Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site.

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Vol.41, No.2 JOURNALOFVIROLOGY,Feb. 1982, p. 542-556

0022-538X/82/020542-15$02.0O/0

Long

Terminal Repeat of Murine Retroviral DNAs: Sequence

Analysis,

Host-Proviral Junctions, and Preintegration Site

CHARLESVAN BEVEREN,1 ELAINE RANDS,2 SISIR K.CHATTOPADHYAY,2 DOUGLASR. LOWY,2ANDINDER M.VERMAl*

TumorVirologyLaboratory, TheSalk Institute, San Diego, California92138,1and DermatologyBranch, Laboratory of Tumor Virus Genetics, National Cancer Institute, Bethesda, Maryland202052

Received 27 July1981/Accepted25 September1981

The nucleotide sequence of the long terminal repeat (LTR) of three murine

retroviral DNAs has been determined. The data indicate that the

U5

region

(sequences originating from the 5' end of the genome) of various LTRs is more

conserved than the U3 region (sequences from the 3' end of the genome). The

locationand sequence of the control elements such as the 5' cap, "TATA-like"

sequences, "CCAAT-box," and presumptive polyadenylic acid addition signal AATAAA inthe various LTRs are nearly identical. Some murine retroviral DNAs

containaduplication ofsequences within the LTR ranging in size from 58 to 100

basepairs. Avariant ofmolecularly cloned Moloney murine sarcoma virus DNA

inwhichoneofthe two LTRs integrated into the viral DNA was also analyzed. A

4-base-pair duplicationwas generated at the site of integration of LTR in the viral

DNA. The host-viraljunctionof two molecularly cloned AKR-murine leukemia

virusDNAs (clones 623 and 614) was determined. In the case of AKR-623 DNA, a

3- or4-base-pair direct repeatof cellularsequences flanking the viral DNA was

observed. However, AKR-614 DNA contained a 5-base-pair repeat of cellular

sequences. Thenucleotide sequence of the preintegration site of AKR-623 DNA

revealedthatthe cellular sequences duplicated during integration are present only

once.Finally,astrikinghomology between the sequences flanking the

preintegra-tion site and viralLTRs wasobserved.

During

thelifecycle ofretroviruses, the viral

genomic RNA is transcribed toDNA, some of

which is integrated into the host chromosome

(4). This crucial function carried out by viral

encoded reverse transcriptase is obligatory for

establishment of infection (42). Although the

mechanismofreversetranscriptionisquite

com-plex (16)and notfully understood, several forms

of double-stranded viralDNA

including linear,

circular, and supercoiled DNAs have been

re-ported in infected cells (41). Both the in

vivo-andin vitro-synthesized viral DNAs have two

typesofgenome-length molecules (2, 6,

15,

20, 32): (i) those that contain 5'-end genomic se-quences (U5) repeated at their 3' end (U3),

formingastructure5' . . .U3U53' and

(ii)

those

that, in addition to having 5' genomic RNA

sequencesrepeated attheir 3'

end,

alsocontain

3'genomicRNA sequences repeated attheir5'

end, forming a structure

5'-U3U5

... U3U5-3'.

However, analysis of the

integrated

viral

DNA shows only structures

containing

U3U5... U3U5 sequences (21, 31). The U3U5

unit is referred to as the long terminal repeat

(LTR).

Thebiological function of theLTRisnotclearly

understood, but its structure warrants several

speculations. TheLTRcontains control elements

for boththepromotion (14a, 44) and termination

of viral RNA transcripts and may mediate or

facilitatetheintegration ofviralDNA into thehost

chromosomal DNA. Nucleotidesequence

analy-sisofLTRsfromseveralproviralDNAs(11, 19, 25, 34, 38) has shown: (i) direct duplication of

sequences at theterminiof viralDNA,i.e., LTRs;

(ii) inverted repeats at the termini of eachLTR;

and (iii) direct repeat of adjacent cellular

se-quences atboth termini ofproviralDNA. These structural attributes ofLTRs are reminiscent of

structures associated with bacterial transposons

(7, 8)TY] elements of yeasts(14),and thecopia

element inDrosophila (13).Inthis

manuscript,

we

have analyzed and compared the nucleotide

se-quences of severalLTRsfrommurine retroviral

DNA.Thehost-viraljunctionsoftwo

molecularly

cloned, integrated, infectious AKR-murine

leuke-mia virus (MLV) DNAs (24) have also been determined. We have also identifiedthe

preinte-gration site ofone oftheintegrated AKR-MLV

DNAs.

542

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MATERIALSANDMETHODS

Recombinant DNA. The construction of recombi-nant DNA clones X-AKR-MLV DNA(clones623and 614) (24), plasmid pMLV-lA containing the uninte-grated form ofMoloney MLV (Mo-MLV) DNA (3, 39), plasmid pMLV-101 containing the 5' halfof an integratedform of Mo-MLV (38), and cloneXAMSV-1 (43)containing unintegrated form ofMoloneymouse sarcoma virus (Mo-MSV) with inverted LTRs have been described. The construction of the Mo-MLV cDNA clone pMLV-201 has also been previously described (38).

To molecularlyclone from uninfected cells a DNA fragmentinwhichthe AKR-MLV DNA had integrat-ed, we used thefollowing approach. About 1 mg of cellular DNA from uninfected NIH/3T3 cells was digested with EcoRI andelectrophoresed on a0.7% agarose gel for20 h at 70 V/cm. The DNA from a portion ofthe agarosegelwastransferredonto nitro-cellulosefilters andhybridizedto aprobe containing the LTR andcellularflankingsequencesof the AKR-MLV DNA clone 623 (pAKR-LC-623), preparedas follows. The X-AKR-MLV DNA clone 623 (X-AKR-623)contains asingle HindIllsite about 2.5kilobase pairs (kbp) upstream from the 5' LTR-cellular junc-tion. TheHindIII-EcoRIfragmentofX-AKR-623 con-tainingthe entire AKR-MLV DNA and someflanking cellular sequences was subcloned in pBR322. Itwas then cleaved with KpnI, and the largest fragment, containing pBR322,2.0kbp of5'cellularsequences, 0.5kbp of5'LTR,0.1kbp of3'LTR, and1.0kbpof3' cellular sequences, was religated and used to trans-form Escherichia coli C600 (seeFig. 4A). Two other plasmidswereconstructedtocharacterize the recom-binant DNA containing the preintegration site. The plasmid pAKR-101 contained a KpnI-digested DNA fragment encompassing about 500 nucleotides of 5' LTRand 2.0kbp of adjacent cellularsequences (see Fig. 4A). Theplasmid pAKR-102 contained a KpnI-digestedDNAfragmentcontaining130nucleotides of 3' LTR andadjacent cellularsequences(seeFig. 4).In both cases, the DNA fragments were tailed with oligodeoxycytidineand annealedto oligodeoxyguano-sine-tailed, PstI-cleaved pBR322 DNA, followed by transformationashas beendescribed(9, 22).The 32P-labeled probes were made by nick translation, and specificactivitiesof3 x10'to5x107 cpm/,Lgof DNA wereusually obtained.

Afterhybridizationof the blot of NIH/3T3 DNAto nick-translatedpAKR-LC-623DNA, asingleband in the size range of 7 to 8kbpwasdetected. The 7- to 8-kbp region ofthe remainderofthe agarosegelwascut, eluted, andretested forhybridization to pAKR-LC-623 DNAand to afragment containing only cellular sequences. The size of thefragment was that predict-ed,since theX-AKR-623DNAis 16.4kbplong and the AKR-MLV DNAis about 8.8kbp. About I to 2 ,ug of thecellularDNAwas ligatedto 5 ,ug of separated arms of modified A phage Charon 4a as describedpreviously (5). Theligated material was packaged in vitro and assayed for viable phage. One positive clone was isolated after screening10,000 to20,000 plaques. The primary plaques were purified by two rounds of screeningtoobtain a pureplaque population.

Sequence determination procedures. The chemical modificationmethodofDNAsequencing as described

byMaxam and Gilbert (26) wasused.Sequence lad-ders weredisplayed on 6, 12, or 18% polyacrylamide-8 M urea gels 0.04 cm thick.

RESULTS

Comparison of sequences of LTRs. Since the LTR sequences may beinvolved in the integra-tion and transcripintegra-tion ofviral DNA, we were interested to determine whether differentmurine retroviral LTRs share some common features. Sizeand sequence heterogeneity of the LTR of murine C-type viruses have been predicted by Rands et al. (28) on the basis of restriction

endonuclease mapping of ecotropic MLV

DNAs. We haverefinedthisanalysis furtherby

determining the nucleotide sequence ofseveral

MLV LTRs. Figure 1 shows a diagrammatic

sketch of the comparison of nucleotide

se-quencesof LTRs from various murine retroviral DNAs (11, 29, 38). The complete nucleotide sequence of several of these LTRs is given below (see Fig. 6). Several generalizations can be made. (i) The size of each LTR is different,

rangingfrom 519 nucleotides (Mo-MLV ofBalb/

Mov-1 locus)to 626 for AKR-614 or AKR-623

DNA. (ii) In comparing Mo-MLV and AKR

LTRs, the U5 region isconserved, whereas the

U3region shows considerable variation.A

com-parison ofthe LTRsof various Mo-MSV isolates

andthatofMLV-1A suggeststhat cloneml

Mo-MSVismoreclosely relatedtoMo-MLVclone1

than is Mo-MSV clone 124. (iii) The 5'-cap

nucleotide islocated atapproximately thesame

position inall cases.(iv)In all cases,a

transcrip-tional control signal like theRNA PolII

initia-tion site (TATA-likebox)ispresent-25 to-31

nucleotides fromthe RNA 5'-capnucleotide. A

similarsequence atpositions+46 to +52 maybe

involved in polyadenylation ofRNA (27).

An-other control

signal

CCAAT, located around

position-80andimplicated intranscription (10,

17), is presentinallcases at a similar position.

(v)SomeLTRs, forinstance those of AKR-614,

pMLV-lA,andpMSV-12reportedhere andthat

ofml-MSV(11), contain an internalduplication

of sequences ranging from 58 to 100 base pairs

(bp).However, other LTRs likepMLV1-101(38)

or pMLV-201 (36), containing 3' LTR, do not

containduplication ofsequences.(vi) All

provi-ralLTRshavelost two A residues at the 5' and

3'termini,whereas the LTRsfrom unintegrated

viralDNAs retain the terminal two A residues. Itshould be noted that the loss of two A residues in the 3' LTR sequence derived from cDNA clone pMLV-201 probably occurred during S1

nuclease treatment (36). (vii) The inverted re-peatsatthetermini ofall MLV- orMSV-related

LTRs have the same sequence, namely

TGAAAGACCCC . . . GGGGTCT'TCA.

To determine whether the LTRs encode a

41,

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544 VAN BEVEREN ET AL.

5'-CAP

U3-"CAT"

Box

IR

Li

'TATA" Box

--

a s d - iSsll lz lS,.Il|- *-l.. ffi|fis ulmllSi

i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~rr|nZY-nHOM /Z , 1..7, ..I -11. lm

9, , " , , | , , a

'MI

"' '"

-. . _ ,,.a.I..

.

_._._...

,

0

0 100 200 300

I.

*ii

II I

400

poly(A)

.a

D

500

IR

I

!pMLV-iA

-pMLV1

-pMLV-201

AKR-614

pMSV-12

MSV-124

KA iieA4%1

600

NUCLEOTIDES

FIG. 1. Schematiccomparison of various murine retroviral LTRs. The nucleotide sequence of the LTR of pMLV-IA (unintegrated Mo-MLV [39]) is compared to those of the integrated 5' LTR from theMov-1 locus (pMLVr-101[38]),the Mo-MLV cDNA clone of the 3' LTR (pMLV-201; see text), AKR-614 (24), pMSV-12 (Fig. 2,structureII),anindependentrecombinant cloneof MSV clone 124 (29), and a recombinant clone of Mo-MSVclone ml (11).Comparisonswerecarried out using the ALIGN program (M. 0.Dayhoff, W. C. Barker, and B.C.Orcutt,personalcommunication) with a unitary matrix and a gap penalty of 3. Symbols: 0,deletions versus pMLV-lA; A, insertions; and 1, single base changes. Control signals: I.R., inverted repeat 5'-AATGAAAGACCCC-3'; CAT-box (10, 17), 5'-CCAAT-3'; TATA-box (10, 17), 5'-CAATAAA-3' for all except AKR-614(5'-CTATAAA-3');poly(A), possible polyadenylation signal (27) 5'-AATAAA-3'.

proteinthe three possibleframes of translation

ofMo-MLV,Mo-MSV, and AKR in the 5'--* 3' directionareshown in theappendix. Thelargest possible open reading frame is foraprotein of about10kilodaltons encoded by AKR-614LTR. In the direction opposite tothe genomic RNA

transcription, the largest possible protein

syn-thesized is less than 7 kilodaltons. Since no mRNA encoded by a murine type C retroviral LTR has been reported, the role ofsplicing in generation ofan RNA molecule which can en-codeaprotein cannotbe ruledout.

Inversion of LTR in Mo-MSV. Although the LTR appears to be structurally analogous to movable genetic elements, no transposition of LTRs has been observed in the infected cell

DNA. Shoemaker et al. (35) have, however, reported variants of molecularly cloned Mo-MLV DNAin whichoneofthe LTRsintegrated into the viral DNA, thuscreatingamolecule in which the two LTRs are not adjacent to each other.Asimilar situation hasnowbeen encoun-tered in amolecularly cloned Mo-MSVDNA.

We havepreviously described the molecular

cloningofthecircular form ofunintegrated

Mo-MSV DNA containing two LTRs in

bacterio-phage ) and its subsequent subcloning in pBR322 (43). Since the circular DNA was cleavedattheHindlIl site,themolecular clone was permuted with respect to the linear viral DNA.However, the two LTRsshouldbe pres-ent adjacent to each other as shown in Fig. 2 J. VIROL.

_ _

,

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[image:3.490.55.452.63.393.2]

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(structure I). Out of a total of three clones

analyzed,twoclones appearedtohavestructure

I, whereas in thethird Mo-MSVDNAclone,

A-MSV-1 (43; subcloned in pBR322, pMSV-12),

the LTRs are not adjacent but separated by a

stretch of 321 nucleotides (structure II). Further-more,the 5' LTR is inverted from the expected

orientation in Fig. 2 (compare structures I and

II). Several conclusions can be made from the

sequenceof inverted LTR in pMSV-12. (i) The

5'. i .,l

'I

U3 U5

5'-LTR

_OR

Hind

m

Us

U5

I

3-LTR

GAGA

CGAG

AACC

U5

PB~~~

3-LTR/M I5MMLTA

t It 0

3'-LTR

5'-LTR

I.

IR

*

TT

CTCGTCTC

ss s7 s~~~1 H

I

IR

*jTTCA

ICGAG

AACC

U3 l H

3'-LTR 5'-L-TR

D:.

FIG. 2. Structure of recombinant clones of Mo-MSV clone 124. Thegenomic RNAgivesrise to

double-stranded DNAhavingone(not shown)ortwoLTRs in either linearorcircularconfiguration.Thearrangementof

recombinant clones ofsupercoils,isolated fromproductivelyinfected cellsand clonedattheunique HindIII site, isgenerally that shown instructureI(e.g., clonepMSV-1L [39a]). Inonerecombinantclone, pMSV-12, the

regionindicatedbythearrow,encompassingthe 5' LTR and the 321nucleotides downstream from it(including the tRNAprimer binding site [PBS]),has been invertedtogivethe arrangement shownin structureII.Thetarget

tetranucleotideCGAG,foundonceinstructure I,is foundrepeatedatthe end of the inversion in structureII.

H

I

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5' LTR and 321 nucleotides downstream from it areinverted with respect to their orientation in pMSV-1L (structureI)(39a).(ii)The 321 nucleo-tides between the two LTRscorrespond to se-quencesrepresentingthetRNAbindingsite and downstream viralsequences. Theinversion pre-sumably occurred during viral DNA synthesis

andis not an artifact ofcloning (35). (iii) Two terminal A residues from the U3 region of 5' LTR and two terminalT residues from the

U5

region of 3' LTR are lost. (iv) A 4-bpinverted repeat(Fig. 2)is observedattheU3 terminusof

theinverted 5' LTR, andatthejunction ofthe

additional 321-bp sequences and

U5

terminus of the 3' LTR. It may benotedthat in contrast to theproviral DNAdescribedbelow(Fig.3and5)

inwhich there isdirectrepeatofadjacent

cellu-lar sequences, the 5' LTR and the additional 321-bp unit are inverted; thus, the flanking se-quences display an inverted rather than direct

orientationbecause the molecule has

integrated

into itself. (v) The 4-bpinvertedrepeatispresent once in the parental DNA atthe site of

inver-sion.

Host-viraljunctions of AKR-MLV 623and614.

A hallmark of structures resembling bacterial

transposons is the direct repeat ofcellular se-quences atbothtermini oftheintegrated mole-cule. We have recentlyreported the molecular

cloning of two infectiousintegrated moleculesof ecotropic AKR-MLV DNA (clones 623 and614) from a chronically infected NIH/3T3 cell line (24). We wanted to determine the nucleotide sequence of host-viral junctions of these two proviral DNAs. In particular, we wanted to determine whether the viral DNA has any pref-erencefor host sites. Figure 3 shows the nucleo-tide sequence of the junctions of AKR-MLV DNAs and mouse cellular DNAs. The inverted repeats at the termini of an LTR define the boundary of the viral sequences. In the case of AKR-623 DNA, the 5' LTR and host junction is ACAA, whereas the 3' LTR and host junction is ACAT. In all the retroviral DNAs analyzed so

far,the terminal two nucleotides are lost during the integration process. Assuming that similar rules apply to AKR-623 DNA, there appear to be either 3- or 4-nucleotide direct repeats of cellular sequences. If one out of the two terminal Aresidues is retained at the 5' junction, then the repeatis3nucleotides.Alternatively,the repeat is 4 nucleotides, and 1 nucleotide at either the 5'

or3'junctionhas undergone transversion.

In the case of AKR-614, the direct cellular repeatis5nucleotides (Fig. 3). Again, the termi-nal two Aresiduesare lost during integration. It

is interesting to note that the two AKR MLV

DNAs, depending on their site of integration,

A) AKR-623

left junction

right junction

5'

- C A A T T T C

5'

- G T C T T T C

B) AKR-614

left junction

right junction

5'

- A G G A A A T

5'

- G T C T T T C

--I

T

A

C

A AIT

G A A A G A C

-I,4 ,7 7-.,7 ,ro,~,- /r~

A

A C A

T|G

A A T A T G C

-T

G

T G

ACT

_{_--}

G A A A G A C

AIG

T

G

A C

A

3 '

3'

-

3'

A T C T C C C -

3'

FIG. 3. Nucleotide sequenceatjunctions of viral AKR and cellular DNA. Thefragments sequencedfor the junctions of AKR-623 are given in the legend to Fig. 5.Todetermine thejunctions of AKR-614, the 12.9-kbp EcoRI insert ofX-AKR-614wassubclonedinto theEcoRIsite ofpBR322. The leftjunctionwasdeterminedby labelingthePstIsite in the 5' LTR, cleaving the 2.5-kbp PstIfragmentwithEcoRI,andsequencingthe1.75-kbp fragment. The right junction was sequenced by labeling the SmaI site in the 3' LTR, cleaving the 9.1-kbp fragmentwithBamHI,andsequencing the 2.9-kbp fragment. Duplicate nucleotidesareenclosed in openboxes, andthe11-nucleotide inverted repeatsatthetermini of the LTRsareindicatedbyhatched boxes.A,Junctions in cloneX-AKR-623;B,junctions in clone X-AKR-614.

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generatedirect repeats of cellular sequences of

either3 or 4bp asinthe caseofAKR-623 DNA

or a5-bprepeatinAKR-614DNA.

Nucleotide sequence ofpreintegration site. In

theproposed models for integration of movable genetic elements, it is imperative that the direct

cellular repeat sequences be present only once

at thesite of integration (33, 35). Since we were not sure whether the directcellularrepeat is 3 or 4nucleotides in the case of AKR-623, we deter-mined thenucleotidesequence of the

preintegra-tion site forAKR-623. Figure 4 shows the

char-acterization of the recombinant DNA clone, X-NIH-623, containing the preintegration site. The ethidium bromide stainingpattern(Fig.4B, lane a) shows that after cleavage with EcoRI, twoinserts of an average size of 7.0 and 7.8 kbp, in addition to the A Charon 4a arms, can be identified. However, when the DNA from the same gel is transferred tocellulose nitrate and hybridized to a nick-translated probe made from pAKR-LC-623 DNA, only the 7.8-kbp insert can

beidentified(Fig. 4B, lane b). The same 7.8-kbp

band hybridizeswhen thefilter is hybridized to

nick-translated X-AKR-623 DNA (Fig. 4B, lane

c). Inaddition, however, the A arms and some uncutDNA can also be identified. The A-AKR-614 DNA does not hybridize to the X-NIH-623

insertDNA (data not shown). Lanes d through i

of Fig. 4B display the analysis of X-NIH-623 DNAbyhybridization to 101 and pAKR-102 DNA. TheBamHI (Quint and Berns, per-sonalcommunication), KpnI, and EcoRI

restric-tion endonuclease map of AKR-623 DNA is shown in Fig. 4A, which also shows the se-quencesin plasmids pAKR-101 and pAKR-102. The restriction map of X-NIH-623 should be

identical to that of AKR-623 DNA minus the

AKR-MLV DNA sequences. To test this

predic-tion,we cleavedX-NIH-623 DNA with BamHI,

EcoRI, andBamHI plus

EcoRI

and hybridized

to labeled pAKR-101 and pAKR-102 DNA as

probes. In both cases, digestion with

EcoRI

shouldyieldaninsert of 7.8 kbp(Fig. 4B, lanes d

andg).DigestionwithBamHIand hybridization

topAKR-101 DNA should show a major band at about 2.8 kbp and a 6.0-kbp band containing NIH-623 and X right arm sequences (Fig. 4B, lanee). On the other hand, hybridization of the

BamHI digest topAKR-102 DNA should show

only the 6.0-kbp

fragment

(Fig. 4B, lane h).

Digestion withBamHIandEcoRIand hybridiza-tion to pAKR-101 DNA should yield the 2.8-kbp band and a smaller 1.4-kbp

fragment

because EcoRIcleaves at the junction ofNIH-623 andX

rightarm(Fig. 4B, lane f). However,

hybridiza-tion of pAKR-102 DNA of the

BamHI-EcoRI-digestedDNAshould yield onlyonefragmentof

1.4 kbp (Fig. 4B, lane i). Thus, it appears that the 7.8-kbp insert of X-NIH-623 DNA has a

restriction endonucleasepattern similar to t-hat

of the flankingcellular sequences of X-AKR-623

DNA.

Thenucleotide sequence of the preintegration site and flanking sequences is shown in Fig. 5. A sequence ACAT which constitutes the direct cellular repeat of X-AKR-623 can be identified to occuronce. This sequence is flanked at its 5' and 3' ends by sequences whichoverlap with flank-ing cellular sequences of X-AKR-623. It is

diffi-culttodecidewhether thedirect cellular repeat

inAKR-623is 3 or 4 nucleotides.

A striking feature of the sequences flanking thepreintegration site of AKR-623 DNA is the apparenthomologywith viral LTR sequences. A sequenceTTCC at the 5' end and TGAA at the 3' end of the preintegration site have homolo-gous sequencesin the inverted repeatsof 3' and 5' LTR sequences. No such homology can be

observedin the caseofAKR-614DNA.

DISCUSSION

Establishment of infectionbyretroviruses

re-quires the integration of viral DNA into host

chromosomes. Usually more than one copy of

the vital DNA is

integrated, although

not all

proviralDNAsaretranscribed. The mechanism

ofintegration remains

totally

obscure.

Howev-er, all proviral DNAs have aunique structure,

namely, U3U5. . .

U3U5.

Detailed nucleotide

sequence analyses of the structure of several

retroviral DNAs

provide

a strong

analogy

with

the structure of

transposable

genetic

elements

likebacterialtransposons, the Tyl elements of

yeasts, and copia in

Drosophila

(reviewed

in reference 19). The retroviral DNA structure is

characterizedbythe presenceof LTRs. The size

ofthe LTRs is

variable,

ranging

from 273

bp

for

avian endogenous

provirus (ev-1) (19)

to

1,327

nucleotides for mouse mammary tumor virus

(12). The murineretroviruses vary insize from 500to650

nucleotides,

whereas the avian retro-viruses have a widersizerange,from 273

nucle-otides forev-1virusto330 nucleotides for

RSV-SR-A(37), 350nucleotides forRSV-SR-D

(23),

and 569bp forspleen necrosis virus

(34).

Each direct repeat has inverted repeatsatits termini. The number of nucleotides in the inverted re-peats rangesfrom 3 for

spleen

necrosis virusto 11 forintegrated Mo-MLV(38). As

pointed

out by Hishinumaetal.(19),the 5' and 3'termini of

allproviraland manytransposableelementsare

always TG ... CA. The LTRs described here are no exception to this rule. In the case of

unintegratedviralDNAs,however,the 5'

termi-nusisAATG,andthe 3' terminus is CATT. The

last 2 nucleotides at each terminus are lost

during integration.

Presumably,

the

staggered

cuts are madeatAA l TG and CA

t

TT

(33,

35).

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w-AK~AR MLV

(8 8kb)

H B Ri B

f4w

uright

arm

t 5 6 7 (kb)

K

L _.f

pAKR-1I0 pAKR-102 KpnI-KpnI (Kpn I-Eco Rl

2.8kb / 09kb

A

RI B B B

I..

Si

IS

1 t 2 t3 4

K K

H K RI

5'I

I--.S..,4

3' pAKR LC-623 (Hind I -EcoRI

3.5 kb J

M.W.

(kb)

9.6

-6.6

-a

b

c

d

e

f

e0

g

h

9.6- t

6.6

2.4-

2.1-

2.4-

[image:7.490.73.421.55.552.2]

2.1-aw

FIG. 4. Characterization of recombinant clone X-NIH-623.A,Predicted restrictionmapofthe7.8-kbp EcoRl fragment containing the AKR-623 preintegration site. Lengths are shown in kbp. Restriction enzymes: RI, EcoRI; B, BamHI; K, KpnI; H, HindIll. (Datafor BamHIarefromQuintandBerns, personal communication.) Regions ofhomology with AKR-623 recombinant subclones pAKR-101 and pAKR-102 are indicated with

brackets. Thelocation of the AKR-623integrationis shownbythe closed box. Thestructureof theinsertinthe AKR-623 subclonepAKR-LC-623,from theHindIIIsitetothe 5' LTRKpnI site, ligatedtothe 3'LTRKpnI site, andthroughtotheEcoRI site is alsoshown.B,Restrictiondigestpatterns ofX-NIH-623 DNA.X-NIH-623 DNA

wascleaved with EcoRI (lanesa,b,c,d, and g), BamHI (laneseandh),orEcoRI plus BamHI (lanesf andi).In

lanea,theDNAwasstained withethidium bromide. Inall othercases,theDNAwastransferredontocellulose nitrate sheetsand annealed tovarious nick-translated[32P]DNAs,and theresultant bandswere visualizedby autoradiography (3). The [32P]DNA probes usedwere:pAKR-LC-623 (lane b),X-AKR-623(lane c),pAKR-101 (lanes d,e,andf)andpAKR-102 (lanesg,h,andi). Wild-type phage DNAdigestedwithHindIIIwasused for size markers.

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FIG. 5. Preintegrationsite and cellular-viraljunctions of recombinant clone X-AKR-623.All sequencesare

read from bottomtotopinthe 5'to3' direction. The nucleotides enclosed in boxesarethose sharedbyallthree sequences,with thepossiblefourth nucleotide enclosed inadotted box. a,Leftjunction.DNA sequenceladder ofthe fastermigratingstrand ofa 5' terminally labeled410-bpSau3A fragment ofthe 920-bpBamHI-KpnI fragmentof subclonepAKR-101. Reactions(26): G,Galone;A,A>C; T,T+C;C,C alone. Partialdigestion

productswereseparatedon a12%polyacrylamide-8Mureagel.b,Preintegrationsite. DNA sequence ladder of the slower migrating strand ofa 5' terminally labeled 255-bp Sau3A partial digestion fragnent ofa925-bp

BamHI-EcoRIfragmentof cloneX-NIH-623. Chemicaldegradationreactions and sequencegelwere asdescribed above. c,Rightjunction. DNAsequenceladder of the800-bpKpnI-EcoRIfragmentof subclonepAKR-1023' terminallylabeledattheKpnIsite. The lanes have been labeledtoallowdirectreadingof thecomplementary

strand. Partial chemical degradation products were separated on a 6% polyacrylamide-8 M urea gel. d,

Alignment of 5' and 3' junction sequences with the preintegration site. Sequences shared between the preintegration site and the inverted repeats (I.R.)of theLTRsareshownbybrackets.

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A comparison of the nucleotide sequence of several integrated andunintegrated murine

re-troviral DNA LTRs allows certain

generaliza-tions. (i) TheU5 regionof the LTR is relatively conserved when compared with the U3 region,

which undergoes rather extensive changes,

in-cluding deletionsand substitutions (Fig. 1).The

heterogeneity in the U3 region is extensive

enough in that we were able to prepare a

Mo-MLVspecificprobefromtheU3 sequences(40).

Similar conclusionswere reached when the

nu-cleotide sequences of several avian retroviral

DNAs were compared(23, 37). (ii) The

impor-tant regulatory signals like the 5' cap, RNA

polymerase II initiation sites (TATA-like

se-quences), and CCAAT box sequences are

pres-entessentiallyatthe samerelativepositionwith

aminimum of change. Wehaverecently shown

thattheTATA-likesequences presentfrom -25

to -31from the5' capof bothunintegrated and

integrated Mo-MLV DNA are involved in the

initiation ofRNApolymeraseII(14a).Asimilar

region for RNA polymerase II initiation has

previously been identified in the avian sarcoma

viral LTR(44). (iii) Theinverted repeats atthe

termini of theLTRareidentical inallcases.(iv)

TheU3 regionof severalLTRs(e.g.,AKR-623,

unintegratedMo-MLV,Mo-MSV-ml, etc.)

con-tains a 58- to 100-bp repeat. However, other

LTRs(e.g.,

pMLVI-101

andpMLV-201)contain

only one copy of sequences involved in the repeats. The nucleotide sequence of SV40

shows a 72-bp repeat about 300 nucleotides

upstream from the 5' capforthe late mRNA's (18, 30). It hasbeen shown that whenonlyone

copy of the 72 bp is present, no effect on the

transcription of simian virus 40is observed(1),

whereas when bothcopies of the 72-bp repeat

are removed, no transcription is detected. We

have previously shown that

pMLVI-101,

con-taining onlyonecopyofrepeat sequencesinthe

5'LTR, isinfectiouswhenligatedto aMo-MLV

DNAcontainingthe 3' half ofthe molecule (3).

One of the salient features of transposable

elementsisduplication of hostsequencesatthe

site ofintegration. The proviralDNAs havealso been shown to generate duplication of the host DNA as witnessed by the presence of direct repeatofcellularsequencesatitstermini(11,19, 25, 34). The number of bases involved in the

duplicationrangesfrom 6bpin MMTV andev-1

proviral DNA to S bp in the case of spleen

necrosis virus (SNV) and 4 bp in the case of severalmurineretroviral DNAs. Twointegrated

AKR-MLV DNAs have been analyzed in this

report.One of them, AKR-623, generates a 3- or

4-bp repeat, whereas the other, AKR-614, cre-ates a5-bpdirect repeat. Because the sequence at the 5' junction is ACAA and that at the 3'

junctionisACAT, there isanuncertaintyin the

precise assignment of the size of the direct

cellular repeat in the case of AKR-623. The

preintegration site in the case of AKR-623 is

ACAT,similar to that of the 3' junction. Asingle

base mutation at the 5' junction couldaccount for thedifference. Alternatively, it may be that thedirect repeat is only three nucleotides and thatonlyoneinsteadoftwo Aresiduesislostat the 5' terminus. Another explanation may be suggested as a result ofsequence analysis of a two-LTR clone ofMo-MSV (39a). This clone hasthe sequence TTAAA at the junction of the

LTRs, so that a loss of the terminal two A

residues from such a clone would still leave a 5'-terminal A on the 5' LTR. Since all murine retroviral DNAs analyzed so far, including the inverted LTR variant reported here, show a4-bp

repeat, itis tempting to suggest that AKR-623

also has a4-bprepeat.However, in the case of AKR-614, the direct repeat appears to be 5bp.

Thus, AKR viral DNA seems to be anexception

among murine retroviral DNAs, being able to integrate either by generating 3- or 4-bp repeats orby creating a 5-bp repeat of host sequencesat its termini. It appears from various retroviral

DNA data that the virus and not the host deter-mines the number ofnucleotides duplicated at the site ofintegration. For instance, Mo-MSV

growninheterologousmink cellsalso shows a 4-bp repeat like that of other murine retroviruses. Among avian viruses, SNV and ev-1 generate

different-size direct cellularrepeats, namely, 5

and 6 bp, respectively. The observation that

AKR-MLV DNA integrated at two different

sites in the same host generates a different number ofduplicatedsequencesis in agreement withthisnotion.

Animportantconstraint in themechanism of

integration oftransposable elements is that

du-plicatedcellular sequences can be present only

once atthesiteofintegration(33).Thishas been

demonstrated in the case of MMTV and ev-1

virus (19, 25). Inthis manuscript, we have also shown that the cellular sequences constituting

the directrepeat in the case ofAKR-623 DNA

arepresent only once in the preintegration site. Inthe case of MMTV and ev-1, there seems to be no apparenthomology between theflanking

cellular sequences and viral LTRs. However, in the caseofAKR-623, onenucleotide to the left of the preintegration site is a sequence TTTC

homologous to TTTC at the 3' terminus of3'

LTR(Fig. 5). Similarly, a sequence TGAA

im-mediatelytotheright ofthepreintegrationsite is

homologoustoTGAA presentatthe 5' terminus

of 5' LTR(Fig.5). Thus,hadacircular form of AKR-MLV been the precursor to theAKR-623

clone, there would have been considerable

se-quence homology between cellular DNA and viral LTRs atthe siteofintegration.

J. VIROL.

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The retroviruses have a strong structural homology with transposable elements. The mechanism of integrationalso suggests thatthey behave likemovablegeneticelements. Further-more,like transposons,theyeastTylelementor the copia element of Drosophila, retroviral DNAs integrate at multiple sites in the host chromosomal DNA. Unlike the bacterial tran-sposons,however,the retroviral DNAs have not been shown to move from theirintegration site in the chromosome. The inverted LTR variant reported here and before (35) offers a direct proof that LTR can integrate like transposable elements by creating a duplication of the se-quences atthe siteofintegration.

APPENDIX

The completenucleotide sequence of the LTRs and the possible open reading frames of the following murine retroviral DNAs have been determined (Fig. 6):

(i)pMLV-lA. Thecircularform ofunintegrated Mo-MLV DNA containing one LTR was molecularly cloned asdescribedpreviously (3).

(ii) pMLV-201. The construction and nucleotide sequence of the viral DNA contained in pMLV-201 haspreviouslybeendescribed (36). There were report-ed tobe afewnucleotide changes in the LTR sequence of pMLV-201 when compared to the LTR sequence of pMLVI-101 (an integrated Mo-MLV DNAclone) (3, 38). Inparticular,atpositions -25 to -31 from the 5'-cap nucleotide, the sequence in pMLV-201 was CAAAAAAascompared withCAATAAA in pMLVI-101. We have independently determined the nucleo-tide sequence of the LTR in pMLV-201 and found only oneof theoriginalthreechanges(atposition88,i.e., -362fromthe5'-cap nucleotide).

(iii)pAKR-614. The13-kbpinsert fromintegrated A-AKR-614 DNA (24) was cleaved withrestriction endo-nuclease EcoRI and subcloned in the unique EcoRI site ofplasmid pBR322. The nucleotide sequence of the LTRof623 DNA is identical to that of AKR-614 DNA.

(lv)pMSV-12. The circular form ofunintegrated Mo-MSVDNAcontaining2LTRs wasmolecularlycloned asdescribedpreviously (43). InpMSV-12,the 3' LTR integratedinto the viral DNA at 463 nucleotides from the 5'-cap nucleotide. Furthermore, the 3' LTR is inverted in its orientation with respect to the 5' LTR as shown in structure II of Fig. 2.

ACKNOWLEDGMENTS

We areindebtedtoJimOstlund for help in the computer analysis of the nucleotide sequences. We thank Janice

Galle-shaw andDoug Murdock forexcellent technicalassistance, andMaureen Brennan for typing the manuscript.

The workreportedherewassupported by grants from the NationalCancerInstitute and the American Cancer Society.

ADDENDUM IN PROOF

Thenucleotide sequence of the LTR ofan indepen-dently isolated clone of MSV-124 (29) was treated separately in Fig. 1 due to the occurrence of 26 differences with respecttothe LTRofpMSV-12 (43). In a recent publication, however, the authors have

made 26 base changes in their 582-nucleotide LTR sequence (E. P. Reddy, M. J. Smith, and S. A. Aaronson, Science 214:445-450, 1981). The revised sequence now has only one difference from that of pMSV-12 (atposition 85). The line in Fig. 1 for MSV-124should, therefore, be almost identical to that for pMSV-12.

LITERATURE CITED

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2. Benz, E. W., Jr., and D. Dina. 1979. Moloney murine sarcoma virions synthesize full genome length

double-stranded DNA in vitro. Proc. Natl. Acad. Sci. U.S.A. 76:3294-3298.

3. Berns, A. J. M., M. H.-T.La, R. A. Bosselman, M. A. McKennett, L. T. Bacheler, H. Fan, E. C. Robanus

Maan-dag,H.v.d. Putten, and I. M. Verma. 1980. Molecular cloning ofunintegrated and a portion of integrated Mo-loney murine leukemia viral DNA in bacteriophage lamb-da. J.Virol. 36:254-263.

4. Bishop, J. M. 1978. Retroviruses. Annu. Rev. Biochem. 47:35-88.

5. Blattner, F. R., A. E.Blechl, K.Denniston-Thompson,H. E.Faber, J. E. Richards, J. L. Slightom, P. W. Tucker, and0.Smithies. 1978.Cloninghumanfetal-y-globin and mouse a-typeglobin DNA: preparation and screeningof

shotgun collections. Science202:1279-1284.

6. Bosselman, R. A., and I. M. Verma. 1980. Genome

organization ofretroviruses. V. In vitrosynthesized Mo-loney murine leukemia viral DNA has long terminal redundancy. J. Virol. 33:487-493.

7. Bukhari, A. I., J. A. Shapiro, and S. L. Adhya, ed. 1977. DNA insertion elements, plasmids and episomes. Cold Spring HarborLaboratory, ColdSpring Harbor, N.Y. 8. Calos, M. P., and J. H. Miller. 1980. Transposable

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9. Chang,A. C. Y., J. H. Nunberg, R. J. Kaufman, H. A. Erlich, R. T. Schimke, and S. N. Cohen. 1978. Phenotypic

expressionin E. coli of a DNA sequence coding for mouse dihydrofolate reductase. Nature (London) 275:617-624. 10. Corden, J., B. Wasylyk, A. Buchwalder, P.Sassone-Corsd,

C.Kedinger,andP.Chambon. 1980.Promoter sequences ofeukaryotic protein-coding genes. Science 209:1406-1414.

11. Dhar, R., W. L. McClements, L. W. Enquist and G. F. VandeWoude.1980. Nucleotide sequencesofintegrated Moloneysarcomaproviruslongterminalrepeatsand their

hostandviraljunctions. Proc. Natl.Acad. Sci. U.S.A. 77:3937-3941.

12. Donehower, L. A., A. L. Huang, and G. L.Hager.1981. Regulatory and coding potential of the mouse mammary tumorvirus long terminal redundancy. J. Virol. 37:226-238.

13. Dunsmuir, P., W. J. Brorein, Jr.,M. A.Simon,andG.M. Rubin. 1980. Insertion of theDrosophila transposable element copia generatesa 5 basepairduplication. Cell 21:575-579.

14. Farabaugh, P. J., and G. R. Fink. 1980.Insertion of the

eucaryotic transposableelementTylcreates a5 base pair

duplication.Nature(London) 286:352-356.

14a.Fuhrman,S.A., C. VanBeveren,and I. M.Verma.1981. Identification ofanRNApolymerase II initiation site in thelong terminal repeat ofMoloneymurine leukemia viral DNA. Proc. Natl. Acad. Sci. U.S.A. 78:5411-5415. 15. Gilboa, E., S. Goff, A. Shields, F.Yoshimura,S. Mitra,

and D. Baltimore. 1979. In vitro synthesis ofa9 kbp terminally redundant DNA carrying the infectivity of

Moloneymurine leukemia virus.Cell 16:863-874. 16. Gilboa,E.,S. W.Mltra,S.Goff,andD.Baltimore. 1979.

A detailed model of reverse transcription andtests of crucial aspects.Cell 18:93-100.

17. Grosschedl, H., and M. L.Birnstlel.1980.Identification of

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