Complete nucleotide sequence of a milk-transmitted mouse mammary tumor virus: two frameshift suppression events are required for translation of gag and pol.

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0022-538X/87/020480-11$02.00/0

Complete Nucleotide Sequence of

a

Milk-Transmitted

Mouse

Mammary

Tumor

Virus:

Two

Frameshift

Suppression

Events

Are

Required for Translation of

gag

and

pol

R. MOORE,' M. DIXON,'R. SMITH,' G. PETERS,2 AND C.

DICKSON'*

Imperial Cancer Research FundLaboratories, London WC2A

3PX,1

andImperialCancer Research FundLaboratories, St. Bartholomew's Hospital, London ECIA 7BE,2 United Kingdom

Received 1 August1986/Accepted 30 October 1986

We sequencedtworecombinant DNA clones constituting a single provirus of the milk-transmitted mouse

mammarytumorviruscharacteristic of BR6 mice. Thecomplete provirusis9,901basepairslong,flankedby 6base-pair duplications of cellularDNA atthe site ofintegration. Five extensive blocks ofopenreading frame corresponding to thegaggene, thepresumedprotease, thepolandenvgenes,and theopenreadingframeorf withinthelongterminal repeat of theproviruswerereadilydiscernible. Translationofgag, protease,andpol involved three different translationalreadingframes toproducethe threeoverlapping polyproteinprecursors

Pr77,Pr110,andPrl60found in virus-infected cells. Synthesis ofthereversetranscriptaseandendonuclease therefore requiredtwo separateframeshifts tosuppress thetermination codons at the ends of the Pr77 and PrllO domains. Direct evidence is presented for translational readthroughof bothstopcodons in aninvitro protein synthesis system.

Retroviruses are widely dispersed among vertebrate

spe-cies butareunifiedby their genomeorganization andmode of replication. An essential step in their replication is the reverse transcription of the viral RNA into a double-stranded DNA intermediate that becomes integrated as a

proviruswithin thechromosomalDNAofthehost cell. Asa

result, analyses of retroviralgenomes haverecently focused on theisolation and characterization of molecular clonesof

proviral DNA to the extent that complete nucleotide se-quences arenowavailable for several avian and mammalian retroviruses (18, 40, 42, 44, 46-49). Suchtechniquesand the

opportunity to perform site-directed mutagenesis have also

ledtotheidentification ofpreviouslyunrecognizedorpoorly

understood viral functions over and above the well-characterized components encoded bythe gag, pol, andenv genes. These includea protease, requiredforprocessing of theviralpolypeptide precursors; anendonuclease, required

for proviral integration; and specific sequences needed for

the initiation ofviral DNAsynthesis, the integration of viral

DNA, and the packaging of genomic RNA (5, 12, 16, 24, 27, 31, 32, 43, 50, 55). Whereas direct evidence for these

functionsis restricted to a few specific examples, the general

uniformity of retrovirus architecture and conservation of

particularlocalized sequence motifs suggest that most

retro-viruses conform, with only minor embellishments, to a basic prototype.

Mouse mammary tumor virus (MMTV) is unique among theRetroviridae because it represents a distinct

morpholog-icalsubclass (Btype) and is one of the causative factors in a

specific epithelial neoplasm (9, 29). However, despite being

the first mammalian retrovirus isolated, MMTV has re-mained among the most refractory to molecular analysis. Two reasons can be citedfor this: its strong tropism for the mammary alveoli, which may underlie the difficulties en-countered intissueculture manipulation of the virus, and its apparentresistance to molecular cloning as a complete DNA provirus (2, 11, 13, 25, 26, 53). Although large segments of

* Correspondingauthor.

the genome have been previously characterized (6, 11, 13, 14, 20, 25, 26, 37), here wereport the successful isolation andDNA sequencing of recombinant phage clones consti-tuting what we believe to be an entire milk-transmitted MMTVprovirus. This provides the first complete compari-sonbetween thegenome organization of MMTV and that of other retroviruses and clarifies the disposition of the open reading frames within the gag and pol domains. Previous experiments from our group and others (for a review, see reference 9) indicating threeoverlapping polyprotein precur-sorswere confirmed by the DNA sequence and extendedby the direct demonstration of two independent translational frameshifts in vitro.

MATERIALS AND METHODS

Cloningof MMTV proviralDNA. High-molecular-weight DNA from BR6 mouse mammary tumors was digested to completion with EcoRI, for which there isa single cleavage site in the MMTV provirus, and ligated into the purified armesofthe XgtWES X B vector(33).Packaged phage was plated on Escherichia coli LE392 and screened by

hybrid-izationtoaprobefor the MMTV long terminal repeat (LTR)

by standard procedures. Positive phage was then plaque

purifiedandhybridizedto5'-and 3'- specific MMTV probes

to distinguish the respective virus-cell DNA junctions (33).

Fromone particular library prepared from the tumor desig-nated W26(30), we recovered recombinants corresponding to both halves ofa single integrated provirus. These were matched initially by preparing restriction fragment probes

specificfor the cellular DNA adjacent to the viral sequences

in eachclone. The procedures used for the labeling of probes and blot hybridization have been adequately detailed else-where (33). Although the 3' junction fragment was readily transferred intoaplasmid vector to facilitate further

analy-sis, difficulties were encountered in subcloning the

corre-sponding5'junction (la).

DNA sequence analysis. The 6.7-kilobase (kb) 5' junction

fragment,containing around 1 kb of flanking cellular DNA,

and thecorresponding 6.4-kb 3' fragment were excised from 480

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their respective vectors with EcoRI and recovered by

pre-parative agarose gel electrophoresis. The fragments were

then self-ligated,randomly sheared by sonication, and

blunt-end ligated into the SmaI site of the M13 vectors mp8 and

mp9 (30). Recombinant M13 phage was grown in small-scale

liquidcultures to preparesingle-strandedDNA templatesfor

dideoxynucleotide chain termination sequencingprocedures

(30). DNA sequences were compiled by the DBUTIL com-puter program(51) and, apartfroma smallsection of the 3' LTR, were obtained for both DNA strands.

In vitrosynthesis ofMMTV-specificRNAsandproteins. A segment of MMTV proviral DNA extending from the PstI

site at nucleotide 2580 to the EcoRI site atnucleotide 5803

wasinsertedinto the polylinkerofthepSP65 plasmidvector (Promega Biotec, Madison, Wis.). Theorientationwassuch

that MMTV-specific RNA ofpositive sense could be

tran-scribed in vitro from the SP6 phage promoter with the

SP6-specific RNA polymerase (28). Conditions for RNA

synthesiswereessentiallyas describedbyMeltonetal.(28).

RNAs of different length were obtained by cleavage of the

plasmid at the specific KpnI, BglII, and EcoRI sites in the

MMTV proviralDNA(see Fig. 3). The sizeand

integrity

of

each RNApreparation wasverifiedby Northernblot

analy-siswithanMMTV

specific probe

(not

shown).

After

synthe-sis, the products were treated with RNase-free

DNase,

phenol extracted, and ethanol

precipitated.

The

protein

coding capacity ofeach RNA wasthen assessed

by

transla-tion in vitro ina nuclease-treated

reticulocyte

lysate (8, 10).

The analysis of the resultant

[35S]methionine-labeled

prod-ucts by acrylamide gel electrophoresis, immune

precipita-tion, and tryptic peptide

mapping

followed

protocols

de-scribed in detail

previously

(7,

8, 10).

RESULTS

Cloning and DNA sequencing of the MMTV

provirus.

A

recombinant phage library of

EcoRI-digested

DNA from a

BR6 mouse mammary tumor was screened with a

probe

specific for the MMTV LTR

(33).

Since EcoRI cuts the

MMTV provirus at a

single

site,

each

positive plaque

iden-tifiedwasexpectedtorepresenta

junction

between viral and

cellular DNA sequences.

During plaque

purification,

these

recombinantswere

hybridized

with

probes

specific

foreither

the 5' or 3'

portions

ofthe

provirus

(defined

relative to the

EcoRIsite). In contrastto our

experience

and that

of otherlaboratories in

cloning

MMTV DNA fromGR and

C3H mouse mammary tumors, we recovered 5' and 3'

junction fragments

with

approximately

equal

frequency

from BR6 tumors (11,

25, 26,

53). Moreover,

many of these

recombinants

represented

proviral

DNA of the

milk-transmitted MMTV characteristic ofBR6 mice rather than

endogenous

sequences

(35).

This was

initially

shown

by

preparing

unique

sequence

probes

specific

for the cellular

DNA

adjacent

to each

provirus

as describedelsewhere

(33,

35). Such

probes

also

permitted

the

matching

of

correspond-ing

5' and 3'

junctions,

since the

probes

identified

signature

restriction

fragments

derived from the

unoccupied

site in

normal DNA.

Asaresultofthese

analyses,

we were in a

position

for the first time to determine the

complete

sequence andgenome

organization

of a

single,

potentially

infectious

provirus

of milk-borne MMTV. Thederived sequencefortwomatched

EcoRI halves and the cellular DNA

immediately flanking

the LTRsare

depicted

in

Fig.

1. Six base

pairs

of cellular DNA were

duplicated

at the site of

integration,

confirming

the

linkagebetween these two

junction

fragments.

It should be

stressed, however,

that

although

the sequencewas obtained

for both DNA

strands,

it did not cross the EcoRI site at

nucleotide

5803,

and we therefore cannot

formally

exclude

additional sequence at this

position

(e.g.,

two

closely

ap-posed

EcoRI

sites).

Other features of the sequence are

described below and inDiscussion.

Open reading

frames in the MMTV

provirus.

From the MMTVDNAsequenceand knownfeatures of the LTRs

(11,

13, 14,

20, 21,

26),

we deduced that the viral genome RNA

must extend for

8,585 nucleotides,

beginning

at nucleotide 1196

(Fig.

1).

Since retroviralRNA is in the

positive

sense

the sequence as

presented

was

directly

translatable into the viral

proteins.

A

computed

translation of the MMTV

provirus

in the three

reading

frames is

depicted

in

Fig.

2,

indicating

the

positions

ofall

potential

terminationcodons. It was

immediately

apparentthatthe viral genome RNA could encompass five substantial

protein-encoding

domains. The three

largest

were

presumed

to encode the viral gag,

pol,

andenv

functions,

as

expected

ofa

prototype

retrovirus.An

additional

segment

ofopen

reading

frame characteristic of

MMTV and

designated

orf began immediately

proximal

to the

boundary

ofthe 3' LTRas

previously reported

(10, 11,

14, 20, 26,

37).

However,

the

presumed

gag and

pol

do-mains,

nominally

ascribedto

reading

frames 1 and

2,

respec-tively,

werenot

contiguous,

and itwasclear that

continuity

in the

generation

ofa

gag-pol

precursorwould

require

the

inclusion of amino acids encoded in

reading

frame 3. The

situation therefore

paralleled

that described

recently

for

Mason-Pfizer

monkey

virus,

bovine leukemia

virus,

and

human T-cell leukemia virus

types

I

(HTLV-I)

and II

(HTLV-II)

in which the viral

protease

bridges

the gag and

pol

domains in adifferentframe

(38, 40, 44, 46,

49,

60;

see

below).

The data also concur with

experiments

in

which three

overlapping

precursors were identified with

antisera to the viral structural

proteins

(for

a review, see

reference

9).

Designated

Pr77,

PrilO,

and

Prl60,

these precursors

probably

share the sameamino

terminus,

initiat-ing

atthemethionine codonatnucleotide1508 and

terminat-ing

atnucleotides

3281,

4087,

and

6771,

respectively

(Fig.

2).

Synthesis

ofthe

Prl60

pol

precursorwould therefore

neces-sitate two

separate

-1 translational

frameshifts,

switching

from frame1to3atthePr77-PrllO

boundary

andfromframe

3 to 2 atthe end of

PrilO.

Demonstration of translational

frameshifting

in vitro. We and others have

previously

shown that rabbit

reticulocyte

lysates

programmed

with

purified

MMTVgenome RNAcan

support

synthesis

of

Pr77,

PrilO,

and

Prl60

in

roughly

the

same relative

proportions

as detected in

lysates

from

in-fected cells

(8, 45).

However,

these studies could never

rigorously

exclude the presence of minor RNA

species

in

virion

preparations

which

might

direct

synthesis

of the

longer

readthrough

products.

To circumvent these

prob-lems,

wefollowedthe

example

ofJacks andVarmus

(19)

and

constructed recombinant

plasmids

in which

appropriate

seg-mentsofMMTVRNAcouldbetranscribedin vitro with the

specific

RNA

polymerase

of

phage

SP6

(28).

Thus,

a seg-mentof MMTV

proviral

DNA

extending

fromthe

PstI

siteat

nucleotide 2580to theEcoRI site at 5803 was inserted into

the

polylinker

ofthevector

pSP65 (Fig.

3).

Linearizationof the resultant

plasmid

at either the

KpnI,

BglII,

or

EcoRI

sites within the MMTV sequences would be

expected

to

yield

RNA

transcripts

of

0.9, 1.8,

and 3.2

kb,

which could

discriminate between the

proposed

translationalframeshifts

attheendsof

Pr77,

PrllO,

and

Prl60.

Although

this

fragment

lacks thenormal initiationcodonatthestartofthegaggene,

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tltgtgtgtgtgtgtgtotacaccttggaggggggagcggctgegttctcctgec&gtcatcagggggtggggtgcgggtggggtollg9t9tcccc99tctoggalgggccockottccgtcag

___1CT6CA2CASAAA7665Tt

AACTCCC6A6A6T6TCCTACAC5TA666A6AACA6CCAA6666T76TT5CCCACCAA66AC5ACCC6tC16C6CACAAAC66AT6ABCCCATCA6AC

12S

AAA6ACATACICATTCTCT6CT6CAAACTT66CATA6CTCt6Cl7T76CCT6666C7AtTT6666AASTT6C66TTC6T6CTC6CASSSCTCTCACCCTT6ACTCTTTtAtA6ACTCTTCT6T6CA

250

A6ATTACAATCTAAAC6ATtC66A6AACTC6ACCTTCCTCCT6A66CAAS66ACCACASCCAACTTCCICtTACAA6CC6CATCAACCTTSTCCTTCA6AAATASAAATAAZAAT6CTT6CTAAAA

375

AT7AtATfTTTACCAA?AA6ACCAATCCAATA66tA6ATTATIA6TTACTAT6T1AA64AAA16AATCAT7ACCTlTTASTACIAlTTTTACTCAAATTCAAA66TTA6AAAT666AATA6AAAAT

500

A6iAAA6A6ACACTCAACCTCA6TT6AA6AACA66T6CA666ACTAA666CC7CA66CCTA,6AA6TAAAAA6666AAASA66A6t6C6CrT6TCAAAATA66A6ACA66T66T66CAACCA666AC

625

T7ATA6666ACCTtACACTCACA6ACCAACA6AT6CCCCCTTACC6TAtACA66AA6ATAT6ACCTAAAlTTT6ATA66TS666TCACA67CAAT6BCTATAAA6T6TTATACA166TCCCTCTCCT

750

T7C6T6AAA66CTCSCCA666C7A6ACCTCCTT66T6TAT6TT6ACTCA66AA6A6AAAAAC6ACAT8AAACAACA66TACAT6iATTAtATrTATTT666AACA66AAT6A6CA6CArTT6666A

875

AA8AtT7TTCATAICCAA66A6A66ACA6T66CT6CACIAATABACACTATTCT6CAAA6ACTTATSAT66At6TTATlAT6ATTA6CCTTTATTA6CCCAA7CrT6T667T=CAA66rTTAA6

l000

tAS6TTCAT66TCACA6ACt6T7CrTTAAACAASSAt6t6AACAA6xT6TTC7l6ACTT66ET7667ATCAAA6nT7T6ATCTAA6CTCTAAAT6CTCTAAbCCTCCTA76TTCtTTT86ATT

112

*cap

CtATMcAA6TTTTAt6tAAAt6CTTAt6tAAACCAT6AIAtAAA6A'666CtAAATTTTT6A67AAACTT6CAACA67CCTAACATTCACtCTCC6T676TT76T67CT6TTC6CCATCCC6TCT

1250

pbs

MC7C67CAC6TTATtCCTTCACTTTMC*6A666TCCCCCC6CA6ACCCC66T6ACCCTCA66tC66CC6ACT6

CT66C6CCC6AACA666ACCCTC66ATAA6T6ACCCTT6tCTCTAT

1375 nTCTACTATTT76t6TTC6TCTt6TTTT67CCtACtATCtTAC66C TATTATCACAA6A6C66AAC66ACTCACCACA66SAACT6CA6tCTC6CCTACA6A6AA6A66TA66TTAC66T6A6CC 1500

AT7664AAAT6666TrCTC666ATCAAAA666CA6AAACICTT76TFTCT6TtCTACAAA6ACTCCTCTCA6A6A66667CTlCAT6T6AAA6A6A6CA6T6CAATA6A6TTTTATCA6TtCCTAA

1625 I10-H 6 Y S 6 S k 6 0 K L F V S V L I R L L S E R 6 L N V K E S S A I E F Y I F L I

TAAA66tC7CtCClT6rTt7CCC6AA6AA66A66ATtAAATTTACAA6ATT66AAAA666T666AA6A6A6AT6AA6A66tAC6CA6CA6AACAT666AC66AtA6tATACCAAA6CA66CTTAC

1750

K V S P N F P E E 6 6 L N L I D N K R V 6 R E N K R Y A A E H 6 t D S I P K Q A Y =

CATTt66CTTCA6TT66A6A6AATACT6AtA6A6CAATCA6ACTT66TTTTr6TATCT6CA6AA6CCAA6TCC6T6ACT6AA6A66AArTA6A66AA66TTTAACC66ACTAC7ATC6ACAA6

1875

P I N L I L R E I L t E I S D L V L L S A E A K S V 7 E £ E L E E 6 L 7 6 L L S t S

TTCACA66AAAAAACTTA1666ACTA66664ACA6CATAT6CA6AAA7A6ATACA6A66TA6ACAA6Ct6tCT6AACAtATTTAT6A76AACCAATA6AA6AAAA66A6AA66CA6ATAAAAAT6

2000

S Q E K T Y 6 t A 6 t A Y A E I D t E V D K L S E H I Y D E P V E E K E K A D K 0 E

A66AAAA66ACCAT6TTA6AAAAATAAA6AA66TA6TACAAA6AAAA6AAAATA6T6A666TAA6A6AAAA6A6AA66AlTTCAA66CCTTTTTA6CCACA6Att66AAC6AT6AT6ACCT6TCC

2125

E K D H V R K I K K V V Q R K E N S E 6 K R K E K D S K A F L A 7 8 V N D D D L S

CCT6A66ATT666AC6ATFTA6A66AACAA6%P66CACArTATCAT6A76AT6AT6A6CtAIATCCTTCCA6TAAAAA66AA66T66TTAA6AA6AAACCTCA66CACTCA6AA66AAACCCCT6CC

25 P E D 0 D D L E E O A A H Y N D D D E L I L P V K R K V V K K K P I A L R R K P LP

IM66T666TTT76CA66A6C6A766CA6A66CCA666AAAAA66A6ATTT6ACTTt7AC6TTTC:T6TA6TTTTTAT666A6A6A676AT6AASAT6ACAC6CC76tTT666AACC6Ct6CCAT

2375

P V 6 F A 6 A N A E A R E K 6 D L T F 7 F

fP'

V F N 6 E 6 D E D D 7 P V N E P L P L

t6AAAACCTTAAA66AATT6C^AAC66CA6TTA66ACCAT666ACCAtCT6CTCCCTACACCCT6CA66T66TA6ACAT66T66C7A6TCAAT66CTCACCCC6A6t6ACt66CACCAAACA6CC

2500

K t L K E L I S A V R t N 6 P S A P Y t L Q V V D

0

V A S Q N L 7 P S D N H Q t A

A6A6CTACCT76TCtCCT6A6ACTAT6tTTTAT66A6AACT6AATAT6AA6A6AAAA6tAAA6AAAT66TACAAAA466CT6CA66CAA6C6AAA666CAA66TCTCTCrT6AtAT6TTACT666

262S R A 7 L S P 6 D Y V L 0 R 7 E Y E E K 6 K E N V- Q K A A 6 K R K 6 K V S L D N L L 6

CACT66CCAAltCC6TNCCCCTTCTTCTCA6ATAAAATT6TCTAA66AT6TCrTTAAA6AT6TCACCACAAAT6C76T6TTA8CAT66A666CCATTCC6CCTCCT66A6TTAAAAAA6ACT6tAT 2750

7 6 I F L S P S S I I K L S K D V L K D V t t N A. Y L A N R A I P P P 6 V K K t V L

TA6CA66ArTAAAACA666AAAT6.AA6A67CTTAt6A6,ACTTTCATTTCAA66CTC6A66AA6CT6TTTACC6AAT6AT6CCCA6A6666AA6667C66ATAtATT6ATCAAACAArT616C6T66 2675

A 6 L K Q 6 N E E S Y E t F I S R L E E A V Y R N N P R 6 E 6 S I I L I K Q L A N

B6AAT6CAAAITtATt6T6TC*6AtCtCATCC6CCCAAtACST^AAAACA66AACTATAtCA66ATTA7ArTC676CTT6TCT86AC6CTTCTCCC6CA6T767TCA1666tAT66CATAT6CA6C 3000

E A N S L C O D L I R P I A K t 6 t I D. D Y I R A C L D A S P A V V Q 6 N A

YFAA

A6CCAT6A6A66ACAAAA67T7tCtACCTTT6TAAA6CAAACAIAT66T66666AAAA66A66TCAA66A6CA6AA666CCA6TT767TTTTCCT6T667AA6ACA66ACACATCA6AAAA6ACT 312S

A N R 6 I K Y S t F V K I t Y 6 6 6 K 6 6 Q 6 A E 6 P V C F S C 6 K t 6 H I R K D C 6tAA66AT6AAAA666CTCAAAAA6666CCCICC7666CtCT6CCtCC6AT67AASAAA66CtA7CACT66AA6A6T6A6t67AAATCTAAATTT6ACAAA6AT666AATCCACTTCCTCCCTT6 325

K D E K 6 S K A A p f I L C P R C K K 6 Y H 0 K I E C K S K F D K D 6 N P L P P L

MCT*AtgATBCT7MTT6i6ACT1A6CA67tCCCCt6Ct6CCAAAA66666AT66A6TtA A466WCTP.A6AXTTZATClC1';AAGCACttCT7*TCALATA6TTT76 33 E T N A E I S K 0 L 0

31- F K K L V K I I 8 P 8 P A I K 6 D 6 V K 6 S 6 L k P I A P P F 7 la N D L

FIG. 1. Completenucleotide sequence ofamilk-transmitted MMTVprovirus.TwoEcoRIfragmentsrepresentingtheS'and 3'ends ofa singleMMTVproviruswere recoveredasrecombinant clones andsequenced by shotgun cloninginto M13 vectorsanddideoxynucleotide

chain terminationprocedures(30, 33).The sequenceacrosstheEcoRI sitewasnotdetermined, but withtheexceptionofasmallsection of the 3' LTR,allsequenceswereobtained for both strands. Shownare9,901 nucleotides ofproviralsequence,numberedfrom base 1 of the S' LTR and flankedbysegmentsof thecellular DNAadjacenttotheprovirus(show'nin lowercase).Theboundaries of the LTRsareindicated

by rectangularbrackets,and the six bases of cellular DNAduplicatedduring proviral integrationandunderlined(-).Thebeginningsof

longopenreadingframes arealso shown (D-, accompanied byanumberreferring tothetranslation frameused). Other sequence features indicatedarethe S' end of the viral genome RNA(cap);thebindingsite fortRNA31Y'(pbs);thesplicedonor(sd)andacceptor(sa)sites for

envandorfmRNAs;the knownamino-terminal sequences ofp27,p14-p30, gp52,andgp36( r' ),and thepotentialinvertedrepeatsequences distaltotranslational frameshift sites(overlined) (11, 13, 14, 17, 20, 21, 25, 26, 34, 37, 54, 57).

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PRST BSAS DLSS KDLI SLED YSLV

TTASTLAPCC

ECC6MATAE'7

MtC6A66CACCCCTS6A6T6C:E6rTT7ACCT6TCATCACASAAZ6A6ATTTCTCC7tCTCtAA6AA66AT6ATATCArT6tSACCCAC6t6AA6ACCCTCCt

666.G P A 6 t P 6 S A 6 L I L S 6 I K D L I L S L E D 6 V S L V P T L V K 6 t L P E 6 T 7

T66ACTAATAATABSTA6AA6TTCTAATTATAAAAA666ACT76A66TTTTACCA66A6TCATT6ACTCC6ATTTCCAA6b6ASAMtCA66T7AT66TTAA66M6CiAAAAAT6C66TCATCA

3625 6 L I 1 6 R S SUY K k 6 L E V L P 6 V I D S D F 0 6 E I K V N V K A A K N A V I I TTCACAAA86A6AAA6^AtA6CACAAC

cT676T76jC76CCATATTTAAMTTACCCMATCCT6TATCSsAA66AASACA66CTCA6AS6CTTC66ATCAACAA67CAT6T6cATT666T6CA6

3750

N K 6 E R I A I L L L L P Y L K L P I P V 1 K I EfR 6 S E 6 F 6 S T s H V H U v Q

6AAATAA6T6ACTCCA6ACCCA76CTTCACATTTACTTMAT66AA6

6ATTM7C66TCtCT766A7ACC6666CA6ATAAACTT6CATA6CA66CA6A6ACT6.6cCSCTAATT66CCCAT 3875 E I S D S A P N L H I Y L N 6 R R F L 6 L L D T 6 A D K 7 C I A 6 R I N P A N N P I

TCACCAAAC76A6A6TTCTCtTtCAA66T76CA766CCTS6T666ST66C6C6TA6TASTCA6CCACTCC6TT66CAACAT6A66AT^AAATCA66AATTATACATCCUTT6tT6ATCCCTACAC

4000 N O 7 E S S L Q 6 L 6 N A C 6 V A R S S I P L R V Q N E I t S 6 1 I N P f V I P t L 76CCTTTCACCTTAT6666AA6A6A7ATTAT 6AAA6ATATA AA66TCA

atTTAA6AMACTACATATCACCB%nAlTTA16AT46CM16A6CAAI

TC CTTT6CA6ACCAAA 4125

P F T L 1 6 R 9 I N K D I I t A L # 7 I S P I D S I D L # #

210-F T S F N I 6 A I E S N L F A D Q I TATCTT66AA6TCAAC6AMSATJsJ*T#ntBXAT

ASTt=

ArAA6AAAtTTA

ACA66CTTTACAAC6TATACASAtACTTACAAT6666C

ACTTAAA6A6A6CAATA6C 4250

S N K S D I t V N L N I V P L K Q E K L I A L I I L V t E Q L I L 6 H L E E S k S

CCtT6AATAC6CC76tTTTT6TCATTAAAAAA6AA1CA66AAAAT66A6ACT6TTACAA6ACC7AC6T6CA6TTAAT6CTACAAt6CAC6ATAt666A6CATTACAACCC66CTT6CC6TCCCC

4375 P H N 7 P V F V I K K K S 6 K N R L L I I L R A V N A 7 H H I N 6 A L I P 6 L P S P

T67A6CA6T6CCCTAAA66AT666AAATAATCATAATA6ATCTACAA6ATT6CTfTTTTAAATAAAAACT6CATCCTSAA6AltTTAATM

6AtTT6CtTTTA676T6CCCTMCCCTAATTTTAA6A 4500 V A V P K 6 N E I I I I D L I D C F F N I K L N P E D C K R F A F S V P S P N f K R

6ACCrTATCAAA6ATTCCAAt66AAA6tTTTTCCCCA666TAT6AAAAATA6CCCTACTTTATSTCAAAAArTT6T66ACAAA6CTATATT6ACT6TAA666ATAAATACCAA6ACTCATATATT

4625

P Y I R F I 9 K V L P O 6 N K N S P T L C I K F V D K A I L 7 Y R D K Y I D S Y I

6T6CATtACA166AT6ACATtCtTtT66CACACCCATCAAi6AtCCATTSTC6AT6AAATACrTAC7?CCAT6ATACAS6CCCTTAACAAACAT66CCT?6tA6TAICCACA6A6AA6ATTCAAAA

4750 V N Y N D D I L L A N P S R 6 I V D E I L 7 S N I I A L N K H 6 L V V S T E K I O K

tAtA6AtAATCTCAAATAtTTT666AACTCAtATACA666T6ATTC46T6tCTTATCAAAAATTACA6ArTA66ACA6ATAAArTAA6AACCTTAAAT6ATTTCCAAAA46CTArTA66AAATATTA

4675 Y D N L K Y L 6 t H I I 6 D S V S V I K L I I 0 t I K L A T L k D F Q K L L 6 N I N

AT766ATAC61CCTTTCTTAAAATTAACTAC666A6A6TTAMACCTCTCrTT6AAATTCTTAAT66A6A77CTAATCC6ATCTCAACAA6AAAACTtAC7CCT6A66CAT6CAAA6rTCttCAA

5"00

N I R P F L K L t T 6 E L K P L F E I L N I D S N P I S t R K L t P E A C K A L Q

TTAAT6AAT6A6A6ACTATCTACC6CTC66ETAAA6A66CtA6ATTTATCACA6CCTTB6tCTCTAT6tAtATTAAA6ACT6AATATACCCCCACA6CAT6CCTCT66CA66AT66A6TTSTA6A

5125 L N N E R L S T A R V K R L D L S I P V S L C I L K 7 E Y t P 7 A C L V Q D 6 V V E

AT66AtACATTTSCCTCATATTTCACCAAA66T6ATTACTCCTTAT6ATATCrTrTTTTACA%AACTTATTATTAA666CC64CACC6tCTCAAA6AATTAtTTA6TAAA6ACCCT6ATTATATTS

5250 N I H L P N I S P K V I t P Y D I F C t I L I I K 6 R N R S K E L F S K D P D Y I V

T76T6CCCTACACCAAA6rTCAATT76ATCTCCtATtACAA6AAAA66AA6ATT66CCTATt7CTTlATTA666TTCTT666A6A66TTCArTTCCATCTTCCAAAA6AcCCCT7T6CTTACATTT

5ms

V P Y 7 K V I f D L L L O E K E D I P I S L L 6 F L 6 E V H F N L P K D P L L t f

ACCCTACAAACT6CCtAlTlTATTCCTCACAt6ACCTCTACCACACCACTASA6AAA66AAT76T6ATTTTTACA6AC666TCA6CAAAt66CC6TTC66TAACATATATACAA66AA666A6CC

5500 t L I t A I I f P N N t S 7 t P L E K 6 I V I F t D 6 S A 1 6 R S V t Y I Q 6 R E P

TATAATTAAA6AAAATACACAAAACACA6CCCAACA66CT6AAATT6T66CASTCATTACA6CCYTT6A66AAT6A66TCAACCCTTTAATTT6ATAACT6ATTCTAAATAT6T6ACA666TT67

5625

I I K E 0 t I N T A O Q A E I V A V I t A f E E V S I P f N L Y T D S K Y V t 6 L f

TTCCC6AAATC6AAACT6CAACTTT6TCACCCA6AACAAAAATTTACACA6AACT6AAACATTTACAAA66TTAATCCACAA6A6ACAA6AAAAATtT7ACATT66TCATATCA6A66ACACACT

5750

P E I E t A t L S P R t K I Y t E L K N L O R L I N K R I E K F Y 1 6 N I R 6 N t

68ACTTCCC667CCTTT66CACA666AAAT6CCTAT6CA6ATTCTTTAACAA6AATTCT6ACC6CTTTA6A6TCA6CTCAA6AAABCCAC6CACTACATCATCAAAAT6CC6:66CCTTA66TT

5075 6 L P 6 P L A I 6 N A V A D S L T R I L t A L E S A O E S H A L H H I N A A A L R f

TCA6TTTCACATCACTC6T6AACAA6C6C6A6AAATA6TAAAATTAT6TCCCAATTSCCCC6ACT66666CAC6C6CC6CAATTA66667AAACCCCA6666CCTTAA6CCCC6A6TTCTAT66C

6000

O F H I t R E I A R E I V K L C P N C P D 9 6 R A P I L 6 V N P R 6 L K P R V L k O

AAAT6,6AYT7tACTCAT6TTTCA6AATTTSSAAAATTAAAAtAT61ACAT6T6ACA6T66ATACTlATTCtCATTTTACTT7C6CIACC6CCC6AAC666C6AA6CAACCAA66ATT61TTACAA

t125

N D V 7 H V S E f 6 K L K Y V N V T V D 7 Y S H F t F A 7 A N t 6 E A 7 K D V L O

UCATT66CTCAAA6CTTT6CATACAT666CATTCCTCAAAAAATAAAAACA6ATAAT6CCCCT6CATAT761TCTC6TTCAATACAA6AATTTCT66CCA6AT664AAAAIATCTCAC6TCAC666

in0 N L A I S F A Y N 6 1 P I K I K t D N A P A V V S R S I I E F L A R H K I S H V t 6

CATCCCCTACAATCCCCAA66ACA66CCATT6TT6AAC6AAC6CACWCAAAATATAAA66CACA6CTlAAIAAACTTCAAAA66CT66AMATAAC7ATACACCCCATCATC76TT66CACAC6CTC

6375

I P Y N P Q 6 Q A I V E R t N O N I K A O L N K L Q K A 6 K Y Y T P N N L L A N A L

tTTTTT6TCT6AATCAT6TAAATAT66ACAATCAA66CCATACA6C66CC6AAA6ACAtTT6666TCCAiATCTCA6CC6ATCCAAAACCTA766TCAtT666AAA6ACCTTCTCACA666TCCT6)6

6500

F V L N N V N N D N I 6 H t A A E A H H 6 P I S A D P K P N V N V K I L L T 6 S V

AAA66ACCC6AT6TCCTAATAACA6CC66AC6A66C7AT6CTT6T6TTTTTCCACA6iAt6CC6AAACACCAAICT666TCCCC6ACC6ATTCATCC6ACCTTTTACT6A6C66AAA6AA6CAAC

"2 K 6 P D V L I t A 6 A 6 V A C V F

R~~

I E t P I I V P D A f I R P F t E R K E A t

sa-3I, P K H O S 6 S P 7 D S S I L L L S 6 K K I R

IICCCACACCT66CACT6C66A6AAAAC6CCSCC6C6A6AT6A6AAA6ATCAACA66AAA6TCCCAA6AATSAATCTA6TCCCCATCAAAMA6A6AC66CtTT6CMCATCT6CA66C6TT6ATC

6750 P 7 P 6 7 A E K t P P R D E K D I O E S P K N E S S P N I R E D 6 L A t S A 6 Y D L

P H L A L R R K A R R E N R K I N R K V P A N N L V P I K E K 7 A V I N L O A L I

tcC6AA6C66A66A66TCCTTAAAACCT>CACAACCCCCAAACCTCTTTACCTTATTlCT76CtT76TT6TCT6TCCTC66cCCCCCC6CCT6t6ACA6666A6A6TTATT666CCTACCTACC

6875 A S 6 6 6 P #

6 E A E E V L K t S I t P I t S L 7 L F L A L L S V L 6 P P P V E S A LP

TAAACCACCTATTCTCCATCCC6T666AT6666AA67ACA6ACCCCAT7A6A6TTCT6ACAAATCAAACCAT6ATTTi666766TTC6CCT6ACTTTCAT666TTCA6AAATAT67TCt66AAT6

7"00

K P P I L H P V 6 V 6 S T D P I R Y L t 0 I t N V L 6 6 S P D F H 6 F R N N S 6 N V

7ACAlTTT6A6666AA6TCT6ATAC6CTCCCCATTT6CCTTTMTCTCTCClTTTCtACCCCCAC666CT6ClTTCAA67A6ACAA6CAA6TATTTCTTTCT6ATACACCCAC66TTlATAATAAT

7125 H F E 6 K S I t L P I C L S F S F S t P t 6 C F I V D K Q V F L S D t P t V D N N

AAACC766666AAA666T6ATAAAA66C6TAT6T666AAClTt6tT76ACTACCTT6666AACTCA6666CCMTACAAMAC766TCCCTATMAAAASAA6TT6CCCCCCAAATATCCTCACT6

7250 K P 6 6 K 6 D K R R N V E L V L 7 t L 6 N S 6 A N t K L V P I K K K L P P K Y P H C

MAAbTC6CCtTTAA6AA66AC6CCT7CT666A666A6AC6A6TCt6CTCCTCCAC66T66TT6CCTT6CBCCttCCCT6ACCA6666T6t6ATTTTCTCCAAAA6666CCCTTS666TTACTTT

7ms

O I A f K K D A F V E 6 D E S A P P R V L P C A F P D C 6 V S F S P K 6 A L 6 L L V

666ATTTCTCCCTTCCCTC6CCTA6T67A6ATCA6tCA6ATCA6ATTAAAA6CAAAAA6AATCTATTT66AAATTATACTCCCCC76TCAATAAAGA66TTCATC6AT66TAT6AA6CA66AT66

7500 D F S L P S P S V D Q S D Q I K S K K N L f 6 N Y t P P V N K E V H R v Y E A 6 k

6tA6AACCTACTT66TTCT666AAAATTCtCCTAA66ATCCCAAT6ATA6A6ATTTCACT6CACTA6TCCCCCATAAAA776TT7CSCTTASTCSCA6CCTCAA6ACATCTTATTCTCAAAAS

7625 V E P t V f N E N S P K D P N D R D F t A L V P H t E L F R L V A A S R N L I L K R

FIG. 1-Continued.

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BCCA66ATTTCAASAACA16A6AT6AlXCCTACATCT6CCT6T61TACTTACCCTTAT6CCATATIATTA66AlT£CTCiA6TTiAA76TATA6A6AAAA6A66ATCTACTTTTCATATTTCCT

P 6 F O E H E H I P T S A C V T Y P Y A I L L 6 L P I L I D I E K A 6 S T F N I S C

6TTCTTCrT67A6ATT6ACTAATl6TTTA6ACTCTTCT6CCTAC6ACTAT6CA6C6AICATA6TCAA6A66CC6CCATAT676CT6CTACCT6TSA6AATT66T6ATBAACCAT66TTT6AT6AT

S S C N L 7 N C L I S S A Y D Y A A I I V K A P P Y V L L P V D 1 6 D E P N F D D S A I I T F R Y A t LL I R A K RJTV A A I I L 6 1 9 A L I A I T7 S F A V AT T A

CT7A6T1AA66A6A76CAAACT6CIACST6TTAArtTAATCTTCAtA66AA76TTACAT7A6CCTTATCT6AACAAC66ATAATA6AMAAATTA6AA6CIAMCTTAAT6CTTTA6A6AA6AA

L V K E N I 7 A T F V 0 k L N R N V 7 L A L 6 E Q R I I D L K L E A R L N A L E E V TASTfT7tA6ATT666ACAA6At6t66CCAATTTAAA6ACCA6AATSTCCACTA667T6CAT6CAAATTA76ACTTTA7Ct6C6T7ACACCTT7ACCCTAtAAT6CtACT6A6AACT666AAA6A

V L E L 6 I I V A N L K 7 A N 6 7 P C H A 0 V D F I C V t P L P V H A 7 E N N E R ACCA666CTCATTTATT666CAlTT66AAT6AtAAt6A6AlTTCA7AIAACATACA66A6T7AACCAACCT6AlTA616A7AT6A6CAAACAACATAT76AT6CAST66ACCTTA6T66CTT66C t R A H L L 6 1 1 N I H E I S Y 0 I 1 E L t N L I C I N 6 K O H I D A V I L 6 6 L A

7750

7,75

3000 8125 8250 .375

ttCATCTTTT6CCAAT66A6T6AA6CTTTA)AATCCATTA6ATT66ACACAATATTTCATTTTTAtA6676TT66A6CCCT6CTTTTA67CAT76tACTTAt6ATTTtMCCATT6TTTTCCA6T 0500

0 S F A N S V K A L N P L I U T C Y F I F 1 6 V 6 A L L L V I V L N I F P I V F I9 a

KCCTT6C6AA6A6CCTT6ACCAA6T6CA6TCA6ATCT7AACT6CTTCTTTTAAAAAA6AAAAAA66666AAlE6CC'6C6CCT6CA6CA6AAATBST766

ACTCCCBA6A6T6TCCTACACTTAiU625

L A K S L I I V I S I L N V L L L K K K K S6 6 A A P A A E H V E L P RVSYYT* 11H P R L 09 K I L N S R E C P t L R

S6A6AA6CA6CCCUS666TT6TTTCCACCAA66AC6ACCE6TCTZCGEACZAACG6AT6A6ECCATCACACAAA6ACATACTCATTC7CTCt6CCAAACTt66CATA6CTC76CTTTGCT66 1750 6 E A A K 6 L F P T K D D P 6 A N K A N 6 P 6 D K D I L I L C C K L 6 1 A L L C L 6 66CtATT66666M6TT6C66TTC676CTCBCA666CTCTCACCCTT6ACTCTTTttAAA6CTCrTCT6T6CAA6ATTACAATCTAASC6ATTC66A6AACTC6AeCTTCCTCCt6A66CM66A

L L 6 E V A V R A N R A L T L D S F N S 6 S V I D Y N L N D S E N S T F L L N Q 6

POPT6SYHWUWW% 16161I 0P6ECIRgHLSI EIYINoT"N""I"LAKNYIFTK"""NMIIn IN III III T K166ITN SPODPRL I MI I RDL LV TN1 I

P Q P 7 6 S V K P H I P C P S E I E I N N L A K N Y I F 7 N K 7 N P I 6 R L L V T I ctTTAAAAYuTrATTArrzrT=TACriAsTTTTirTr^aATir^ALcctT^crAA^t=cAAAAAcAaTcanasrarrArrTrAhrTPACTTcAA&ArAnyacr=rrTAAcalrrT

mtv

3575

9000

aIJr,

a*IIwINR"fDMlo In"l IIIInI§WI INII IIRL ILA" II1IL"I"ARRDI iR"W""""lbombIM6Lbo iboIRAI IWRIk"bAII nb"RLMV " NMLI VBo"I TIAD

L A I E I L t F 1 7 I F 7 O I I R L E N 6 I E N R K A N 6 7 6 V E E O V I 6 L R A 6 MC68CT*'6AA61AAAAASCSAAABA66A6T6C6CTT6TCAAAATA66A6ACA66T66766CAACCA666ACTTATA6666ACCT7ACATC7ACA6ACCAACA6,AT6MCCCTTACC6tATACA 9250

6 L E V K R 6 K A 6 A L V K J 6 1 N I N Q P 6 t V N 6 P Y I Y R P 7 0 A P L P Y t

66AA6AIAT6ACC7AAA7TTT6ATA66T666TCACA67CAA766C7ATAAA6T6TTATACA66TCCCTC7CCTTTC6T6AAA66CTC6CCA666CTABCC7CCTT66T6TAT6TT6ACTCA66 9m 6 A Y b L N F I N H V T V N 6 Y K V L Y R S L 6 F A E N L A N A R P P N C N L t I E A6A6AAAAAC6ACAT6AAACAACA66TACAT6ATTA7AlTTATTT666AACA66AAT6A6CA6CAtTTT6666AAA6ATrTTTCATACCAA66A6A66ACA6766CT6CAIC7AAIABA6CACTATT "00

E K H I 0 K I I V H D Y I Y L 6 T 6 P S S I 9 6 K I F H t K E A T V A A L I E N Y 6 CT6CAAA6ACTTA166CA76A6TTATlA16AT7A6CCTTTAlT7ACCCAATCT76T66TTCCCA66T7AA67AA6TTCA766TCACA6ACT61TCTTAAAACAA66AT6T6A6ACAA6T66T 9US

A K T 6 N S6 Y YDI

Mt6ACT766CT766TATCMAAt67tT6ttbCCTAACCTAAAT6CTCIAACTCCtAT6T7CTtTt76ATTCTAtCC6tTttAT67AAAT6CttAT6tAAACCAt6ATATAAAAST6rKT 9750

975 AC*CCTCAC*6TC86CC8ACTCstcctcgag_

&ccaccgFtgagttag

-gCogagtincgtuedg.ccggtgcttcgtcgcgggttcgctcgctagctatctggatcactccc

FIG. 1-Continued.

we noticed a fortuitous ATG codon 36 nucleotides distal to thePstI site, which was likely to act as an initiator under the

more promiscuoustranslation conditions of the reticulocyte

lysate. The 0.9-kb RNA species should therefore be

trans-lated into a 25-kilodalton (kDa) protein which terminates at

theTAAcodon at the end of Pr77 (nucleotide 3281), whereas

readthrough of this codon should yield a 28-kDa species

terminated at theKpnIsite marking the end of the transcript

(Fig. 3a).Similarreasoning for the 1.8-kb RNA would predict

naturally terminated 25- and 51-kDa species and a 64-kDa

readthrough product ending at the

BglII

site, whereas the

3.2-kb RNA should yield 25-, 51-, and 92-kDa products.

Theresultsobtainedby translating these RNAs in a rabbit

reticulocyte lysate and by analyzing the

[35S]methionine-labeledproducts by polyacrylamide gel electrophoresis (7, 8)

are shown inFig. 3b. Densitometric scanning of the

autora-diographs, with corrections for the relative contents of

methionine, indicated that the predicted products were

pre-sentin proportions consistent with approximately 15 to 25% readthrough of the successive termination codons. With RNA terminating at the EcoRI site, the low level of the

92-kDagag-pol product presumably reflected the

vulnerabil-ity of this longer RNA to degradation in the lysate or premature termination of the translation machinery. A sim-ilar explanation could be proposed for the background in these gels, since breakage of RNA chains permits initiation

atessentiallyanymethionine codon encountered close to the

5' ends of the resultant RNAfragments (8, 10).

Neverthe-less,theindicatedproductswerespecificallyprecipitated

by

antiseraagainst themajorgagdeterminant, p27 (not

shown).

Partial amino acidsequencingof thevirionproteins p27and p14 has established their amino termini at nucleotides2315

and2976, respectively (N. Totty, M. Waterfield,R.

Moore,

M. Dixon,R.Smith, G. Peters,andC. Dickson) sothatthe

in vitro translation products predicted in Fig. 3a should include approximately 60% of theprimarysequenceof

p27.

Trypticpeptide analysis of in vitro readthroughproducts.

To verify the identity of the in vitro translation products

detected in Fig. 3, we prepared fingerprints of the

methio-nine-labeled tryptic peptides from the 25-, 51-, and 92-kDa

proteins(Fig. 4). Previous reportsfrom this laboratory have

described two-dimensional tryptic-peptide maps of

individ-ualMMTVproteinsandofPr77, Prl1O, and Pr160produced

either in vivoorin vitro(7, 8). A schematic representationof thepattern of spots that weobtained from Prl60expressed inareticulocytelysate(8) is shown in Fig. 4d. In thisearlier work, it was established that peptides 1 through 8 were present in Pr77 and peptides 9 and 10 were additionally present in PrllO, whereas only peptide 11 distinguished Prl60 from Prl1O. Although not all of these peptideswere represented in the present work (because of the artificial initiation site generated in the SP6 transcripts), it was clear that peptides 9 and 10 were again acquired in the readthrough from the 25- to 51-kDa species and that peptide AAATTTTT6ASTAAACTTBCAACASTCCTAACATTCACCTCTCSTSTSTTTST6TCTSTTC6CCA7CCC6TCTCC6CTC6TCACTIATCCTTCACTTTCCA6A666TCMCCCSCA64CCM66T

w_,....

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a

LTR gag pol

°

1 2 11 1111 11 11 kb11111111

21

2 3

b Pr77 0.

Prl6O *..:..j *.. ...

Pr731

FIG. 2. Dispositionof openreading frames in the MMTV proviral sequence. (a) Computed translation oftheMMTV DNA sequence of Fig. 1in the three possible reading frames. The positions of all potential termination codons are indicated relative to the linear map from nucleotides 1 to 9901. (b) Open reading frames shown in panel a directly correlated with the precursor polyproteins identified in MMTV-infectedcells andcharacterizedbyimmuneprecipitationandtryptic peptide mapping (9). Thedifferentreading frames are depicted by thesymbols: a,frame 1; [lii,frame 3;mm, frame 2. Thus Pr77 is translated in frame 1,PrllOspans frames 1 and 3, andPrl60uses frames 1, 3, and 2. Theopen reading frame orf in the LTR is not included in this figure, since the predicted protein has not been detected in MMTV-infectedcells (8, 10, 45).

11 was acquired in the readthrough from the 51- to 92-kDa

products. Moreover, theothermajor peptides were entirely

consistentwith the initiation of translation within the coding region of virion protein p27.

DISCUSSION

The majority of previous studies on the MMTV provirus

and sequence have concentrated on the milk-borne viruses

characteristic of the GR and C3H strains of mice or on

endogenouselements. Although there have been numerous

reportsonthe sequenceoftheLTRs, theenvgene,and parts

ofthepol gene,noneofthese hasprovidedacomprehensive

picture ofthe MMTV genome (6, 11, 13, 14, 20, 25, 26, 37).

Oursuccessinobtaining recombinantclones encompassing the 5' half of the genome mayin some measure reflect our

choice ofthe BR6 mouse strain. The exact origin of the

milk-transmitted MMTV in these mice remains unclear,

since they were derived initially from a cross between an

RIII male and a nonviremic C57BL female (15), but we assume that this virus represents the onethat is character-isticofRIIImice. However, theso-called poisonsequences which have hampered many studies on MMTV are not

completely

abrogated in this

strain,

since we encountered

difficultieswhenever attemptswere madetotransfer cloned

5' junction fragments from A vectors into

plasmids (la).

It

was therefore impracticable to reconstruct a complete

provirus byjoiningthetwoEcoRI

junction

fragments,which

thus precluded any tests on the

biological

activity

ofthis

provirus. Nevertheless, all thefeatures of the derived DNA

sequencewouldfulfilthe

expectations

foraninfectious virus andwould be entirely consistent with the known

biochemi-cal data.

The5'LTRandleadersequences. The LTR of theprovirus shown inFig. 1 extends for 1,328 base pairs and is bounded

bytheexpected 6-base-pairinverted repeats (11, 14, 20, 25).

The various elements required for initiation of transcription

and glucocorticoid regulationhave been described in detail

by others(for a review, see reference 39) and are maintained in our sequence with only scattered base changes. Flanking the LTR to the 5' side is the hexanucleotide CGTCAG

duplicated upon integration of the provirus into cellular

DNA(11,20,25), andatthe3' boundary is the18-nucleotide

bindingsite for the DNAsynthesis primertRNA-ys(25, 34).

The sequences distal to the primer binding site are loosely

described asleader sequences, since they precede thestart ofthe gag gene, buttheyencompassthesplicedonor site for

envmRNAat nucleotide1484(Fig.1) andpresumablyallor

partofthe

packaging

signal forMMTV genomeRNA(13, 26,

27, 54, 55).

gag gene. Identification of the ATG codon at nucleotide

1508astheinitiator for the gag gene productsis based on a number ofconsiderations: it is the first ATG encountered

afterthe LTR

(Fig.

1),itconformstotheGNNATG

consen-sus(23), itis followedby590 codonsin frame

(Fig.

1and2),

and itpredictstheaminoterminalMet-Glysequence consis-tent with the observed

myristylation

of the MMTV gag precursor(13, 41). However, weasyethavenoinformation that would allow us to align the amino-terminal virion

protein,

plO,

with the DNA sequence. Partial amino acid

sequencinghas allowed ustomatch the N terminus of

p27,

themajorcore

protein,

with the

proline

codonatnucleotide

2315, and upstreamofthis

point

aretwodomains ofaround

20 amino acids, one of which is

composed

of 50% acidic

residues, the other being 50% basic. This latter

region

is

reminiscent ofthe

highly

basic virion

protein, p8,

described

env

LTR

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[image:6.612.131.475.70.315.2]

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a

P K Bg E

i ~~i

[111111:

_

SP6

K . 0.9kb

2s :.-~I

Q8

7 6

00

90

203 O'2

11 01c)lo

O5

0

4

[image:7.612.65.311.57.510.2]

01

FIG. 4. Tryptic peptide maps of in vitro translation products. Two-dimensional fingerprints were prepared of tryptic peptides from the 35S-labeled in vitro products depicted in Fig. 3b (7, 8). Shown are the 25-kDa (a), the 51-kDa (b), and the 92-kDa (c) productsand aschematicdiagram of thepreviously reported finger-print of in vitro synthesized Pr160 (d) (8). Numbered peptides correspondtothose discussed in earlierpublications andare repre-sentedaccordingtothe framesymbols used in Fig. 2. The symbols A and A indicate additional peptides found in the 51-kDa and 92-kDaproducts, respectively.

464

_M 451

"428

a

425 *425

_451

25

_, 2 5

FIG. 3. Translationalframeshiftingin vitro.(a) Partial restriction

mapof MMTVproviral DNA. The positionsofsignificant (butnot

all) PstI(P), KpnI (K), BgIII (Bg),and EcoRI(E)sitesareindicated.

The segment from PstI to EcoRI depicted by the bold line was

subcloned into the polylinker of the pSP65 vector such that

tran-scription of the resultant plasmid DNA by the SP6 phage RNA polymerase initiated at the specific promoter (*) and copied the MMTVsequences into positive-sense RNA(19, 28). Linearization of the plasmid at the KpnI, BglII, orEcoRI sites yielded specific

RNAtranscripts (.--) of 0.9, 1.8, and 3.2 kb, respectively. When thesewereusedtodirect protein synthesis inarabbit reticulocyte

lysate (8, 10), the expected products had the sizes (in kilodaltons) and composition shown in the diagram. The symbols for each translationalframe correspondtothose usedin Fig. 2. Termination occurred eitherat the normal stopcodons oratthe 3' end ofthe specific RNA transcripts. (b) In vitro translation productsgenerated fromRNAs terminating attheKpnI (K), BglII (Bg), orEcoRI (E) sites in the MMTV sequence. These products were analyzed by electrophoresis in 10% polyacrylamide gels (7, 8), labeled by the addition of[35S]methionine tothereticulocyte lysates, and visual-ized by autoradiography. The positions and molecular sizes (in kilodaltons) of the relevant products are indicated. The No RNA

lane shows the background of protein synthesis observed in the

previously (7, 9). The amino-terminal sequences ofvirion protein p14 and the minorprotein p30 both align perfectly with the DNA sequence beginning at nucleotide 2996 (N. Totty, M. Waterfield, R. Moore, M. Dixon, R. Smith, G. Peters, and C. Dickson, unpublished results). P14 is the majornucleic-acid-binding protein ofMMTV, a conclusion that issupported bythepresenceoftwoof the motifs(N,N + 3, N+ 13)ofcysteineresidues identified in othersystems (1, 4). However, any function ofp30 remains a mystery, since to our knowledge, no equivalent protein has been described for other retroviruses. As p30represents a

trans-frameprotein, sharingan amino terminus withp14 in frame 1 andacarboxyterminus withPrllOinframe3,it willclearly be interesting to determine the complete amino acid

se-quence of p30 to define exactly where in the nucleotide

sequence the frameshiftoccurs.

Protease. One consequence ofexpressing viral structural proteins in the form ofprecursors is the requirement fora

specific protease to process these polypeptides into the mature forms. It is therefore hardly surprising that

retrovi-ruses encode theirown enzymes toperformthis role, butit has become apparent that they have adopted various strat-egiesforimplementingthisfunction. Avianleukosisviruses, for example, include the protease within the gag gene; in murine andfeline leukemiaviruses, the enzymeisformally

nuclease-treatedlysate. We notedthat several of the major in vitro products migratedastriple rather than single entities in acrylamide

gels,butwehavenoexplanation for this phenomenon,norhavewe

been abletodistinguishdifferences between the various species.

a

_ ... ...

b

Bg 1..kb

25 ..-..

C d

E 3.2kb

25

b

NO

RNA K Bg E

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VOL. 61, 1987

MMTV

aMPMV

RSV MoMLV HTLV-1 VTS N A

MYTVt MPMV

R SIV"

MoMLV HTLV-1 VISNA

MMTV

MPMV RSV MoMLV HTLV-1

VISNA MIVI MPMV RS, V MOML.V MT L V-1 VISNA

b

MNTV% MPMV

MloM LV HTL'V- 2.

VISNA

M TV MPMV PRSV MoMLV MTLV -1 VISNA

MMTV MPMV

MoMLV H L -A VISNA

MMTV DNA SEQUENCE

DPLTNDKLAAAJLV QZQMEASITKSs$ L

DUWLPEGILV&LTWV EKEL4I£PS

LISW

IVWVZRKA WSY

ALLHRAVU4

KQYRVSAR LGSIKPHI Q L.LEQtrLVPCQSMU LLPV*PGNNYRPVGS

.JEVN

QPVPFPgUlQHL RKALEAQUIXYT G?GN?VFPVPLWA NGTISF TM

AQI WTUEGtKEIVDRLEKEGKVGF¶AFPHWTCWNTIIFCZIW 151"MLLIDFREL"U

.AV

Kf$KlflsvnSPM.AlVrarJ

AKLTVPF AVQQAPVLSAPA WPLMVLD&KCWSIPLAEQORE A PAPTVfNQA

LRvED:MrilHPTV PN PYNLLSGLPS FPS HQWYVLDLKIDAPFC LRF,L SQF' nEWR D?EMS:' S LT'IDLSSSSPQPPDLSSLPrTLAH LQTWb>A"QI PLPKQFQPYIWTVW rQYG KQTEDLAEAQLSHPGGLQ RKKHVTILRIGDAYVTIPLYEPYFQY'TCFTMLPN'ULG

487

Vt

flQKFVP%ALLTVWfDKYQWYlSWYXPZLL&MFWtZVUZ

P!siMJIKn?QAIbtonYvAsATA;HA kn AWkQMY-IwLIAGK:;GQQVLQ

PARVtKVLJT I0TVVGQ\ EPLRLKHF..CML*I£LLAASSH LEA

SGQLT W#TRRf$

_S

C;QT

~~~~~~~~~~~~bT

DAHl~:RQHPODLILLYvRL4AtSEI.D

FRI?EP>LTC _L ; F E LFAaXv4 SE }i

PGNAYWKtWAH.L Z, P R- PWf,vFI].L4wA-V

PCvatYYIKVL WRLS#A;YQFTMQIK:TLF.JIEErPRT W M : QFGWY:GV DLLEA-, L,3S EHF.

CFDQLKQEL"T AAHI

APS.,VQLQEDP

YTYWFELNGP AG-E E VISSTLE RA TI SPDKVQREEPS VQflYKLGST

GTR ALL TLG NLRx'AA KKAQICQQVKIITtLAY KEG LSEATMASLI SDSLPVIENKT,QQZKTPC;M-KFIAQIZSPN

GI7TLVN F LASYrAQYSFlMPyE.CQEJYP AKWLSFL H?E

ZLT*AIEBSAQKSEALHQNAAALRFQFHITREQAREIKVKLCPNCPDWGRAPQL GVNPR INfNL&% A4Hr, NAC77L T-TMP .1IFIt

MAR

I VW-YCP Cp'9 L GVNPR

QAYErL?EvAKLMTALML 3PF;.ALSKAThNIZXQARE V&?CPF CNS^ PAL.EA GVNPIR THLSF S.MKA ..EFSWHS2r'lYMLNFDFNI..C>CAASA.^G'P. P P L, L.;S; AE:RESXTEB ALTTLQ,a A T:TT:& C AeC fH.- w =^A.G7@ -FWR

WP ALrSFEAHFI H TA L.LIECriIGT?A- 5C sQ ' FR

GLKPRVL WQMDVTHVSEPGKL KYVHVTVDTYSHFTFATARTGQATKDVLQJLAQSFA QLFPM' WQMDVTh"'SEflNL KYtNV:H;: DTwS PAT1G"TrTK .T HL.z-;V: Fs GLGPLIWT-T-DFT FF3 WLA.-,?Y£VTVDTA.SJw s/W}T';- -;. fi;HA?A

GHRPPS-TH WE DFTE IF-n3L_Y frKYI.VFY D--FIDTFS A E -KvTrKLYF GLS1P H 9WQ D :TN F;-.IV K NTr L FHVAVDT?S AIX.T K 3:5JLA:A

GSN DxR=ID1WQ.DYTEx.rf-, LP TNSs' .AerG: .- rhVxMKWYA

YMGIPQKZKTDNAPAYVSRSIQEFLARWKXSHVTGI PYNPQOQAIVERTHQNIKAQLNKL :AIBLPKQW1KTDlM PGP-YTYSVVrQEFW½CI2--eVI-VH:TGITPYNPQQQ:I--VLRAHLKT??:E K: VLG^RPKAIKTDNIGJ>_W-LSKS? EnW-ARW 2It*^H.TGi;P *eNLQGQAMVKRCEA\Esi .K21F IRVL

1-. PKAI NTnw DNff.:3`_Y-FSKS.--E'r..-LARW I P. TGyI P:: N,- .'*vRIJsVK

F FGMPQ r_TD-3PAN'-AFVSi` I It.-.C YPQ'QKLVJ - IKL'

HL_GKP'SYINTDNSPAYSCW..'HKT>PYN?`x3/K.

lgFAPrK.3EZDXN.3PA FV AEES', .J--;-. ..iTGIPA"NP.QA"H VKRTHQt-'K\ BK

FIG. 5. Amino acid sequence homologies amongretrovirus polgenes. Sequence comparisons are presented between thepolgenes of

MMTV(thispaper), Mason-Pfizer monkeyvirus(49), Roussarcomavirus (42), Moloney murine leukemia virus(47),HTLV-I(44),andVisna virus(48). Residues homologous to the MMTV sequence are shaded. The distinction between polymerase (a) and endonuclease (b) domains isbasedonthefeaturesof these proteins in Rous sarcoma virus and Moloney murine leukemia virus.

partofthepol gene, occurringdownstream of the termina-tion codon separating gag and pol; and inHTLV-I,

HTLV-II, bovine leukemia virus, and Mason Pfizer monkey virus, the proteaseis encoded in a separate reading frame between the gag and pol genes (5, 18, 19, 38, 40, 42, 44, 46, 47, 49,

58-60).

Thesituationin MMTV most closely parallels that of the last virus group in that the sequence LLDTGADK, which is homologous to the consensus active site of retrovi-ral and some cellular acid proteases (52), occurs in frame 3

(centered onnucleotide 3821)in thebridgebetween the gag

and poldomains (Fig. 1and 2). Although we are not yet in a

positiontodefine the boundariesor exactsize ofthe MMTV

protease, it is clear from these data that its expression

requiresatleast onetranslational frameshift.

Polymerase and endonuclease. Characterization of the MMTV reverse transcriptase has been and remains a con-tentious topic, with conflicting reports ofa 100-kDa mono-mer or 85- and 55-kDa dimers as the active moiety (for a

review, seereference 9). Now that the fullDNAsequenceis

known, it should bepossibletoresolvethis issueby

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a

Frame 1

Frame 3

b

Frame 3

Frame 2

Pr77 /PrllO READTHROUGH

3260

GCTGAAAATTCAAAAAACTTGTAAAGGGGCA

A E N S K N L

* K F K K L V K G Q

PrllO/Prl6O READTHROUGH

4069 4

GATGATTCACAGGATTTATGATAGGGGCCAT

D D S Q D L

* F T G F M I G A I

FIG. 6. Sequences involved in translational frameshifting. The

DNA sequences spanningthe boundaries between Pr77 and PrllO (a)and between PrllO and Prl6O(b) are displayed in a simplified

form.Theaminoacidsencoded in therelevant frames areindicated by the single letter code, with stop codons also shown (i). The sequence motifs discussed inthe textare underlined.

ing and aligning terminal amino acid sequences. A similar strategy will be required to characterize the endonuclease domain, since the presence of poison sequences has pre-cluded the types of mutagenesis studies applied to other

retroviruses (12, 16, 32, 43). Asnoted by others, the coding domains ofpolymerase and endonuclease are nevertheless readilydiscernible intheMMTVsequence simplyby homol-ogiestootherknown sequences (3, 6, 38,40, 42, 44,46-49).

The degree of conservation is most striking within the polymerase itself(Fig. 5), particularly between MMTV and

Mason-Pfizervirus,wherethehomologyintheregionshown

is around65% (Fig. 5) (49).

envgene.Wepreviously reportedtheenvgene sequenceof

the GR strain of MMTV and discussed its implications in

terms of the viral glycoproteins and their precursor Pr73 (37).Thenucleotide sequenceof thecorresponding region of

the BR6provirus differs at60positions, reflectedin only10

aminoaciddifferences. Five ofthese changesarelocalized

withinthe leaderprecedingthegpS2domain(17, 26, 37), but

theirsignificanceisunclear,since wedonotyetknowwhich

ATGcodonserves astheinitiatorforPr73(26, 37). None of

theessentialfeatures ofthe two matureglycoproteins, such as the number and position of glycosylation sites or the

natureofthe transmembraneanchor, isaffected.

The3'LTRandorf.The mostsignificant featureof the3'

LTRofMMTVistheexistenceofasubstantialopenreading

frame designated orf, which begins at an A residue in the

polypurine tractimmediatelypreceding the boundary of the

LTRandcontinuesfor962residuesinthe U3 portionof the genome(8, 10,11, 14, 20, 26, 45). Wepreviouslyargued that

themaintenanceoforfinseveralendogenousand milk-borne

strains ofMMTVindicates it hasafunctional role (9). The sequence presented in Fig. 1 extends this argument, since

the integrity ofthe reading frame is maintained despite 94 single-base differences between this sequence and the GR

virus LTR. Moreover, the putative splice acceptor site mapped approximately50nucleotidesupstreamofthe LTR isalsorepresented in oursequence (54, 57) (Fig. 1).

Suppression by translational frameshifting. Although it is

stillunclearhoworwhy orfmay beexpressed, synthesisof theenvgenefromaspliced, subgenomicmRNApresumably reflects the need for high levels ofthe viral glycoproteins, roughly equivalent to the levels ofthe gag structural

pro-teins. In contrast, the protease,

polymerase,

and

endonucle-ase are nonstructural components, and

although

they

may occurin

virions,

they

are

required

in

only

catalytic

amounts. It has long been

recognized

that infected cells express the

gag-pol

precursor atonly around 5% ofthe level of thegag precursor, and it has

recently

become apparent that

retrovi-ruses haveadoptedanunusual strategy,

namely

suppression

of termination

condons,

for

controlling

the levels ofthese

functions (19, 36, 58-60). InMMTV, there arethree

precur-sors, nowidentifiedas

pr77gag

prllogag

pro,

and

Pr160gag

propol,

so that two termination codons must be

bypassed

during

synthesis ofthe

pol

gene.This

required

twoframeshifts

(Fig.

2),and bothevents couldoccurinrabbit

reticulocyte

lysates

primed withdefinedsegmentsof MMTV RNA

(Fig.

3and

4).

From the ratios of readthrough

products

to terminated

products, we estimated that each frameshift occurred with

an efficiency of around 15 to 25% in

vitro,

which is

reason-ably consistent with the levels of Pr77,

PrilO,

and

Prl60

observed for infected cells (7, 9). However, these studies cannot formally exclude the

possibility

of

self-splicing

or

splicing catalyzedby components ofthe

reticulocyte

lysate.

The sequences at which frameshifting occurs are dis-played in more detail in Fig. 6. At present, we have insuffi-cient information to determine where within the

overlaps

between frames1 and 3in

Fig.

6aandbetween frames 3and

2 in Fig. 6b the actual switches occur. Resolution ofthis issue awaits protein sequence data for the virion

p30

and protease, the twolikely candidates for products which span these frameshifts. However, wenote thatboth these

frame-shift sequences comply with precedents set by other retro-viruses, in that one occurs at or near the sequence ATTTA, as in avian sarcoma virus (19,

42),

and the other probably

involves the AAAAAACsequence identifiedasthe potential frameshift site in bovine leukemia virus,

HTLV-I,

and

HTLV-IIand paralleled in some procaryote systems (22, 38,

46, 56, 60). Moreover, both frameshift sites are closely

followed by potential stem-loop structures in the virion

RNA. Although we have not assessed the thermodynamic

stability of such secondary structural features relative to

othersin thesequence, it is interestingtospeculatethatthey maycontribute in cis toframeshifting (38, 46, 60). Whether suchprocesseshavewider significance in the generalcontrol

ofgene expression remains an intriguing possibility.

ACKNOWLEDGMENTS

We thank S. Brookes for additional technical help; L. Crawford, J. Wyke, and J. Witkowski for comments on the manuscript; and A. Kessler for its preparation.

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