsl

groupofmutants seemtobemorepotentthan theCas-Br-E

strain of MuLV (they cause paralysis in every one of the

mice injected, and their latent period is relatively shorter

than that ofthe Cas-Br-E strain of MuLV); and (iii) this

groupofparalytogenic mutants share a common

character-istic in their inability to process the precursorPr8Oe'7'(20). If

the accumulation ofPr80e'l' polypeptides can be correlated

with theinductionof paralysis in vivo, it will provide a basis

*Correspondingauthor.

for further investigation into the molecular mechanism of

this retrovirus-induced paralysis.

Toidentifythe mutation(s)inthe genomesof the

paralyto-genic mutants which confer temperature sensitivity,

ineffi-cientprocessing of

Pr8Oehz',

and theabilityto causehind-limb

paralysis, we constructed chimeric genomes between tsl

and MoMuLV or MoMuLV-TB. Using this approach, we

have found that replacing the pol-gp7O 1.6-kilobase-pair (kbp) HindIII-BamHI DNA sequence from nucleotide (nt) 4895 to nt 6537 oftsl with thehomologous sequence from

either MoMuLV or MoMuLV-TB was sufficient to correct

temperature sensitivity and inefficiency in the intracellular

processing ofPr8Oe'7' in tsl-infected cells. In addition, the

constructsalsofailedtoinducehind-limbparalysis.

Further-more, hybrid viruseswhose genomescontained

the

1.6-kbp

HindIII-BamHI tsl sequence from nt 4895 to nt 6537

re-mained temperature sensitive and inefficient in processing

Pr8Oe"'. This 1.6-kbp tsl subgenomic fragment is also

nec-essaryfor the induction ofhind-limbparalysis. The

follow-ingis areport onthese studies.

MATERIALSAND METHODS

Cells. Viruses werepropagated in mouseTB

cells,

a

thy-mus-bone marrow cell line derived from CFW/D mice (1),

andassayedon 15Fcells,amurinesarcoma-positive,

leuke-mia-negative cell line (23). NIH 3T3 cells were used for

transfection experiments. All cell lines were maintained in

Dulbecco modified Eagle medium supplemented with 8%

fetal calfserum.

Viruses. The strain of MoMuLV used in this study, MoMuLV-TB,wasisolatedasdescribedby Wongetal.

(19)

from the tissue extract of a sarcoma produced in an

MoMuLV-infected BALB/c mouse provided by Dr.

Mo-loney. Since itsisolation, it has beenpropagatedin TB cells. MoMuLV-TB has been single-virus, single-cell cloned on

severaloccasions. CloneLV30, usedin the present

studies,

364

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A R U5 R US U3 III P15

p121|-p30--.plOk-

pOl

*-i--

9p70

-4.Pl5E

-1

IU3

l

Ulf

0 1 2 3 4 5 6 7 8 Kb

P Sm K PSA Bg SXHPSM Kp S

p

1) ( (\ P Xh o A SC K SP H x 9BC >X

Sc Sc

BSc A

PXh A Sc K \ SP

1 I1

B0 BgaKSm Sm Bg K

Xh

BScA

PXh AB Sc K \\SC SP II 1111

11S

p Sge eSmx Sm

II

I

H LTR H DNA

k

.

H

pKC7

II

PS

H

LTR

+ HindIII

PstI

PS

Ps

pUC9

+ HindIII

H

J

Pstl

H Ps

I _ I HI PS

+ ligase

H-C +HindIII

La S;m

n

liga

H

Sm H

+ HindlII

ft

Pst

I

H H PS H PS

A4

1

,Is

FIG. 1. (A)EndonucleaserestrictionmapsofMoMuLV(I),MoMuLV-TB(II),and tsl(III). (B)Schematicpresentationofthe molecular

cloningof thetsl, MoMuLV,andMoMuLV-TBSmaI-HindIl sequencefromnt31to nt4894 intopKC7 (I) and the HindIII-PstIsequence

fromnt4895to nt8264 and nt1to nt567intopUC9 (II). Detailsaredescribedinthetext. (C) Constructionofchimericgenomeswith the

HindIII-PstIsequenceof tsltotheHindIII-SmaI sequencesofMoMuLV-TB and MoMuLVand-viceversa.Detailsaredescribed in thetext.

Abbreviations: A, Aval; B, BamHI; Bg, BgII; C,ClaI; H, HindlIl; Hp, HpaI; K, KpnI; P, Pi'uII; S,Sall; Sc,Sacl; Sm, SmaI; X, XbaI; Xh,XhoI.

p Sm PS

P Sm PS

~\) (K \

Sc

B

K p Sm

a () Hp C(tX K

K p Sm

el

>p

Hr

C (

')I

g

Sc

I i t

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1---- I

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was one of the isolates obtained in the most recent clonal

isolation. It hasbeen shown by Shields and co-workers (14) and confirmed by us (unpublished data) that MoMuLV-TB

can be distinguished from standard MoMuLV by the

elec-trophoretic mobilityof the p30protein.Inthis report the two

strains ofMoMuLV will be referred to as MoMuLV and MoMuLV-TB.

tsl is a spontaneous temperature-sensitive mutant of

MoMuLV-TB isolated as described by Wong and

co-workers (19). The tsl strain used in the present studies was also recently purified by cloning TB cells infected with tsl at amultiplicity of infection of 0.01.

Virus assay. The 15F virus assay has been described

previously (18, 21).

Viral DNAs. The permuted genomesof tsl and

MoMuLV-TB were molecularly cloned into Charon 21A as described

by Yuen et al. (22). The infectious recombinant viral DNAs

tsl-19, tsl-20, and wt-25, each with one copy of the long

terminal repeat (LTR), were used in the studies reported here. Plasmid p8.2, an infectious permuted MoMuLV

ge-nome with one copy of the LTR cloned into pBR322 at the

HindIll site (12), was a gift from D. Baltimore. The viral

DNA ofp8.2is referredto as wt-8.2.

Isolation,purification, and analysis of viral DNAs.

Restric-tion fragmentswere separated byagarosegel electrophore-sis. The desired fragments were electroeluted into dialysis bags. The DNA was filtered through glass fiber to remove

agarose debris and then concentrated by ethanol

precipita-tion. The DNA pellet was suspended in TE buffer(10 mM

Tris,1 mM EDTA,pH 8.0) and then extracted with

phenol-chloroform, phenol-chloroform, and ether.

Restriction enzymes were purchased from Bethesda

Re-search Laboratories, Gaithersburg,Md., and the conditions

forendonuclease digestionwerethose recommended bythe

supplier. Gel transfer and filter hybridization were carried

outbythe method of Southern (16).

DNA transfection. The calcium-phosphate precipitation

method of

Graham

and van der Eb (4) was used. Details of

theprocedures were as described in Yuen et al. (22).

Recombinant plasmid and chimeric genome construction.

Restriction fragments were cloned into pUC9 (17), pKC7

(10), or pBR322. Transformations into Escherichia coli

HB101 werecarriedoutby standardprocedures. Construc-tion of recombinant plasmids and chimeric genomes is describedbelow.

Metabolic labeling, immunoprecipitation, and SDS-PAGE.

Immunoprecipitation of intracellular virus-specific proteins

and sodium dodecyl sulfate-polyacrylamide gel electropho-resis (SDS-PAGE)were performed asdescribed previously (20,21). Gelswerefluorographedand exposed to X-ray film at -70°C.

Mouse strain andinoculationprocedure. Theinbred CFW/D

mice and the inoculation procedureused in this studywere

described in Yuen et al. (22).

RESULTS

Construction of chimeric genomes between molecularly

cloned tsland MoMuLV or MoMuLV-TB DNAs. The

restric-tion maps of themolecularly cloned tsl, MoMuLV-TB, and

MoMuLV,

designated

tsl-20, wt-25, and wt-8.2,

respec-tively, are shown in Fig. 1A. These molecularly cloned

genomes,

together with tsl-19, a tsl genome obtained in a

separate cloning experiment (22), were used in the studies

reported here.

As a first step in identifying the mutation(s) in the tsl

genome responsible forthe temperature-sensitive function,

1 2 3 4 5 6 7 8 9 111213141516.1718 Kb

23-4.3

2.3_- 2.0- 1.3-

0.6-FIG. 2. UV fluorescence photograph of the ethidium bromide-stained gel of the endonuclease digests of wt-25, p14, p12, and p18. The DNAs(restriction endonuclease) used in each lanewere: 1and 11,wild-type A(Hindlll)and4X174 (HaeIII);2, wt-25, a recombi-nantCharon21Awith thefull-sizeMoMuLV-TB genome(HindIII and PstI);3, 3.9- and4.1-kbp fragments of wt-25 (PstI);4, pUC9

(HindIII); 5, p14, a recombinant pUC9 plasmid with the 3.9-kbp

HindIII-PstIsequenceofwt-25(HindIII and PstI); 6,p14 (HindlIl); 7, p14 (HindlII and XhoI); 8 and 9, p14and pUC9, respectively

(HindIII andBamHI);12, pKC7(HindIll); 13,p12,apKC7

recom-binant plasmid with the full-size wt-25 genome (HindlIl); 14, p12 (SmaI); 15, p18, a pKC7 recombinant plasmid with the 4.9-kbp SmaI-HindIIIwt-25 DNAsequencefromnt31 to nt 4894(HindlIl);

16,p18 (SmaI);17,p18 (HindlIl andSmaI);18and,p18 and pKC7,

respectively (KpnI andHindIll).

the inefficiency in processing Pr80en,', and the ability to

induce paralysis in mice, chimeric genomes were

con-structed by exchangingthe entire env gene between tsl and

the wild-type virus genomes. Since the permuted tsl,

MoMuLV-TB, and MoMuLV proviruses were cloned into

Charon 21A or pBR322 at the HindIll site, we decided to

fragment each genome into halves: (i) a HindIII-PstI

frag-mentfromnt4895 to nt 8264andnt 1 to nt567,consistingof

the3' end of thepol gene, the entire env gene, the LTR, and

the amino-terminal portion of the gag gene (5' noncoding

sequence), and(ii)aSmaI-HindIllfragmentfrom nt 31 to nt

4894, consistingof part of the Rregionof theLTR, U5,the

gaggene, and the major portion of thepolgene.

Molecular cloning ofthe HindIII-PstI sequence from nt

4895 to nt 8264 and nt 1 to nt 567 oftsl-19, tsl-20, wt-25,and

wt-8.2isshown inFig. 1BII.Thevarioussteps in the actual

cloning of p14, pUC9 recombinant plasmid with the

HindIII-PstI sequence ofwt-25,arepresented inFig.2.The

permuted genome ofwt-25 was restricted from its Charon

21A vector atthe HindlIl site and further cleaved with PstI

(lane 2). The purified 3.9-kbp fragment from nt 4895 to nt

8264andnt1 tont567 and the4.1-kbpfragmentfromnt744

to nt 4894of the PstI-restricted wt-25 genomeare shown in

lane 3. These DNA sequences were not separated before

ligation with PstI-HindIII-restricted and dephosphorylated pUC9 DNA (lane 4). The ligated DNAs were transformed

intoE. coli HB101. Recombinants with the HindIII-PstI 3'

halfof the viral genome could bedistinguished bythesize of

the viral insert, which was about 3.9 kbp (lane 5), the

absence of an XhoI site(lanes6and7), and thepresence of

asingleBamHI site. Due to the presenceofaBamHI site in the polylinkerofpUC9 10 ntfrom theuniquePstI site, the

viral HindIII-PstI fragment was cleaved into two

subfrag-ments of about 1.6 and 2.3 kbp after BamHI restriction

(lanes 8 and 9). Recombinant plasmids pl, p5, and p15,

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+ ligase

J./

H X B LTRPs

L _ ~~p15

x x

p14

Sm

construct

1,2

3,4

5

6

gag

0 1 2

H X B

x

'WI _V ...I . . .o. m

ub

mmm mommmmmm

---x x

__s- --_ _ _1 _ _ WE, Xx x

Ps Virus

IJ tslwt-1,2

tSlwt-3,4

tslwt-5

ts1wt-6

pol

gp7OI1L5E

wLTR

3 4 S 6 7 8 Kb

plasmid DNA -MoMoNuLV-TB DNA

----MoMuLV DNA

ts1 DNA

FIG. 3. Chimeric viral genomes constructed from the 3' pol-env-LTR and the 5' U5-gag-pol sequences of tsl, MoMuLV-TB, and MoMuLV. Infectious viruses tslwt 1 and tslwt 2 were recovered from NIH 3T3 cellstransfected with constructs 1 and 2, which were

constructed by ligating the SmaI-HindIll sequence ofp18 (MoMuLV-TB) to the HindIII-PstI sequence ofpl (tsl-19) or p15 (tsl-20), respectively. Infectiousvirusestslwt 3 andtslwt 4wereproducedwhenconstructs3 and4,obtainedbyligatingthe SmaI-HindIIIsequence ofplO (MoMuLV) with theHindIII-Pstlsequenceofpl(tsl-19)orp15 (tsl-20),respectively,weretransfected into NIH 3T3 cells.Infectious

viruses tslwt5and tslwt 6wereobtainedby transfecting the reciprocalconstructs5and6, generated by ligatingtheSmaI-HindIIIsequence ofp17 (tsl-20) and the HindIII-PstI sequenceofp14(MoMuLV-TB)orp5(MoMuLV), respectively. Abbreviations:B,BamHI; H, HindIll; Ps,PstI; Sm, SmaI; X, XbaI.

carrying the HindIII-PstI sequence oftsl-19, wt-8.2, and

tsl-20, respectively, wereobtained similarly.

Molecular cloning of the SmaI-HindIIl nt sequence of

tsl-20, wt-25, and wt-8.2 is shown inFig. 1BI.The

proxim-ity of the two PstI sites at the 5' end of the gag coding sequence necessitated adifferent strategy for clonally

puri-fyingthegag-polhalves of the genome sothat the full-size

genome could be restored when the cloned 5' halves were

ligated tothe HindIII-PstI 3' halvespreviously cloned(Fig.

1C). The permuted genomes of tsl-20, wt-25, and wt-8.2

wereisolatedandpurified from theirrespectivevectorsand

cloned into pKC7 at the HindIII site. The recombinant

plasmids obtained were restricted with SmaI to determine

the orientation of the viral insert. Recombinant plasmids

with the viral insert in the desired orientation (Fig. 1B la)

werecharacterized bythepresenceofa9.5-kbp SmaI-SmaI

fragment, which consisted of the viable portion of pKC7,

including the ampicillin resistance gene, and the 4.9-kbp

SmaI-HindIII 5' half of the viral genome. Therefore, to isolate the 4.9-kbp SmaI-HindIII viral sequence,

recombi-nantplasmidswiththecomplete viral genomein the correct

orientation were restricted with SmaI, and the 9.5-kbp

fragment was isolated, purified, religated at the SmaI site,

and transformed into E. coli HB101. Ampicillin-resistant colonies were screened for the presence of the 9.5-kbp

plasmids. Recombinant plasmids containing the

SmaI-HindIII viral sequence were then purified for chimeric

genome construction.

Restriction analysiswasperformedonp12, arecombinant

plasmid with the permuted wt-25 genome in the desired

orientation, andp18,adeletion mutantofp12withonlythe

SmaI-HindIll 5' half of the wt-25 genome (Fig. 2, lanes 12

through 19). The full-size wt-25 genome restricted from its

pKC7vector attheHindIIIsites isshown in lanes 12 and 13.

OnSmaIdigestion, p12wasfragmentedintoalarge 9.5-kbp

andtwosmallerfragmentsofabout 2kbpeach(lane 14).The presenceofunique HindIII (land 15)andSmaI(lane 16)sites in deletion mutant p18 caused it to be cleaved into two

fragmentsof about 4.8 kbpeach afterdoubledigestion with

SmaI and HindlIl (lane 17). AfterHindIII and KpnI

diges-tion, p18wascleavedinto threefragments, consistingof the

deleted pKC7 vector with a few nt from the virus genome andtwo viral sequencesof about2.0 and 2.9kbp (lane 18).

pKC7 did not have a KpnI site (lane 19). Recombinant

plasmids plO and p17, with the SmaI-HindIII sequence of

wt-8.2 and tsl-20, respectively, were similarlyisolated. The chimeric genomes between tsl and MoMuLV or MoMuLV-TB were constructed asshown in Fig. 1C. Each of these constructs (Fig. 3) had an overlapping sequence from theSmaI siteat nt30tothe PstI siteat nt567,derived from both tsl and MoMuLV or MoMuLV-TB DNA. The

ligated DNAs were transfected into NIH 3T3 cells without

pl8

p17

Sm H

_ -- ___0-_ - I_l

40--i

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further treatment. Infectious viruses obtained from each

construct werecharacterized. The mechanism for generating

the infectious viruses after transfection of these hybrid

DNAs into NIH cells is not known. Although a complete

LTRsequence is at the 3' end, only part of R and the entire

US region are present at the 5' end of the genome. A

recombination of theoverlapping region (nt 30 through 567),

which is duplicated in the constructs to generate the

pro-moter atthe 5' endofthe genome, may be necessary. If this

is the case, the parental origin of the overlapping region in the resulting viruses may vary from virus tovirus.

Characterization of the infectious reconstructed viruses.

The reconstructed viruses tslwt 1, 2, 3, 4, 5, and 6 were

testedfortemperature sensitivity, abilitytoprocess Pr80en'

at the restrictive temperature, and ability to induce

hind-limb paralysis in mice (Table 1). tslwt 1, 2, 3, and 4,

producedby the constructscomposed ofthe 3' halfofthe tsl

genome and the 5' half ofthe MoMuLV or MoMuLV-TB

genome, remained temperature sensitive. NIH 3T3 cells

transfected with any of these four viruses accumulated

Pr80en", with protein profiles indistinguishable from that of tsl-infectedcells (see Fig. 8). In addition, 100%of the mice

injected with tslwt 1, 2, 3, or 4became paralyzed. The latent

period oftslwt 3 was also examined because ofthe large numberofmiceinjected with this virus. More than80% of

themice becameparalyzed by60dayspostinjection (Fig. 4), suggestingthat tslwt3 may have alonger latentperiodthan

eitherthe cloned or noncloned tsl. The significance ofthe

apparentlyextended latentperiodshownbytslwt3 needs to

be investigated further.

Incontrast, tslwt 5 and 6, produced byconstructs

com-posed ofthe 5' halfofthe tslgenome and the 3' half ofthe

MoMuLV-TB orMoMuLV genome, respectively, were not

temperature sensitive and processed Pr80en' like wild-type

virus-infected cells (see Fig. 8). In addition, tslwt 5 and 6

failed to inducehind-limb paralysis in mice. Of the CFW/D mice infected with tslwt 6, 100% died oflymphoma 3 to 5

monthspostinjection. Noneof the 10CFW/Dmice infected

withtslwt 5 showed any sign of disease atthistime (97 days

postinjection). Therelativelyshort latentperiodrequired by

tslwt 6 to induce lymphoma is in agreement with that

reported for the standard MoMuLV (9) but in contrast to

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that reported forMoMuLV-TB (7).

TABLE 1. Characterization of virusesproduced by chimeric genomes intransfectedNIH-3T3cellsa

Virus

_(34aC/39aC)y

Titer ratio _processingbPr80e"' No. of mice %of mice injected paralyzed'

wt-25d 1.4 P 20 0

tsll9d 3.0 x 103 NP 28 100

tslwt 1 1.5 x 103 NP 16 100

tslwt2 2.5 x 103 NP 14 100

tslwt 3 1.2 x 102 NP 52 100

tslwt4 1.4 x 102 NP 22 100

tslwt5 4.3 P 10 0

tslwt 6 1.2 P 22 0

aAppropriatelydiluted virus was inoculated into two setsofplatesseeded with 15F cells and allowed to adsorb for 45 min at34°C.One setofplateswas

incubatedat 34°C and the other at 39°C. The assay was read 4 to 5days postinfection.

bInfectedcells grown at39°Cwerepulsedfor 15 min with[35S]methionine

andchased for 3 h. Cell extracts wereimmunoprecipitatedwithantiserumto MoMuLV gp7O, analyzed by SDS-PAGE, fluorographed, and exposed to Kodak X-ray film. P, Pr80M processed togp7Oandp1SE;NP, Pr80M""not

processed.

cCFW/Dmice within 48 h of birth were inoculated intraperitoneallywith

105to106 infectiousvirus in 0.1 ml ofgrowthmedium. d Parentalvirus.

40

20

~0

N

(U

E

-0

0

40

20

40-tsl

ts1-19

tsl wt-3

201

20 40 60 80

Latent period

(days)

FIG. 4. Histogram ofthe percentage of mice paralyzed by the

non-molecularly cloned tsl, the molecularly cloned tsl-19, and hybridvirustslwt3, with the indicated latentperiods.

The above results indicated that the 3' half ofthe tsl

genome contained the sequence(s)

responsible

forthe

inef-ficiency

in

processing

Pr8Oenl' atthe restrictivetemperature,

the induction of paralysis, and the

temperature-sensitive

function.Theobservationthat tslwt1, 2, 3, and 4,

although

possessing

theU5and the 5'

portion

ofthe gag geneofboth tslandwild-typegenomes,remained

phenotypically

like tsl

suggeststhat thisoverlapping

portion

ofthe constructfrom

the SmaI site at nt30 to the PstI site at nt 567 may not be

involved in producing the

phenotypic

differences observed

between tsl and wild-type virus.

Constructionof chimericHindIII-PstI sequences of tsl and

MoMuLV-TB or MoMuLV. To further delimit the DNA

sequence of the tsl genome which confers temperature

sensitivity,

inefficiency in

processing Pr80ens,

and

ability

to

induce paralysis, chimeric genomes between tsl and

MoMuLVorMoMuLV-TB (see

Fig.

7) were constructed.

The isolation and purification of the HindIII-BamnHI and

BamnHI-PstI sequences of the tsl, MoMuLV-TB, and

MoMuLV genomesareshowninFig.5and 6. Todistinguish

constructs p29 and p32, which consist of a

wild-type

HindIII-BamHI sequence and a tsl BamHI-PstI sequence,

from constructp31, which consists ofatsl HindIII-BamHI

sequenceandawild-typeBamHI-PstI sequence

(Fig.

7), the

chimeric HindIII-PstI

fragments

wererestricted with XbaI.

We have previously shown (22) that the HindIII-PstI

se-quencesoftsl,MoMuLV-TB,and MoMuLV may be

distin-guishedbyrestriction with XbaI. tsl has lost theXbaI siteat

nt8113 in the U3regionof MoMuLV-TB and MoMuLV.In

addition, the XbaI site at nt 5325 in the

carboxy-terminal

portionof thepol

coding

sequenceof the MoMuLV genome

is absent in both thetsl and MoMuLV-TB genomes.

pUC9

had noXbaI restriction site

(Fig.

6, lanes 8 and

9).

Whereas

p31

(lanes 10and 11)wascleaved

by

XbaI intotwo

fragmentsof about 2.3 and4.4

kbp,

XbaI restricted

p29 (lane

12) into two fragments of about 6.3 and 0.4

kbp

and

p32

(lane 13) only once.

To restore the complete genome, the HindIII-PstI viral

sequences isolated from p29,

p30,

and

p32

were

ligated

to

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B Ps

p1

p5

p14

B

A +BamHI

B H B B\Ps B

B

pBR322

+ligase pU9 +ligase

'B

B Ps B

p~~ ~ ~ ~ ~~~B p26

p4 p22 p27

B

B

I+BamH I

+ ligase )

Ps B

B

p29 Ps p31

B p32

~~~~B

[image:6.612.138.470.81.572.2]

correct orientation

FIG. 5. Schematicpresentationof themolecularcloning of the HindlIl (H)-BamHI (B)(panel A)and BamHI-PstI(Ps) (panel B)sequences

oftsl,MoMuLV-TB, and MoMuLV, and the construction ofachimericHindIII-PstI viralsequence.Recombinantplasmidspl,p5,andp14, containingthe HindIII-PstIviralsequencesoftsl, MoMuLV, and MoMuLV-TB, respectively,wererestrictedwithBamHI, generatingtwo

fragments of about 2.2 and 4.1 kbp (see Fig. 6, lane 2). The 2.2-kbpfragment consists of the BamHI-PstI viralsequenceand 10ntfrom the polylinker of pUC9. The4.1-kbp fragment consists of the viral HindIII-BamHIsequenceandpUC9. The4.1-kbp fragmentwasreligatedat

the BamHI site, transformed into strain HB101 cells, and generated recombinant plasmid p19 from pS, p21 frompl, and p40 from p14. The 2.2-kbp fragment derived from pl wascloned into pUC9at the BamHI site, and the 2.2-kbp fragment from p5 and p14 was clonedinto

pBR322, alsoatthe BamHI site. The recombinantplasmid with the BamHI-PstIsequencefromtslwasdesignated p22, and the recombinant

plasmids with the MoMuLV and MoMuLV-TB BamHI-PstI sequences weredesignated p26 and p27, respectively. Recombinant plasmids p22, p26,andp27wererestricted with BamHI.TheBamHI-BamHI fragment containing the BamHI-PstI viralsequence wasseparated by agarose gel electrophoresis and isolated by electroelution. Recombinant plasmids p21, p19, and p40, carrying the HindIII-BamHI viral sequence of tsl, MoMuLV, and MoMuLV-TB, respectively, were restricted with BamHI and dephosphorylated with calf intestinal phosphatase.TheBamHI-BamHI tslsequencefromp22wasligatedtop19orp40, and after transformation into strain HB101 cells generated the6.7-kbp recombinants p29 and p32. Similarly, recombinant plasmid p31wasconstructedby ligating p21tothe BamHI-BamHI fragment

of MoMuLV.

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1 2 3 4 5 6 7 8 910111213

94 -6.6 -4.3

1.3

-0.6

-FIG. 6. Endonuclease restriction analysis of cloned viral se-quences. Lanes: 1, HindIll digest of wild-typeXandHaeIII digest of 4X174DNAs; BamHIrestriction ofpl(lane 2),p21 (lane3), p22 (lane4), pUC9(lane 5), p27 (lane 6), and pBR322 (lane 7); pUC9 restricted with HindIll (lane 8) and doubledigested with Hindlll and XbaI (lane 9); XbaI restriction of p31(lanes 10 and 11), p29 (lane 12),and p32 (lane 13). Sizes are shown in kilobases.

the HindIll-restricted and dephosphorylated plasmid plO,

p17, or p18(recombinant plasmidswith theSmaI-HindIII

5'-U5-gag-pol sequence of MoMuLV, tsl, and MoMuLV-TB,

respectively) to generate the constructs shown in Fig. 7.

pl8

p17

p10

Sm H

- -Jwwww^""

L

TABLE 2. Characterization of viruses produced by chimeric genomesintransfectedNIH 3T3cellsa

VTiterratio Pr80en%,

No.

of % of

Virus

(34TC/390C)

processing mice mice

injected paralyzed

tslwt 7 2.1 P 12 0

tslwt 8 2.5 P 62 0

tslwt 9 1.8 P 27 0

tslwt10 1.6 P 24 0

tslwt 11 100-1,000 NP 20 NAb

tslwt 12 100-1,000 NP 10 NA

tslwt13 100-1,000 NP 22 PAC

a See Table 1, footnotes a and b. Data for the parental viruses were the

same asin Table1.

bNA, Notavailable. Mice 40 to 120 days postinjection with tslwt 11 or 12 haveasyet shownnosignofparalysis.

C PA,Partially available. Of 22 mice injected with tslwt 13, 4 have begun to showsignsofparalysis.

Infectious viruses

produced

aftertransfection ofthe

recon-structedviralgenomes were

designated

tslwt 7

through

13.

Characterization ofinfectious viruses tslwt7

through

13. The infectious viruses tslwt 7 through 13 were tested for

temperature

sensitivity, efficiency

in

processing

Pr80env at

the

nonpermissive

temperature, and

ability

to induce hind-limb paralysis (Table

2).

tslwt

7,

8, 9, and 10 were not temperature sensitive and processed Pr80eMv

intracellularly

H X B

.-x .-x

= p32

. p29

x_-ouD-OJ.X _p31

+

ligase

4f

_{H X B}

_

_.

_

X X

._ -1

xx

-- -0%0 I aumummmmmun~mu

,1 _

J--!

tslwt-8

_-' tslwt-9

ml tslwt-10

x x

- - 04-04-04 IL I. .IdmmmuuuJ

x X

'W_N- _- - -

--L---tslwt-11

tslwt-12

....

X.&1

tsIwt-13

b gag pol gp70p15E, LTR

0 1 2 3 45 7 8

las0plasmid

DNA

-MoMuLV-TB DNA

----MoMuLV DNA

ts1 DNA

FIG. 7. Schematic presentationofconstructs tslwt 7through 13. B,BamHI; H, HindIll; Sm, SmaI; X, XbaI; Ps, PstI.

construct

7

Sm s Virus

tslwt-7 8

9 10

1 1 12

13

Pe

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[image:7.612.113.259.73.211.2] [image:7.612.322.564.95.194.2] [image:7.612.129.484.360.707.2]

1 2 3 4

[image:8.612.116.239.75.247.2]

Pr80O W

5VT

_gp7O

FIG. 8. Metabolic labeling and immunoprecipitation with anti-gp7O ofintracellularviralproteins of cellsinfected withtsl(lane 1),

tslwt 8fromtwo separatetransfectionexperiments (lanes 2 and3),

and MoMuLV-TB (lane4).

at the nonpermissive temperature. The intracellular viral

proteins ofcells infected with tslwt 8, a representative of

these constructs, were immunoprecipitated with anti-gp7O

(Fig. 8). At thetimeofwriting,miceinjected withtslwt7, 8, 9,or10were130to150dayspostinjection,andnoneof them

had become paralyzed. In contrast, tslwt 11, 12, and 13

remainedtemperature sensitive andfailedtoprocessPr80en'

intracellularly atthenonpermissive temperature.Theresults

ofimmunoprecipitation were similar tothose shown in Fig.

8 andthereforeare notshown. Atthetime of writing, 4 of 22

CFW/D mice 39to95days postinjectionwith tslwt 13, have

beguntoshowearlysignsof paralysis. However, mice 40to

120 days postinjection with tslwt 11 and 12 have as yet

shown no sign of paralysis. It is possible that the latent

periodinthese hybrids isprolonged. Thedifferencebetween thegenomesoftslwt13andtslwt11or12isthat thegag-pol

region oftslwt 13 is derived from tsl, whereas the same

genomic segment of tslwt 11 and 12 is derived from

wild-type virus. Whetherthissegmentof thegenome playsarole

inmodulating theonsetofparalysis in tslwt 11 and 12 needs

to be investigated further.

DISCUSSION

Toidentify the mutation(s) in thegenome of the

paralyto-genic mutant tsl which confertemperature sensitivity,

inef-ficiency in processing Pr80enl', and the ability to cause

hind-limb paralysis, we constructed an assortment of

chi-meric viral DNAgenomes between the molecularly cloned

genome of tsl (22) and those of its parental wild-type MoMuLV-TB or MoMuLV. Viruses obtained from these

chimeric genomes were tested for their temperature

sensi-tivity, ability to process Pr80en', and ability to induce

hind-limb paralysis in mice. We showed that the

HindIll-PstI sequence from nt 4895 to nt 8264 and nt 1 to nt 567, which comprises the 3' end of the pol gene, all ofthe env

gene,andthe LTR, containsthe sequence(s)responsible for

the temperature-sensitivefunction, the inefficiency in

proc-essing Pr80env at the nonpermissive temperature, and the

induction of paralysis in mice. These results are in

agree-ment with the observations ofDesGroseillers and

co-work-ers(2) thatthe 3.9-kbpSaIl-ClaI fragment comprising the 3'

endofthepoland all ofthe env sequenceofthe neurotropic

Cas-Br-E MuLVwassufficienttoconferthe

paralysis-induc-ing

potentialto the virus.

We have extended the above studies and showed that

constructs with the 1.6-kbp HindIII-BamHI sequencefrom

nt 4895 tont 6537 of either MoMuLV orMoMuLV-TB are

nottemperaturesensitive, process Pr80e"vininfectedcells at the restrictive temperature, and do not cause hind-limb paralysis. This HindIII-BamHI sequenceconsists of the 880

base pairs (bp) of the carboxyl terminal ofthe pol coding sequence and 660 bp ofthe amino-terminal portion ofthe

gp70 coding sequence. Conversely, hybrids with the

HindIII-BamHI sequence derived fromtslremained

temper-aturesensitiveandinefficient in theintracellular processing

of Pr80eMv at the restrictive temperature. At the time of

writing, 4 of 22CFW/D mice 60 dayspostinjection withone

of these hybrids, tslwt 13, have begunto showearlysigns of paralysis.

Our observations clearly indicate that the 1.6-kbp

se-quence not only controls the processing of Pr80env and

encodes the temperature-sensitive function, but is also

nec-essary for the induction of paralysis.

Restriction mapping of tsl, ts7, and the wild-type

MoMuLV-TB genomes with 11 endonucleases failed to

show any difference in the restrictionpattern of the 1.6-kbp

HindIII-BamHI sequence. It is entirely possible that the

phenotypic differences may have resulted from mutations

which were not detected at this levelofrestriction analysis.

Alterations in a single base which lead to a change in

phenotype and lossofinfectivity have beenreported(8). To

determine whether one or more mutations haveoccurred in

the tsl genome, acomparison of the nt sequences between the 1.6-kbp HindIII-BamHI sequences of tsl and those of MoMuLV-TB is now inprogress. Ifmorethanone mutation is found, chimeric genomescould beconstructed by exchang-ing subfragments of theHindIII-BamHI sequence.

In vivo studies (submitted forpublication) oftsl-injected CFW/D mice showedthatthe titer ofinfectious virus

recov-ered from the plasma and spleen was maximal by 10 days

postinjection. In contrast, the infectious virus titer in both

the spinal cord and brain cells gradually increased and

reached a level which exceeded the maximal concentration

ofvirus foundintheplasma at 25 to 30dayspostinjection.At

this time, paralysis became evident in tsl-infected mice, whereas the wild-type virus-infected mice appeared to be

normal; the virus titer was about 2 orders of magnitude greater in tsl-infected spinalcord samplesand about 1 order of magnitude greaterintsl-infectedbrainsamplesthaninthe same tissues infected with the wild-type. The infectivity detected in the tsl-infected spinal cord and braincells could

not be due to the presence of endogenous viruses, since

these infectious viruses were found to possess the

pheno-type of the tsl virus, i.e., they were temperature sensitive andimmunoprecipitation ofthe intracellular protein ofthese

cells with anti-gp7O showed that Pr80env had accumulated.

These findings clearly indicate that tsl virus can infect and

replicate muchmore efficientlythanwild-typeMoMuLV-TB

in the central nervous system of infected mice. This

en-hanced neurotropism of tsl virions was further indicated

when it was found that tsl grew to high titers in primary

cultures of neurons from CFW/D mice, in contrast to the

barely detectable infectivityfound in similarprimaryneuron

cultures infected with wild-type virus. It may be speculated

that alterationsin theconformationofgp70in tslmay enable

tsl virions to gain entry into neurons. Alternatively, the

failure to process PM80env efficiently, which results in

re-duced amounts ofgp7O and piSE on the virion envelope of

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tsl (20), may allow tsl virions to bypass the blockage at the

cell surface and gain entrance to the neurons. Once tsl is

able to enter the neuron and establish replication, the

accumulation ofPr80env in the infected neuron may disrupt

its normal function, resulting in paralysis. In addition, tsl and wild-type virus may differ in their ability to integrate in the nerve cells, or a change(s) in the tsl genome may render

its replication in nerve cells much moreefficient than that of

wild-type virus. Experiments to resolve these possibilities are under way.

As yet no mammalian retrovirus protein has been shown

to possess the protease activity for processingPr80en'.Thus,

it has been assumed that Pr80env processing is carried out by

a cellular enzyme. The present studies have shown that a

mutation(s) in the nt sequence of tsl, extending from the

HindIll site (nt 4894) at the 3' end ofpol to the BamHI site

(nt 6537) of gp7O, retards the processing of Pr8Oen" at the

nonpermissive temperature. These findings suggest that the

HindIII-BamHI sequence of tsl may encode a heat-labile

env protein cleavage enzyme which functions inefficiently at the nonpermissive temperature or, alternatively, that the

sequence encoding gp7Ois altered so that the precursor has

an altered conformation which cannot be cleaved efficiently at the nonpermissive temperature.

As pointed out by Shinnick and co-workers (15), thepol

gene has a coding capacity much larger than necessary to

encode the 80,000-dalton reverse transcriptase molecule.

Recent studies bySchwartzberg and co-workers (13) showed

that mutants carrying deletions at theSacl(nt2558) andStuI

(nt 2927) sites released virus with no detectable reverse transcriptase activity, in contrast to mutants with deletions

at the HindlIl site (nt 4894), which produced virions with

normal reverse transcriptase and RNase H activities. These

findings demonstrated that the reverse transcriptase

mole-cule is encoded at the 5' end of the pol gene. Thus, the

sequence extending from somewhere between the StuI and

HindIIIsites to the beginning of the env sequence encodes a different function.

It has also been demonstrated by Kopchick and

co-work-ers (6) that Rauscher MuLV virions contain endo-DNase

activity in a 40,000-dalton protein, p40. Kopchick and

co-workers suggested thatp40 may be involved in integration of

the viral genome by making staggered cuts in the host and

viralDNAs. Recently, Donehower and Varmus (Proc. Natl.

Acad. Sci. U.S.A., in press) reported that the integration efficiency of the proviruses of two MoMuLV mutants, one with a base substitution at nt 4950 and the other with deletion mutations at nt 4950 and nt 4951, was reduced

10-fold. Thus, it appears that the 3' end of the pol gene

encodes the 40,000-dalton endonuclease. However, as

pointed out by Kopchick et al. (6), the reverse transcriptase

andendonuclease molecules together still do not accountfor

all the coding potential of thepol gene. The demonstration in

the present study that replacing the HindIII-BamHI

se-quence of tsl with the homologous wild-type sequence

corrected the defect in the processing ofPr80etn suggests the

possibility that the 3' end of the pol gene may in addition encode a protease responsible for cleaving the envelope precursor protein. The alternate explanation for our

obser-vationsis a conformational change in Pr80en' resulting from

a mutation(s) in the 5' end of the gp7O coding sequence

which renders it unable to be processed to gp7O and

p1SE.

To test these possibilities, we are currently making

con-structs by replacing the HindIII-XbaI (nt 4895 to nt 5766)

sequence of thepol gene or theXbaI-BamHI (nt 5767 to nt

6537) sequence of gp7O with the homologous sequence of tsl

andvice versa. Inaddition, werecently isolated a

rccombi-nant inCharon21A with adeletion mutationof 50 to 100

bp

at nt 5580. Characterization of these constructs and of the

deletion mutant should be helpful in resolving these

ques-tions.

ACKNOWLEDGMENTS

WewishtothankM. M.Soong forsomeof theproteinanalysis. We also thank the Word Processing Center of the University of Illinois College of Medicine at Urbana-Champaign fortyping this manuscript.

This investigation was supported by Public Health Service

re-search grantCA36293, awardedbytheNational Cancer Institute. LITERATURE CITED

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