Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and cutting.

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Vol. 64, No. 10

Analysis of Mutations in

the

Integration Function of

Moloney

Murine Leukemia

Virus: Effects

on

DNA

Binding and Cutting

MONICA J. ROTH,1 PAMELA SCHWARTZBERG,2 NAOKO TANESE,2t ANDSTEPHEN P. GOFF2*

DepartmentofBiochemistry, University of Medicine andDentistryofNewJerseylRobert Wood Johnson Medical School,

Piscataway, New Jersey 08854,1 andDepartments of Biochemistry and MolecularBiophysics and Microbiology,

Columbia University College of Physicians andSurgeons, 630 West168th Street, New York, New York100322

Received5April 1990/Accepted 20June 1990

The3' terminus of thepolgene of Moloney murine leukemia virus encodes the integration (IN) protein,

required for the establishment of the integrated provirus. A series of six linker insertion mutationsand two

single-base substitutions were generated within the region encoding the IN protein. Mutationswereinitially

generatedwithinanEscherichia coliplasmid expressing the IN protein, and the resulting variantswereassayed

for DNA-binding activity. Mutations which altered conserved cysteine residues within a potential DNA

finger-binding motif resulted in lowerorvariable DNA binding, which appearedto be the resultof variable

protein folding. Upon renaturation, these proteinswereabletononspecifically bind DNA ina mannersimilar

tothat of the othermutantINproteins and theparent.Whenreconstructed back into full-length virus,seven of theeight mutations werelethal.All mutantsproducedastable IN protein in virions and mediated normal

conversion ofthe retroviral RNAtoits three DNA forms. Fine-structure analysis of the linear double-stranded

viral DNA indicated that all seven lethal alterations within the IN protein blocked the formation of the3'

recessedterminithat normally precedes integration.

The key step in the retroviral life cycle that ensures the

persistence of the viral genome in the infected cell and its

transmission todaughter cells is the integration ofa

double-stranded DNAcopyofthegenomeintothe DNAof the host

cell (for recent reviews, see references 21, 40, and 53).

Integration of the viral DNA is an efficient and orderly

process: specific sites neartheends of the viralgenome are

joined to the host DNA, so that every integrated provirus

has a similarstructure and organization (9, 25). These sites

consist ofshortinvertedrepeats,oftenimperfect,presentat

the terminiof thelongterminalrepeats(LTRs)that flank the

viral genes (55). During the integration reaction, two

char-acteristic events occur: the two terminal base pairs ofthe inverted repeatsare lost, and a small numberofbase pairs

from thetargetsequence areduplicated andultimatelyflank

the integrated provirus (24, 51, 52). These features are

sharedby retroviruses, otherretroposons, and many other

transposable elements (50).

A single viral gene product, the integration protein (IN),

has been identified as essentialforthe establishment of the

integrated provirus (11, 41, 44, 49). The IN function is

encoded nearthe 3' end of the viralpolgene; mutations in

this region have no effect on reverse transcription butcan

block integration of the viral DNA and subsequent

expres-sion of viralgenes. The IN region is initially expressed as

part of the large gag-pol precursor protein (28, 29). In the mammalian retroviruses this precursor is cleaved during

virion assemblyto generateaseparateINprotein of about 40

to45kilodaltons(kDa) (58). Thisproteinhas been shownto

exhibitatleastnonspecific DNA-binding activity (34, 46). In the avianretroviruses,thegag-polprecursorisonly partially

cleaved, and the IN function exists both as a free protein

termed pp32 and as part of the beta subunit of reverse

*Correspondingauthor.

tPresent address: Department of Biochemistry, University of

California, Berkeley,CA 94720.

transcriptase (14, 48).Thepurified avian proteins have been

shownto exhibit DNA binding (22, 39) and DNA

endonu-clease activity (18-20, 23, 32, 59), with specificity for the termini ofthe viral LTR (7, 12, 13, 27). Thepresence ofa

zincfinger motif-a cysteine-containingsequencecapableof

folding around a chelated zinc-has been noted and

pro-posedasimportantfor DNAbinding (26). Mutagenesisof the

Moloney murine leukemia virus (M-MuLV)IN protein has

been previously described, but detailed analysis of the

biochemical effects of thesemutationshas notbeen carried

out(10).

Recent work in the M-MuLVsystemhasprovideda more

detailed definition of the structures and sequences of the DNA intermediates in the integration reaction. Direct

anal-ysisof theunintegratedlinearviral DNA has shown that the

two ends are a mixture oftwo structures: full-length blunt

ends, and termini with the 3' ends recessed bytwo

nucleo-tides (5, 45). Kinetic analysis suggested that the terminal nucleotides on the 3' ends are cleaved in vivo to form recessed 3' ends and that the IN function isrequiredfor this

step. More information has followed from in vitro reactions

which recapitulate the integration of viral DNA, either

endogenousorexogenous,into added target DNA(3, 4, 15,

16). These studies have shown that the linear form of the viralDNA,notthe circularforms,is the directprecursorto

the provirus; that linear DNAs with recessed 3' ends are

activeprecursors; and that these 3' endsaredirectly joined

totargetDNA in the strand transfer step, leaving protruding

5' endsonthe viral DNA.

Tohelpdefine the role of the INproteininintegration,we

generateda series of linker insertion andsingle-base

substi-tutionsthroughout the domain of thepolgene encodingthe

M-MuLVINfunction. The effects of these mutationsonthe

in vitro DNA-binding activity of the protein and on the

formation of the recessed 3' termini of newly synthesized

linear viral DNA were examined. Mutations located in the

cysteine region, affecting the proposed zinc finger domain,

apparently altered the folding of the protein but did not

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completely abolish the nonspecificDNAbinding activityin

vitro. Most of these mutations were lethal to virus

replica-tion, blocking early events in infection. All mutations that

blocked viral integration werefoundtoblockthe formation

of the recessedtermini on the linear DNA formed in vivo.

MATERIALS AND METHODS

Cells, viruses,andinfections.NIH 3T3and Rat2cellswere

grown in Dulbecco modified Eagle medium (GIBCO)

sup-plemented with 10% (vol/vol) calf serum (Hyclone).

Wild-type virus was derived from clone pTll (33) orpNCA (8).

MutantsofM-MuLVweretested forreplicationcompetence

by the XC assay (47) andby reverse transcriptaseassay of

the supernatant medium (17) after transformation ofNIH

3T3 cells by the DEAE-dextran method (37) with cloned

DNA (0.25 ,ug per 105cells). dl5401viruswasobtained from

an existing NIH 3T3 producer line (49). Cloned Rat2 cell

linesexpressing IN- mutant genomeswereestablished after

either Polybrene- or calcium phosphate-mediated

cotrans-formation(61) withpSV2neo DNA(54). Individualcolonies

were selected for growth in thepresence ofthedrug G418

(GIBCO; 400 ,ug/ml) and assayed for release of reverse

transcriptase activity (17). Virus used for infection was

collected from 10-cm dishes containing 5 ml of medium

every 12 h. Infections ofRat2 cells were carried out in the

presence of Polybrene (8 ,ug/ml).

Bacteria, plasmids, and extracts. Escherichia coli HB101

was used as the host for propagation of all plasmids. The

bacterial IN protein expression plasmid pSCB150-18 was

described previously (46). Preparation of lysates and

isola-tion of insoluble protein fractions ofE. coli were done as

describedpreviously (57).

Electroblot transfer and binding assay. Southwestern

(DNA-protein) blot analysis was performed as described

previously (46).Insomeexperiments,afterelectroblot

trans-fer,thenitrocellulose filtercarryingtheproteinswassoaked

in buffercontaining 50 mMTris hydrochloride(pH 8.3), 50

mMdithiothreitol, 1 mMEDTA, and 8Mguanidine

hydro-chloridefor1htodenature theproteins.The filterwasthen

washed for 1 h at room temperature in dilution buffer (50 mM

Tris-HCl[pH 7.5], 2 mMEDTA, 2 mMdithiothreitol,0.5 M

NaCl, 0.1% Nonidet P-40, and 10% [wt/vol] glycerol),

fol-lowedby anadditional wash at 4°C for at least 24 h in the

samebuffer, to permitrenaturation.

Mutagenesis of pSCB150-18 plasmid DNA. (i) Bisulfite

mutagenesis. Plasmid pSCB150-18 DNA was partially

di-gested with BspMI in the presence of 1 ,ug of ethidium

bromideper ml (42), and the full-length linear DNAs were

isolated after electrophoresis (60). The DNA was treated

with 2 M sodium bisulfite for 3 h at 37°C and dialyzed as

described before (43). The plasmidDNAwas recircularized

by ligation and used to transform E. coli BD1528 (ung).

Colonies were screened for loss of either the ApaLI or

Hindlll sites which overlapped the BspMI cleavage site in

the pol gene.

(ii) Insertional mutagenesis. After limited digestion of

plasmidpSCB150-18 withAluI,thelinear DNA was isolated

by agarose gel electrophoresis. Then, 12-base-pair (bp)

EcoRIlinkers(sequence CCGGAATTCCGG; New England

BioLabs)wereadded to the termini with T4 DNA ligase. The

DNAwas digested with EcoRI, treated with T4 DNA ligase

to form circles, and used to transform bacteria. Mutants

werenamedby the nucleotide position of the mutation, using

thenumbering system in which the left edge of the left LTR

is nucleotide 1.

0 2 4 6 8 kb

Wild type

b15346-1

3-12 1 f * j557-12

247-12 /

ThrCy*LysAb Cys Al ---CCTCAAA GCT T,GT GCA C

-BspMI Hindlil ApaLI Val

---- ACCTGCAAAt TGT GCA C

-T r

b15349-1 ----ACCTGCAAAGCT T

GCAC---Ala GlyIh Pro Ala

in5345-12 AccGGATaC AAA-ATTCOcrG q TGT GCA C -EcoRI

FIG. 1. Positions of mutations affecting the IN protein. M-MuLV proviralDNAisshown at the top; thetwoboxedregions

atboth ends of theDNArepresentLTR sequences.Enlargement of

theINregion is shown below.Open triangles above the line indicate positions of 12-bp EcoRI (RI) linker insertion mutations. Aminoacid

and DNA sequences in thecysteineregion areshown inthelower half of thefigure. RT,Reverse transcriptase; PR,protease.

Antisera and immunoprecipitations. Anti-Pol serum was

prepared in rabbitsby immunizationwith thebacterialfusion

protein encoded by plasmid pSC1(58). Goatanti-M-MuLV

(serum 81S-107) was obtained from the National Cancer

Institute. To detect virion proteins, cultures were labeled

with [35S]methionine (Amersham), and the virions were

collected, pelleted through a 25% sucrose cushion, and

immunoprecipitatedasdescribedpreviously (58).Prestained

molecular weight markers were purchased from Sigma

Chemical Co.

DNA electroblotting and hybridization. Electroblotting of

DNA fromsequencing gels and hybridizationwith

oligonu-cleotide probes were performed as described before (45).

The U5 probe D contains the sequence 5'-GAGGAGACT

CACTAAC-3'.

RESULTS

Construction of linker insertionmutations in the IN domain

andanalysis of theirDNA-binding activity. Inpreviouswork,

we haveexpressedtheIN protein ofM-MuLVinanE. coli

expression system and demonstrated that both the

bacteri-ally derived protein and the natural gene product exhibit

DNA-binding activity (46). To identify regions of the IN

protein required forbindingtoDNA,weconstructeda setof

derivatives of bacterialplasmid pSCB150-18 expressingthe

INprotein byinsertinga12-bpEcoRI linker into AluI sites.

Six clones with different inserts in the IN region were

identified andmapped (Fig. 1). The presence ofonlyasingle

linker in each mutantwasconfirmed by detailed restriction

mapping, ensuring that eachmutationresulted in the

inser-tion ofonly fouramino acids(Table 1).

To testthe the ability of the mutant plasmids toencode

stableproteins,extracts werepreparedafterinduction of the

trp promoter, and proteins in the insoluble fraction were

analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide

gel electrophoresis (Fig. 2). Ineach case, the IN protein of

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M-MuLV IN PROTEIN MUTATIONS 4711 TABLE 1. Summary of mutations in IN domain

Amino acids Reverse

Virus Wild Viability' transcriptase

type Mutant activityb

Uninfected Rat 2 -

-cells (control)

in5233-12 QL QPEFRL - +

in5247-12 LSF LSRNSGF - + +

in5345-12 KAC KAGIPAC - + +

in5557-12 KL KPEFRL - +

in5939-12 QAH QAGIPAH - + +

in6161-12 AAW AAGIPAW - ++

bi5346-1 A V + +++

bi5349-1 C Y - +

Wild type + +++

a Viabilitywasdeterminedby detection ofXCplaques and reverse tran-scriptaseactivity aftertransient transformationof cells with theindicatedviral

DNAs (seeMaterialsandMethods).

b Reverse transcriptase activity present in culture medium from stable

producer celllinesexpressing theindicated viral DNAs. +++, Wild-type levels;++, 10 to25% of wild-type levels;+, 4 to10%oofwild-type levels; -,

nodetectableactivity.

Mr 46,000 could be readily detected by Coomassie blue

staining (Fig. 2A); in some casesthemobilityof themutant

protein was slightly altered from that of the wild type,

suggesting that significant structural changes had been

caused by themutation (e.g., Fig. 2A, lane6).

Each of the mutant proteins was tested for its ability

to bind a mixture ofsingle- and double-stranded DNA by

the Southwestern blotting procedure (2, 38). Briefly,

pro-teins were separated by electrophoresis, blotted to

nitro-cellulose, and challenged with labeled DNA. Most of the

mutant proteins retained the parent protein's ability to

bind DNA, either with added

Mg2+

as the divalent cation

(Fig.2B1) or in the presenceof EDTA(Fig.2B2). Ingeneral,

there was stronger binding with EDTA than with

Mg2+.

There were significant variations in the amount of DNA

bound by differentmutant proteins, notcorrespondingwith

the amount of protein produced by different clones. The

protein from one mutant, in5557-12, consistently bound

more DNA than those from the other mutants and the

wild-type parent (lanes 6). Only one mutant, in5345-12,

showed a significant defect in binding when normalized to

the amountofprotein(lanes 5). Similar resultswereobtained

when the binding was performed in higher saltconditions,

although here weak binding by mutant in5345-12 could be

detected (Fig. 2C1 and C2). The mutation in this construct

resulted in the insertion offour new amino acids nearthe

N-terminus, increasing the spacing between two cysteine

residues from two six amino acids. This region is highly

conserved among homologous retroviral pol proteins (26)

and has the potential to form a zinc finger DNA-binding

structure, although not a canonical finger structure (1).

These results suggest that this region maybe important for

binding activityinthisassay,affectingeither therefolding of

theIN protein orits intrinsicbinding activity.

Constructionof twopointmutations near the5'end of the

IN region and further analysis of DNA-binding activity.

Be-causeanalysis ofthe insertion mutations suggestedthat the

amino-terminal region of the IN protein was potentially

involved in formation ofa DNA-binding site, point

muta-tions in this area were also tested. Two mutants were

constructed: bi5346-1, bearingahighlyconservative

Ala-to-Val substitution between the two cysteine residues

men-tionedabove, andbi5349-1, carryingachange of the second

Cysresidueto aTyr

(Fig.

1).The

proteins

encoded

by

these

new mutantplasmids, by thelinker insertion

plasmids,

and

bythe parentwereanalyzedfor

DNA-binding

activity

after

electroblotting

(Fig.

2Dand E). The IN

proteins

of thetwo

point mutants bound DNA

well,

both with

(panel D1)

and

without (panel D2) divalent cation; the Cys residue was

therefore not essential for this activity.

Experiments

with

mutant in5345-12 gave variable results,

depending

on the

particular

preparation

ofelectroblotted filters

(Fig. 2).

The

variation in the binding

activity

ofthis mutantcould reflect

variable refolding ofthis

protein; depending

onthe

experi-ment, the mutant demonstrated no

binding

(panels

B1and

B2), weak

binding

(panels Cl and

C2),

or

good

binding

(panels Dl andD2).

To test whether variations in the renaturation of the

proteinswere responsiblefor these results,

duplicate gels

of

the variousmutant proteins were

prepared

and

electroblot-ted as usual. One copy was incubated

overnight

and then

used directlyin a

binding

assay, while anotherwas treated

with guanidine

hydrochloride

and thenexposed to

renatur-ation conditions before the assay. Controlled

experiments

done either

directly

(Fig.

2E1) or after denaturation and

renaturation

(Fig.

2E2) showed that the IN

proteins

with

alterations in the

cysteines

did not refold

correctly

to

re-cover

activity.

We conclude that under some

conditions,

thesemutant

proteins

are

capable

of

binding DNA; however,

the ability of the in5345-12

protein

to renature,

compared

with that of the proteins from the wild type and the other

mutants, is sometimes

impaired.

Whenfolded

correctly,

the

mutant proteins still exhibitbinding

activity.

Viability ofviralgenomeswith mutations in the INdomain.

Totest the

infectivity

of viral genomes

carrying

site-specific

mutations in the IN

region, fragments carrying

each

muta-tionwereexcisedfromthe

expression

constructsandusedto

replacethe

equivalent fragment

ofa

full-length

clone of the

viral genome. Theresulting cloned DNAs were introduced

into NIH 3T3 cells by the DEAE-dextran method

(37).

Production of

replication-competent

virus was detected

by

the XC

plaque

assay (47) and

by

measurement of

virion-associated reverse

transcriptase

activity (17).

None of the

12-bp insertion mutants gave rise to viable viruses after

transfectionat

37°C,

as

judged by

theXC

plaque

assayorthe

reverse transcriptase assayof the medium (Table 1).

Trans-fection

experiments

with thetwo

point

mutantsshowed that

the

bi5349-1

mutation, which resulted in the change ofthe

cysteine

residue to

tyrosine,

was

similarly

lethal.

However,

mutant

bi5346-1,

with theconservativechange ofanalanine

residue to valine, was

viable;

the number of XC

syncytia

induced after DNA transfection was similar to that of the

wild-type virus. The level ofreverse

transcriptase

activity

detected in the medium was

indistinguishable

from that in

the medium collected from the

wild-type producer

cells.

These results

suggested

thatalthoughseveral

12-bp

insertion

mutations did not affect the

DNA-binding

activity

of the

fusion

protein

in

vitro,

these mutations were nevertheless

lethal to the virus in vivo. In

addition,

the mutation that

changedthe

cysteine

residueto

tyrosine

was lethal in

vivo;

in the

nitrocellulose-binding

assay of the IN

protein

ex-pressed

in E.

coli,

however, the

cysteine

residue did not

appeartobecriticalfor DNA

binding.

Therefore,

alterations

in the IN

protein

that havelittle effecton the

DNA-binding

activity

as

assayed

in vitro canblock its function in vivo.

The functions of the IN

protein

in

replication

are

clearly

more

demanding

than the

binding

ofDNA as measured in vitro.

Construction of cell lines

producing

IN- virions. To

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4712 ROTH ET AL.

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M-MuLV IN PROTEIN MUTATIONS 4713

FIG. 2. DNA-binding activity ofIN proteins with insertion and point mutations. (AtoC) Insoluble proteinswereprepared fromvarious

bacteriallysates; proteinsweresolubilized inSDS and analyzed by electrophoresisonSDS-polyacrylamide gels. (A)Coomassie stain of the

protein gel. Inlanes 2 to 8,the 46-kDa(kD)INprotein is indicated by the arrowhead. (B andC) Nitrocellulose-binding assayof the IN

proteins. Following electrophoresis,proteins wereblottedtonitrocellulose, denatured,renatured, andassayed forDNA-binding activityin

thepresenceof50 mMNaCl and5mMMgCl2(Bi),50 mMNaCl and10 mMEDTA(B2), 150mMNaCl and 5mMMgCl2(Cl),or150 mM

NaCl and10 mM EDTA(C2). Samples inpanels A toC: lanes1,pATH3; lanes2,wild-typeplasmidpSCB150-18; lanes 3, in5233-12; lanes

4,in5247-12;lanes 5,in5345-12;lanes 6,in5557-12; lanes7,in5939-12;lanes8,in6161-12;lanesM,sizemarkers. Positions of the size standards

are indicated. (D) Analysis of proteinsfrom added mutants. Proteins on blots were denatured, renatured, andassayed forDNA-binding activity inthe presence of 50 mMNaCl and5mM MgC12 (Dl)or 50 mMNaCl and10 mM EDTA(D2).Lanes1,pATH3controlcells; lanes 2, pSCB150-18; lanes3,in5345-12; lanes 4,bi5349-1. (E)Proteinswereelectroblottedasbeforeandincubated indilution buffer aloneprior

totheDNA-binding assay(El)or denatured andrenatured (E2). Each lane containsinsolubleprotein pelletsfrom thefollowing plasmids:lane

1, pATH3;lane 2, pSCB150-18;lane 3, in5345-12;lane 4,bi5349-1;lane 5, bi5346-1.

acterizein more detail the block to replication caused by the

new mutations in the IN domain, we established cell lines

expressingthemutantgenomes. The complete proviral DNA

bearing each mutation was introduced into Rat2 cells by

cotransformation with pSV2neo plasmid DNA (54).

Individ-ual G418-resistantclones were tested forproduction of virus

byassays ofreverse transcriptase activity released into the

medium.Allthe mutant genomes were able to induce release

of virion-associated reverse transcriptase activity,

suggest-ingthat late stages of the life cycle were normal (Table 1).

Virus was collected from these lines and used without

dilutiontoinfectfresh NIH 3T3 cells; the number of virions

in the preparation, judged from the level of reverse

tran-scriptase activity detected, was approximately 5- to 10-fold

less than in a similarpreparation of wild-type virus.

Medium washarvested from the infected cells and tested

for the appearance of progeny virus by reverse transcriptase

assays. Whereas cells infected with wild-type virus became

producers ofprogeny within 1 to 2days, cells infected with

the mutants did notrelease detectable progeny virus for up

to a month postinfection. These results suggested that the

mutant virions were unable toestablish aproductive

infec-tion andwereblockedinthe early phase of the life cycle.

Analysis of the virion proteins from IN- virus. Toexamine

the viral proteins present in the IN- mutant virions,

pro-ducer cellsweremetabolically labeled with [35S]methionine overnight, thevirionswereharvested,and the viral proteins

wereanalyzed by SDS gelelectrophoresisafter

immunopre-cipitationwith an antiserum raisedagainst whole M-MuLV

virions (Fig. 3). The abundant gag proteins of the virus are

the major proteins recognized by this antiserum. In

wild-type virions, the initial gag gene product,

Pr659'9,

is

nor-mally processed to four smaller cleavage products by the

viral protease; in these experiments, high levels of the

mature CA protein were detected, showing that, as

ex-pected, cleavage oftheprecursor was essentially complete

(Fig. 3, lane 10). In most ofthe IN- mutant virions, the

predominantgagproteindetected was also the CA product.

The quantity of gag protein detected was highly variable

from producer linetoline; thisvariation may reflect both the

inherent efficiencyof assembly of the mutant virion and the

levelof expression from the particular integrated provirus.

Inseveralof the IN- mutantvirions,the Pr659'9precursor

proteincouldstillbedetected along with variousprocessing

intermediates(Fig.3, lanes 2 to 6 and9). A reduced rate and

final extent of cleavage of the gag proteins have been

reported previously for several mutants with alterations in

thepolgene (49). For mutantin5557-12, the cleavage was so

limited that the levels of the precursor were higher than

those of the mature proteins. These results indicate that

changes in many portions ofthepol gene can have some

effect on the activity or action of the viral protease. The

inability of these mutant viruses to process every gag

protein in the viral particle, however, is probably not the

causeof the lethality of these mutations. These virus were

capable of successfully infecting and carrying out reverse

transcriptionafter infection of fresh cells (seebelow).

Itis alsoimportant to note that all the mutants produced

functional levels of env gene products. Because of overlap

between the IN coding region and 5' portions of the env

mRNA, the two 3'-proximal mutations constructed here

could have affected env mRNA expression. In particular,

mutation in5939-12 is an insertion of 12 bp precisely at the

acceptor splice site for the formation of the singly spliced

env mRNA(31) and might well have disrupted formation of

the mRNA and protein. The fact that these two mutants

expressed normal amounts of envelope protein and could

entercells and carry out reversetranscriptionshows that the

mutant mRNAs containing the insertions are fully

func-tional.

Analysis of pol gene products of the virus. One trivial

explanationfor the lethality ofthe IN mutationsis thatthe

mutant IN protein might be unstable and not persist in the

CS N N N CY C4 in Co, CzNU)a

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2 27

Anti-Moloney MuLV Sera

FIG. 3. Analysis of virion proteins. Cells were metabolically labeled with [35S]methionine, and virus was pelleted from the

medium. Virion proteins were immunoprecipitated with anti-M-MuLVantiserumandanalyzedbySDS-polyacrylamidegel

electro-phoresisandfluorography. Lane1, Rat2 cells, uninfectedcontrol;

lane2,d15401inNIH 3T3cells;lanes 3 to9, mutantsasindicated;

lane10, wild-typeM-MuLV. Molecularmassofproteinsize mark-ers are shownatright (in kilodaltons).Arrowsatleft indicate the

position ofthe majorcapsid protein CA and the gag polyprotein precursor

PM65gag.

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o 7

L?C1

IOU,c

oT0

f

N N N cN

0) ) - - c0 _

, teCto o

) N N U) 0)

e o e U) U, ID

C)

CQoScE i:s

-1 2 3 4 5 6 7 8 9 10

kD

80-v

_

_0

16_- X

84- u _ - * RT

(P80)

58 _

48 (P46)

_m _o _ *-4 N (P46)

3 6

_t

_

ftw

j

Anti-Moloney PQI Sera

FIG. 4. Analysis of virionpol gene products. Cells were

meta-bolicallylabeled with [35S]methionine, virus was pelletedfromthe

medium, and virion proteins were immunoprecipitated with rabbit

anti-pSC1antiserum and analyzed by SDS-polyacrylamide gel elec-trophoresis. Lane 1, Rat2 cells, uninfected control; lane 2,d15401in NIH 3T3cells;lanes 3 to 9, mutants asindicated;lane 10,wild-type

M-MuLV. Positions of protein size markers are shown at left (in

kilodaltons). Arrows atright indicate the positions ofthe reverse

transcriptase(RT)and IN proteins.

mature virion. To examine this possibility, the pol gene

products within the virions were examineddirectly. Proteins

weremetabolically labeled with[35S]methionine, viral

parti-cleswereisolated, and the virionproteinswere

immunopre-cipitated with sera which recognize both the reverse

tran-scriptase and the IN pol gene products (Fig. 4). Wild-type virions exhibited the expected pattern of an equimolar

amountofthefully-processedreversetranscriptase (ofabout

80 kDa) and mature IN proteins (46 kDa); none of the

gag-pol precursor, Pr200Bg-Pl, was detected (Fig. 4, lane

10). In sixoftheseven mutantvirions,thematureINprotein

was readily detected and was present at approximately

equimolaramountswith themature reversetranscriptase. In

some experiments the IN proteins of both mutants and

wild-type virions migrated as a doublet. The basis for this

behavioris unknown, but itisprobably not due to

phosphor-ylation (58). Forinsertionmutant in5557-12, theprocessing

of the pol precursor was incomplete; in some experiments

theprecursorpredominated andthecleaved INprotein was

barelyvisible (Fig. 4, lane 3), while in others thematureIN

protein was readily detected. These results show that the

new mutant IN proteins are synthesized, are generally

processed correctly, and persist stably inthemutantvirions.

As previously described, virions of the deletion mutant

d15401contained no detectable IN protein (58).

Analysis of the viral DNA synthesized after infection by

IN-virus. Todetermine whether the mutant virions could initiate

an infection and mediated the reverse transcription of the viral RNA into DNA, we examined the viral DNA in recently infected cells. Virus preparations of the various

IN- mutants were collected from the stable producer cells

and used to infect fresh Rat2 cells. The

low-molecular-1 2 3 4 5 6 7 8 9 1 0 1 1

%t=8:1§ FORM 11

_

- 4---Linear

3w "3 4-=8.8 FORM

8.2

FIG. 5. Southern blot analysis of unintegratedviral DNA.Virus

washarvested from producer celllinesand used toinfect freshRat2

cells. Low-molecular-weightDNAwasisolated, separated by elec-trophoresis on agarose, transferred to nitrocellulose, and probed

with M-MuLV sequences. Cells were infected with supernatants from thefollowing lines: lane 1, Rat2 uninfectedcontrol; lane 2, d15401in NIH 3T3cells;lanes 3 to 9, mutants asindicated; lane 10, mock-infectedcontrol;lane11,wild-typeM-MuLVcontrol.Lanes 1 to 3 were a7-dayexposure. Lanes 4 to 11 were a1-dayexposure.

Sizesareindicatedattheright (in kilobases).

weight DNA was isolated 30 h postinfection, separated by

electrophoresis, and analyzed by Southern blot

hybridiza-tion(Fig. 5).

Inawild-type M-MuLV infection, three viral DNA

prod-ucts weredetected:an8.8-kb linearDNA,an8.8-kbcircular

DNA, and an8.2-kb circularspecies (Fig. 5, lane 11). The

linearspecieswasthepredominant formatthisearlytime in

infection; the amount of circular forms increased at later

time points (data not shown). Infection with the deletion

mutant dl5401 generated the same three proviral DNA

forms, although the ratio of circular to linear forms was

higherthanin the wild type(lane2), asdescribedpreviously

(45, 49). All of the IN- linker insertion and point mutants

were capable of infecting cells and synthesizing the three

proviralDNAproducts (lanes3to9). Theratio of circularto

linear DNAspecies againwas generallyincreased, favoring

the circular forms, as for mutant dl5401. Similar

observa-tions have been made after infectionbymutantsdefectivein

integration due to changes in the LTR termini (8). For

mutantin5557-12, theincomplete processing of the gag and

pol geneproducts apparentlyhad littleeffectontheabilityof

the virusto reversetranscribe its RNA genome(Fig. 5, lane

7).Interestingly, althoughthevirions of the insertionmutant

in5345-12 contained high levels of reverse transcriptase

activityaswellas reversetranscriptaseprotein (Fig. 4, lane

7), thismutantconsistently synthesizedadisproportionately

lowlevel ofproviral DNA.

Analysis ofthe viral DNA formed after infectionby the

various mutants showed that all the mutations were still

present. All the viral DNAscontaining the linker insertions

were sensitive todigestion withEcoRI,and the DNAs from

thebisulfitemutants were resistanttodigestionwith ApaLI

(datanotshown).The results show that all the IN- mutants

were genetically stable and at least largely competent to

carry out reversetranscription of all the viral DNA forms.

Analysisof the termini of linear viral DNAsynthesized after

infectionbyIN- virus.Recentanalyses ofunintegratedlinear

1

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retroviral DNAs have demonstrated that a mixture of two

terminal structures are present: some termini are

blunt-endedandothers have 3' ends recessed by two nucleotides

(5, 45). The IN- deletion mutant d15401 was found to be

unable to form the recessed ends, suggesting that the IN

protein was required for and might directly mediate the

cleavage ofthese strands. To test whether the new mutant

IN proteins could mediate this early step in the integration process, we examined the terminal structures of the linear

DNAsformed after infection by the IN- viruses.

The low-molecular-weight DNA from freshly infected

cellswas first isolated and digested with restriction enzymes

to generate small terminal fragments. The resulting

frag-ments werethen separated by electrophoresison a

sequenc-ing gel, electroblotted onto nylon filters, and probed with

oligonucleotides specific for each of the strands at the two

termini of the linear DNA (Fig. 6). DNA from wild-type

virus showed the expectedmixture ofblunt and recessed 3'

ends (lanes 1, 11); deletion mutantd15401 showed only the

blunt ends, as seen previously (lane2). DNA fromeach of

the six linker insertion mutants and the defective point

mutant bi5349-1 showed only the blunt termini, with no

detectable formationofthe 3' recessed ends(lanes 4 to10).

Thus, every IN- mutant was defective in the cleavage

process, the first detectable step leading to the integrated

provirus. No region of the protein was singled out as

dispensable for this early eventbut neededfor later events;

therewere nopartially functionalmutants, as tested at37°C.

In confirmation ofthe Southern blot

analysis above,

the

IN- viruses generally directed the formation of a higher

proportion of theLTR-LTRjunction fragmentthanterminal

fragments (lanes 4 to 10). The

higher

abundance of that

fragment reflects the higher abundance of circular DNAs

overlinear seenin the absence of

integration.

DISCUSSION

Inthese studies,we haveexamined the effects of various

mutations inthe M-MuLV INprotein, bothin vitro, with a

DNA-bindingassay, andinvivo, by measuringthe

ability

of

the mutant viruses toproceedthroughthelife

cycle.

Muta-tions altering the INprotein had

complicated

effectsonthe

binding activity. Most ofthe mutations showed very little

effectonthe potent,

nonspecific

DNA-binding activity

ofthe

protein. Two mutations, both

mapping

in the zinc

finger

region near the N-terminal third ofthe

protein,

did reduce thebindingin some assays, althoughtwoother mutations in this regionhad no effect. Alterations

changing

the distance

between two cysteines (in5345-12) or

eliminating

one of

these two cysteines (bi5349-1) often

profoundly

decreased

binding, suggestingthat the

cysteines

may function in

form-ingaDNA-bindingstructure, buttheir effectswere variable andcritically dependentontheway the

protein

washandled

before assay, as is often seen for renatured enzymes

(30).

Thus, themutations may act

largely by

affecting

the

ability

of the

protein

torenature

rather

than

by

directly

affecting

the

inherent binding of the protein. Furthermore, the

activity

assayed after

blotting

may

require only

partial refolding

and

may not reflect the true sequence

specificity

of the native

protein. Otherassays may be required to define functional

domains ofthe

protein.

The effects of these mutations as

judged by

our other

assays in vivowere more severe. All of thelinker insertion

mutations were lethal to the

virus,

suggesting

that the IN

protein carries out far more

specific

functions than the

simple DNA binding measured in vitro. Of the bisulfite

CD t- N

aN

uV

3: 1-0 c .- ic

) In

-w 4n to

'- zEe E 3

._ n _ N_

1 1

~

4**"-, -- Circle

Junction

-+ Strand

Strong Stop

] LTR

Termini

U5 Probe D

FIG. 6.

Analysis

of termini from a collection of mutant IN-viruses.Wild-typeM-MuLV andmutantIN-viruseswerecollected from

producer

cells and usedtoinfectRat2cells.Cellswere

lysed

30

h postinfection,and terminal DNA structures were

analyzed

after

blotting as described in Materials and Methods. The filter was

probedtodetect the 3' terminus ofUS.For IN-virus,

proviral

DNA

from

approximately

6 x 107 to 9 x 107 cells was

analyzed;

for

wild-type virus,DNAfrom1 x 107to2 x 107cellswasused.Lanes 1and11,

Wild-type

(WT)virus. Lane2,d15401virus(IN-).Lane3, DNA isolated after a mock infection. Lanes 4 to 10, Mutants as indicated. The identities ofthe variousDNA

species,

basedonthe

size ofthe

fragments,

areindicatedattheside of the

panel.

Marker

DNAs derived from Maxam and Gilbert

sequencing

reactions(36) (G+Aand T+Clanes)are atthe left.

mutations,

only

the

extremely

conservative Ala-to-Val

mu-tation had no effecton virus

replication.

In all the mutants,

the

ability

of the virus to enter the cell and

synthesize

the

linear viral double-stranded DNA was not

altered;

integra-tionitselfwas

apparently

blocked. Fine-structure

analysis

of

the termini ofthe viral DNAformed invivo indicatedthat

thelinear molecules from

IN-

mutant viruswereall

blunt-ended. These results

support

the model

(5,

45)

that the IN

protein

is

required

for the formation of the 3' recessed

termini and that thisstructureis inturn

required

for

integra-tionof the DNA.

The process of

integration

seemstobedivisibleintotwo

steps:

cleavage

of the termini of the viral

DNA,

and then

strand transfer ofthe 3' ends into the

target

DNA,

leaving

nicks in the target

(5,

16).

Allof themutants

analyzed

here

wereblocked in the firststep,the

cleavage

of the viral

DNA,

and we have not been able to

separate

these two

steps

by

VOL.64, 1990

W,Oql,

,z,

if

*.

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mutation. The

approach

of

creating

mutations scattered

along

anentirecoding regionhasbeensuccessfulwith other

genes in

defining

functional domains of the

respective

gene

products (6, 35, 56); although

INdomainsmayexist, neither

assay used

here-nonspecific binding

in vitro and cleavage

in vivo-allowed a definition of any such domains with

partial

activities. It is

possible

that the IN

protein

is foldedas

a

single

functional

protein.

Weare

currently

analyzingthese

mutants in an in vitro

integration

reaction to determine

whether

they

areblockedat separate stages ofthe process.

Preliminary

data suggest that most of these mutants are

severely impaired

in their

ability

to

integrate

anexogenous

substrate,

even when the recessed 3' ends are preformed.

Theseresultssuggest that themutants wehave

generated

are

defectivefor both steps of the

integration

process.

ACKNOWLEDGMENTS

Thisworkwassupported byPublic Health ServicegrantCA30488

from the National Cancer Institute.M.J.Risaspecialfellow of the LeukemiaSocietyofAmerica, Inc.

LITERATURE CITED

1. Berg,J.M.1990.Zincfingersand othermetal-bindingdomains.

Elements for interactions between macromolecules. J. Biol. Chem. 265:6513-6516.

2. Bowen, B., J. Steinberg, U. K. Laemmli, and H. Weintraub.

1980. The detection ofDNA-binding proteins by protein

blot-ting. NucleicAcids Res. 8:1-20.

3. Bowerman,B.,P. 0.Brown, J. M.Bishop, and H. E. Varmus.

1989. A nucleoprotein complex mediates the integration of retroviralDNA. Genes Dev. 3:469-478.

4. Brown,P.O., B.Bowerman,H. E. Varmus,andJ.M.Bishop.

1987. Correct integration of retroviral DNA in vitro. Cell

49:347-356.

5. Brown, P. O.,B.Bowerman,H. E. Varmus,andJ.M.Bishop.

1989. Retroviral integration: structure of the initial covalent

productandits precursor, andarolefor the viral IN protein.

Proc. Natl. Acad. Sci. USA 86:2525-2529.

6. Chen, M., and M. S. Horwitz. 1989. Dissection of functional

domains of adenovirus DNA polymerase by linker-insertion

mutagenesis.Proc. Natl.Acad. Sci. USA86:6116-6120.

7. Cobrinik, D.,R.Katz,R.Terry,A. M.Skalka,andJ.Leis.1987.

Aviansarcomaand leukosisviruspol-endonuclease recognition

of the tandem long terminal repeatjunction: minimum site

requiredforcleavageisalsorequiredfor viralgrowth.J.Virol.

61:1999-2008.

8. Colicelli,J.,andS. P. Goff.1985. Mutantsandpseudorevertants

of Moloney murine leukemia virus with alterations at the

integrationsite. Cell 42:573-580.

9. Dhar, R., W. McClements, L. Enquist, andG. Vande Woude.

1980. Nucleotide sequences of integrated Moloney sarcoma

provirus longterminalrepeatsandtheirhost and viraljunctions.

Proc. Natl. Acad. Sci. USA77:3937-3941.

10. Donehower, L. A. 1988. Analysis ofmutant Moloney murine

leukemiavirusescontaininglinker insertion mutationsin the 3'

regionofpol. J. Virol. 62:3958-3964.

11. Donehower,L. A., and H. E. Varmus. 1984. A mutantmurine

leukemiavirus withasinglemissense codon inpol is defective

inafunctionaffecting integration. Proc. Natl. Acad. Sci. USA

81:6461-6465.

12. Duyk, G., J. Leis, M. Longiaru, and A. M. Skalka. 1983.

Selective cleavage inthe avian retrovirallong terminal repeat sequence by the endonuclease associated with the alpha-beta

form of avianreversetranscriptase.Proc. Natl. Acad.Sci.USA

80:6745-6749.

13. Duyk, G., M. Longiaru, D. Cobrinik, R.Kowal, P. D.Haseth,

A.M.Skalka, andJ.Leis. 1985. Circleswithtwotandemlong

terminalrepeatsarespecifically cleavedby pol gene-associated endonuclease fromavian sarcomaandleukosis viruses:

nucle-otide sequences required for site-specific cleavage. J. Virol.

56:586-599.

14. Eisenman, R.N.,W. S.Mason,and M. Linial. 1980. Synthesis andprocessing of polymerase proteinsofwild-typeand mutant avianretroviruses. J. Virol. 36:62-78.

15. Fujiwara,T., and R. Craigie.1989.Integration ofmini-retroviral DNA: acell-free reaction for biochemicalanalysis of retroviral integration.Proc. Natl. Acad. Sci. USA 86:3065-3069.

16. Fujiwara, T., and K. Mizuuchi. 1988. RetroviralDNA integra-tion:structureofanintegration intermediate. Cell54:497-504. 17. Goff,S. P., P.Traktman, and D. Baltimore.1981. Isolation and

properties of Moloney murine leukemia virusmutants:useofa

rapidassayfor release of virionreversetranscriptase. J. Virol. 38:239-248.

18. Golomb, M., and D. P. Grandgenett. 1979. Endonuclease

activ-ity of purified RNA-directed DNA polymerase from avian myeloblastosisvirus. J. Biol. Chem. 254:1606-1613.

19. Golomb, M., D. P. Grandgenett, and W. Mason. 1981.

Virus-coded DNA endonuclease from avian retrovirus. J. Virol.

38:548-555.

20. Grandgenett, D. P., M. Golomb, and A. C. Vora. 1980.

Activa-tionofan

Mg2"-dependent

DNAendonuclease of avian myelo-blastosis virusalpha-betaDNApolymerase by in vitro

proteo-lytic cleavage. J.Virol. 33:264-271.

21. Grandgenett, D. P., and S. R. Mumm. 1990. Unraveling

retro-virusintegration. Cell60:3-4.

22. Grandgenett, D. P., A. C. Vora, and R. D. Schiff. 1978. A

32,000-dalton nucleic acid-binding protein from avianretrovirus corespossesses DNAendonucleaseactivity. Virology

89:119-132.

23. Grandgenett,D.P.,A.C.Vora,R.Swanstrom,andJ.C. Olsen.

1986. Nuclease mechanism of the avian retrovirus pp32 endo-nuclease. J.Virol. 58:970-974.

24. Hughes, S. H., A.Mutschler,J. M. Bishop, and H. E. Varmus.

1981.A Rous sarcomavirusprovirus is flanked by short direct

repeats ofacellularDNAsequence presentin onlyonecopy

priortointegration. Proc. Natl.Acad. Sci. USA 78:4299-4303.

25. Hughes, S. H., P. R. Shank,D. H. Spector,H.-J. Kung,J. M. Bishop, H. E. Varmus, P. K. Vogt, and M. L. Breitman. 1978.

Proviruses of avian sarcoma virus are terminally redundant,

co-extensive with unintegrated linear DNA, and integratedat many sites. Cell15:1397-1410.

26. Johnson, M.S.,M.A.McClure,D.-F.Feng, J. Gray, and R. F. Doolittle. 1986. Computer analysis of retroviral pol genes:

assignment ofenzymatic functions to specific sequences and

homologies with nonviral sequences. Proc. Natl. Acad. Sci.

USA 83:7648-7652.

27. Katzman,M., R. A. Katz, A. M.Skalka,andJ. Leis. 1989. The

avian retroviral integration protein cleaves the terminal se-quencesof linear viralDNA at theinvivo sites ofintegration.J.

Virol.63:5319-5327.

28. Kopchick, J. J., G. A. Jamjoom, K. F. Watson, and R. B. Arlinghaus. 1978. Biosynthesis ofreverse transcriptase from

Rauschermurine leukemia virusbysynthesis and cleavageof a

gag-pol readthrough viral precursor polyprotein. Proc. Natl. Acad. Sci. USA75:2016-2020.

29. Kopchick, J. J.,W. L. Karshin, and R. B. Arlinghaus. 1979. Tryptic peptide analysis ofgagand gag-polgene products of

Rauscher murine leukemiavirus. J. Virol. 30:610-623. 30. Lacks, S. A., and S. S. Springhorn. 1980. Renaturation of

enzymes afterpolyacrylamide gel electrophoresis in the pres-enceof sodiumdodecylsulphate.J.Biol. Chem. 255:7467-7471.

31. Lazo,P.A.,V.Prasad,and P. N. Tsichlis.1987.Spliceacceptor

sitefor theenvmessageofMoloneymurine leukemia virus.J. Virol. 61:2038-2041.

32. Leis,J., G. Duyk, S. Johnson, M. Longiaru, and A. M. Skalka.

1983. Mechanism of actionoftheendonucleaseassociatedwith thealpha-beta and beta-beta forms of avianRNA tumorvirus

reversetranscriptase.J. Virol.45:727-739.

33. Lobel, L. I., andS.P. Goff. 1984. Construction of mutants of

Moloneymurineleukemia virusby suppressor-linker insertional mutagenesis: positionsof viableinsertion mutations.Proc.Natl. Acad. Sci. USA 81:4149-4153.

34. Luc, K.-C., T. D. Gilmore, and A. T. Panganiban. 1987. The

on November 10, 2019 by guest

http://jvi.asm.org/

(9)

spleen necrosis virusint geneproduct expressed in E.colihas DNA binding activity and mediates att and U5-specific DNA multimerformation in vitro. Virology 157:127-136.

35. Lyman, S. D., and L. R. Rohrschneider. 1987. Analysis of

functional domains of the v-fms-encoded protein of Susan McDonough strain feline sarcoma virus by linker insertion mutagenesis. Mol. Cell. Biol. 7:3287-3296.

36. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labeled DNA withbase-specificchemicalcleavages. Methods Enzymol.

65:499-560.

37. McCutchan, J. H., and J. S. Pagano.1968.Enhancement of the

infectivityof simian virus 40 deoxyribonucleic acid with

dieth-ylaminoethyl-dextran. J. Natl.CancerInst. 41:351-357.

38. Miskimins, W. K., M. P. Roberts, A. McCleUland, and F. H. Ruddle.1985.Useofaprotein-blottingprocedure and a specific

DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene. Proc. Natl.

Acad. Sci. USA 82:6741-6744.

39. Misra, T. K., D. P. Grandgenett, and J. T. Parsons. 1982. Avian

retroviruspp32 DNA-bindingprotein.I. Recognitionof specific sequences onretrovirusDNAterminalrepeats. J.Virol. 44:330-343.

40. Panganiban, A. T. 1985. Retroviral DNA integration. Cell 42:5-6.

41. Panganiban, A. T., and H. M. Temin. 1984. The retroviruspol geneencodes aproduct required forDNAintegration: identifi-cation ofaretroviral intlocus. Proc. Natl. Acad. Sci. USA 81:7885-7889.

42. Parker, R. C., R. M. Watson, and J. Vinograd. 1977.Mapping of

cloned circular DNAs by cleavage with restriction endonu-cleases and calibration by agarose gel electrophoresis. Proc. Natl.Acad.Sci. USA74:851-855.

43. Peden, K. W. C., and D. Nathans. 1982. Local mutagenesis

within deletion loops of DNA heteroduplexes. Proc. Natl. Acad. Sci. USA 79:7214-7217.

44. Quinn, T. P., and D. P. Grandgenett. 1988. Genetic evidence

that the avian retrovirus DNA endonuclease domain ofpolis

necessaryfor viralintegration.J. Virol.62:2307-2312. 45. Roth, M. J., P. Schwartzberg, and S. P. Goff. 1989.Structure of

the termini ofDNAintermediates intheintegrationof retroviral DNA:dependenceonINfunction and terminalDNA sequence.

Cell58:47-54.

46. Roth, M.J., N. Tanese, and S. P. Goff. 1988.Gene product of Moloney murine leukemia virus required forproviral integration isaDNA-binding protein. J. Mol.Biol.203:131-139.

47. Rowe, W. P., W. E. Pugh, and J. W.Hartley.1970.Plaqueassay

techniques for murine leukemia viruses.Virology42:1136-1139. 48. Schiff,R.D., and D. P.Grandgenett.1978.Virus-encodedorigin ofa32,000-dalton protein from avian retroviruscores:structural relatedness ofp32 and thePpolypeptideof the avian retrovirus DNApolymerase.J. Virol. 28:279-291.

49. Schwartzberg, P., J. Colicelli, and S. P. Goff. 1984.Construction

and analysisof deletion mutations in the pol gene of Moloney murine leukemia virus: a new viral function required for pro-ductive infection. Cell 37:1043-1052.

50. Shimotohno, K., S. Mizutani, and H. M. Temin. 1980. Sequence of retrovirus provirusresembles thatof bacterial transposable

elements. Nature (London)285:550-554.

51. Shoemaker, C., S. Goff, E.Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore. 1980. Structure of a cloned circular Moloney

murine leukemia virus DNA molecule containing aninverted segment: implications for retrovirus integration. Proc. Natl. Acad. Sci. USA 77:3932-3936.

52. Shoemaker, C., J. Hoffman, S. P. Goff, and D. Baltimore. 1981. Intramolecular integration within Moloney murine leukemia virus DNA. J. Virol. 40:164-172.

53. Skalka, A. M. 1989. Integrative recombination of retroviral DNA, p. 701-724. In R. S. Kucherlapati and G. R. Smith (ed.), Genetic recombination, American Society for Microbiology,

Washington, D.C.

54. Southern,P.J., and P. Berg. 1982.Transformationof mamma-lian cells to antibiotic resistance with a bacterial gene under control of the SV40early regionpromoter. J.Mol.Appl. Genet. 1:327-341.

55. Swanstrom,R., W. J.DeLorbe, J.M.Bishop,and H. E. Varmus. 1981. Nucleotide sequence of cloned unintegrated avian sar-coma virus DNA: viral DNA contains direct and inverted repeats similar to those in transposable elements. Proc. Natl. Acad. Sci. USA 78:124-128.

56. Tanese, N., and S. P. Goff. 1988. Domain structure of the

Moloney murine leukemia virus reverse transcriptase: muta-tionalanalysisand separateexpressionof the DNApolymerase and RNase H activities. Proc. Natl. Acad. Sci. USA 85:1777-1781.

57. Tanese, N., M. J. Roth, and S. P. Goff. 1985. Expression of

enzymatically active reversetranscriptase inEscherichia coli. Proc. Natl. Acad. Sci. USA 82:4944 4948.

58. Tanese, N., M. J. Roth, and S. P. Goff. 1986. Analysis of retroviralpolgeneproducts with antisera raisedagainstfusion

proteins producedin Escherichiacoli. J.Virol. 59:328-340. 59. Terry, R., D. A.Soltis,M.Katzman,D.Cobrinik, J. Leis,and

A.M.Skalka. 1988. Propertiesof avian sarcoma-leukosis virus

pp32-related pol-endonucleasesproducedin Escherichiacoli.J.

Virol.62:2358-2365.

60. Vogelstein, B.,and D.Gillespie.1979.Preparativeandanalytical purificationof DNAfromagarose.Proc. Natl.Acad.Sci.USA

76:615-619.

61. Wigler, M.,R.Sweet,G. K.Sim,B.Wold,A.Pellicer,E.Lacy, T.Maniatis, S.Silverstein,and R. Axel.1979. Transformationof mammalian cellswithgenesfromprocaryotes andeucaryotes.

Cell16:777-785. VOL. 64, 1990