Simple affinity procedure for the purification of mammalian viral reverse transcriptases.

0022-538X/80/08-0555/05$02.00/0

Simple Affinity Procedure for the Purification of Mammalian

Viral Reverse Transcriptases

M. G.SARNGADHARAN,' V. S. KALYANARAMAN,' R. RAHMAN,' ANDR. C. GALLO2*

Department of Cell Biology, LittonBionetics, Inc., Kensington,Maryland 20795,' and Laboratory of Tumor CellBiology,NationalCancer Institute,National Institutes of Health,Bethesda,Maryland202052

Polyguanylic acid was found tobe a potentinhibitor of RNase H associated with mammalian viral reverse transcriptase, indicating a strong interaction

be-tween polyguanylic acid and the reverse transcriptase protein. Based on this observation, wehave developed three simple procedures for the purification of mammalian viralreversetranscriptases. In the first procedure,anucleic acid-free extractofRauschermurine leukemiaviruswasappliedtoa column of phospho-cellulose and thereversetranscriptasewaselutedbyalow concentration (50,M) ofpolyguanylic acid. Polyadenylic acid and polyuridylic acid could not replace

polyguanylic acid forthe elution. Inthe second procedure, a polyuridylic

acid-Sepharose column was substituted for phosphocellulose, and the elution was again achieved by polyguanylic acid. In the third affinity procedure, thereverse transcriptase inanucleicacid-free viralextractwasincubated in the cold with 50 ,uM polyguanylic acid and the complex was adsorbed onto a DEAE-cellulose column. Afterwashing toremoveuncomplexed andweakly complexed proteins, thereversetranscriptasewas eluted inaconcentrated form at0.3 MNaCl with arecoveryofgreaterthan70%.Bypolyacrylamide gelanalysisin thepresenceof sodiumdodecylsulfate, theenzymeappearedtobenearlypure.

DNApolymerasesareknowntohave affinities

toanionic

polymers

(2), such as

phosphocellu-loseand

carboxymethylcellulose,

andtherefore

also interact with variable affinities withmost

nucleic acids becauseofthepolyanionicnature

of the latter. This is in addition to

possible

specific affinitiesbetween

polymerases

and

par-ticular nucleic acids. Several

chromatographic

procedures described for the purification of

DNApolymerases utilize their

affinity

to

phos-phocellulose or to one of several matrix-bound

nucleic acidsto adsorb theseenzymes (7). The

enzymesarerecovered fromthese matrices

by

a

relatively

nonspecific

stepofelutionwithasalt

solution. Most of the efforts to

improve

the

chromatographic

procedures

for the

purification

of DNA

polymerases

havebeen directedat

iden-tifying

affinity

matrices that show

adsorption

specificitytowards one

particular

DNA

polym-erase as

compared

withanotherormatrices that

showwidely varying affinities toward different

DNApolymerases.

An alternative approach to enzyme

purifica-tion in

general

istouse a

specific

effector mole-cule with strong affinity to elute the enzyme from anion-exchangeorsimilaradsorption ma-trix. Substrates, inhibitors, and other effectors bind toenzyme molecules and

produce

substan-tial changes in protein conformation

(often

changing proteinadsorption characteristics

to-ward the chromatographic media), resultingin

elution (5, 8). We examined whether such an

approach was possible for the purification of

mammalianviral reversetranscriptases.

Polyguanylic acid[poly(G)] wasshown to be

a potent inhibitor ofthe RNase H

activity

as-sociated with the reversetranscriptase molecule

ofRauscher murine leukemia virus (R-MuLV)

and simian sarcoma virus (6). In comparison,

polyadenylic acid [poly(A)], polyuridylic acid

[poly(U)], and polycytidylicacid were only

min-imally active or not at all active against this

enzyme (6). Our interpretation of this finding

hasbeenthatthe reverse transcriptase molecule

hasa very strong and possibly specific interac-tion with poly(G). We report here that this

interactionwith poly(G) has proved to be very

useful in developing simpleaffinity procedures

for thepurification ofmammalian viral reverse

transcriptases.

A nucleic acid-free extract of R-MuLV was

prepared from 8 x 1011 virusparticles (2.7 mg)

andappliedto a5-ml column of

phosphocellu-lose. After washing with 0.1 M NaCl until no

moreproteinseluted,thecolumnwas

developed

with 20 ml of 50,uM poly(G) (minimum size,

6S;

Miles Laboratories) in the presence of 0.1 M NaCl plus 0.5 mM

MnCl2.

The eluates were collected in 1-ml fractions and

assayed

for

re-versetranscriptaseactivitywith

(dT)_15. (A).

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556 NOTES

the

primer-template. Any

residualreverse

tran-scriptase

activity

on the

phosphocellulose

col-umn wasrecoveredbyastepelution with 0.5 M

NaCl[withnoadded

poly(G)].

Figure

1A shows

that the

poly(G)

eluate

yielded only

a small amountofenzyme

activity,

and yetnotmuch of theenzyme

activity

remainedonthe

phospho-cellulose column to be eluted

by

the high-salt

wash.Thereasonfor thisapparentlowrecovery

wasthe fact that

poly(G)

has a strong

affinity

for thereverse

transcriptase

protein and that it

inhibits thereverse

transcriptase

activity (9)

as

it inhibitsthe RNaseH

activity (6).

Tomeasure

the true reverse

transcriptase

activity

in the

poly(G)

eluates,

the

poly(G)

hadtoberemoved

from these fractions. The

poly(G)-eluted

frac-tionswere

pooled

andloadedonto a1-mlcolumn

of DEAE-cellulose. All the enzyme

activity

bound to the

column,

confirming

that the

en-zymethatwaseluted from

phosphocellulose

did

not exist as free enzyme

molecules,

but was

complexed with

poly(G).

It is known that free

reverse

transcriptase

does notbind to

DEAE-celluloseatthe salt concentration

(0.1

M

NaCl)

which was present in the

poly(G)

eluates

(4).

The reverse

transcriptase

was

selectively

re-covered fromDEAE-cellulose

by

abatch elution

with 0.3 M NaCl which dissolved the

poly(G)-reverse

transcriptase

complex,

butdidnotelute the

poly(G). Figure

1B shows that theenzyme

appearedas a

sharp peak

inaconcentrated form

representing an increase in

activity

of

several-fold compared to the

poly(G)-containing

frac-tions

(Fig. 1A).

Theseseries of

experiments

dem-onstrated that

poly(G)

elution removedmostof

the reverse

transcriptase

activity

bound to a

phosphocellulose column. There was only a

small amountof

activity remaining

onthe

col-umn after the

poly(G)

elution that could be

recovered

by

a

high-salt

wash

(Fig.

1A).

The elution of reverse transcriptase from

phosphocellulose involvedaspecific interaction

between theenzymeandpoly(G), and this

inter-actionwasstrongenoughtooffset theaffinity of

theenzymefor

phosphocellulose.

The

substitu-tion of eitherpoly(A) orpoly(U) forpoly(G)as an eluant did not elute reverse transcriptase

fromphosphocellulose;

furthermore,

noactivity

wasfound in theeluatesevenafterthe

polynu-cleotides wereremoved by subsequent

DEAE-cellulose

chromatography(Fig.1C,D, E,and F). Allenzymeactivity could be recovered, however,

by subsequent elutionwith 0.5 MNaCl(Fig.1C

andE),orby50,Mpoly(G) (datanotshown).

Since reverse transcriptases bind to

matrix-attached nucleic acids in general, and in fact,

poly(U)-Sepharoseis acommonlyusedaffinity

adsorbent for reversetranscriptase,theinability

ofpoly(U) and poly(A) to elute

phosphocellu-v

I

I

I

FRACTnONVUSER

FIG. 1. Elution of R-MuLV reverse transcriptase fromphosphocellulose bypolynucleotides. Three 4-ml samplesof nucleic acid-freeextractofR-MuL V, pre-paredasdescribed elsewhere (M. Robert-Guroff, V. S.Kalyanaraman, and M.G.Sarngadharan, Int. J. Cancer,inpress) from8x1011 virusparticles(2.7mg ofprotein),weredialyzedagainst50mM Tris-hydro-chloride, pH 8, containing1 mMdithiothreitol, 20% glycerol,0.02mMphenylmethylsulfonylfluoride, and 0.05% Triton X-100(buffer A) and applied to three 5-ml columns ofphosphocellulose equilibrated with bufferA.Afterthecolumnswerewashed withbuffer

A containing0.1M NaCl until no additional UV-absorbing materialwaseluted (25 to 30ml), they were developed with20mlofa 50

,uM

solution ofpoly(G) (A),poly(A) (C), or poly(U) (E) in buffer A containing

0.1 MNaCl and0.5mMMnCl2. Subsequently, the columns were washedfurther with 15 ml of 0.5 M NaClin bufferA to recover anyremaining reverse transcriptase activity. Aliquots of10ulfrom the1-ml fractions collected were assayed for reverse

transcrip-taseactivity (Robert-Guroff et al.,inpress). The

frac-tions frompolynucleotide elutions were separately pooledandapplied to1-micolumns of DEAE-cellu-lose equilibrated with Tris-hydrochloride, pH 7.9, containing 1 mM dithiothreitol, 20% glycerol, 0.02 mMphenylmethylsulfonyl fluoride, and 0.05% Triton

X-100 (buffer B), and any enzyme in the pools re-coveredfreeof thenucleotides in a concentrated form by elution with0.3 MNaCl in buffer B (B, D, and F). Theprimer-template used for reverse transcriptase assaywas(dT)_15.(A)n in all cases, except in fractions containing poly(U), in which case (dG)

(QC),

was

theprimer-template.

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lose-bound reverse transcriptase indicated that

the affinity ofreverse transcriptase forpoly(A)

and poly(U) was weaker than its affinity for

phosphocellulose, and much weaker than the

affinity for poly(G). One would predict,

there-fore,that poly(G)should elute reverse transcrip-tase bound topoly(U)-Sepharose.To verifythis

prediction, a nucleic acid-free extract of

R-MuLV wasappliedtoa 5-ml column of

poly(U)-Sepharose (Pharmacia Fine Chemicals, Inc.).

After the column was washed with buffer

sup-plemented with0.2 MNaCl until no more

pro-teins eluted, it was developed with 50 yM

poly(G) in the wash buffer. Fractions of 1 ml

werecollected, andthe reverse transcriptase

ac-tivitiesweredetermined(Fig. 2A). Atremendous

increase in enzyme activity was observed once

again when poly(G) was removed from the

eluted reverse transcriptase fractions by

chro-matographyon aDEAE-cellulose column(Fig.

2B). After thepoly(G) elution, the residual

re-verse transcriptase activity on poly(U)-Sepha-rose wasrecovered byasubsequent0.7 MNaCl

wash (Fig. 2C). A comparison of the results

showninFig.2B andConce again indicatesthe

effectiveness of

poly(G)

to elute matrix-bound

reversetranscriptase.

In theseinstances, thereversetranscriptaseis

adsorbed

(along

with otherproteins)onto asolid

matrix[phosphocelluloseorpoly(U)-Sepharose]

and elutedwithadilute solution ofpoly(G).Two

levelsofspecificityareinvolvedinthese

proce-dures, the first at the adsorption step and the

second attheelution step, and therefore these

procedures haveadvantagesoverthose

employ-inga

nonspecific

salt elutionsteptorelease the

enzymefrom these adsorptionmatrices. Useof

substrates, inhibitors, and other effectors to

elute enzymes from

adsorption

matrices is

known

generally

toresultinsubstantialenzyme

purification (5, 8)because of the

high

specificity

involved.

Thebasis of theprocedures described above

was the formation of a

high

affinity complex

between reverse

transcriptase

and

poly(G)

whosecharacteristicswere

significantly

different from the

properties

of the free enzyme. The

complex

hadalower

affinity

to

phosphocellulose

and

poly(U)-Sepharose

anda

higher affinity

to

DEAE-cellulose than didthefreeenzyme.Since

poly(G) didnotelute otherproteinstoany

sig-nificantdegreefrom

phosphocellulose,

as

judged

from the protein

profile

when a

sample

was

analyzed by

sodium

dodecyl

sulfate-polyacryl-amide

gel

electrophoresis (data

not

shown),

it

was reasonabletoassume that the property of

forming this

complex

with

poly(G)

was

some-what specific to reverse

transcriptase.

On the

basis of this

rationale,

we

attempted

the

follow-24'

'

21

0

20' DEAE-C.lluloo

Chromatography

Is

C15

00

w

C O.7MNaCIglutton

from

Poly(U)-gaos

5 10 15 20

FRACTION NUMBER

FIG. 2. Elution of R-MuLVreverse transcriptase bypoly(G)fromapoly(U)-Sepharose column. A

nu-cleicacid-free extractfrom 7x 1011particlesof R-MuLV(2.5 mgofprotein)wasdialyzed againstbuffer C(bufferBplus1mMMnCI)andappliedto a5-ml

columnofpoly(U)-Sepharoseequilibratedwithbuffer C.After washing with 30mlofbuffer Ccontaining

0.2MNaCl,thecolumnwasdevelopedwith30mlof

50 LMpoly(G) inbufferCcontaining0.2MNaCl(A),

and the eluates were collected into 1-mlfractions. Thesefractions werepooledand chromatographed

onDEAE-cellulose (B) to removethepoly(G) as de-scribed in Fig. 1. After the poly(G) elution, the poly(U)-Sepharose column was washed with 0.7M

NaCl in buffer Cto remove the residualactivity of reversetranscriptase(C).

ing

simplified

scheme topurifyreverse

transcrip-tase from R-MuLV. Anucleic acid-free extract of R-MuLV(10mg of

protein)

wasincubated in ice for 5 min with 50,M poly(G) and 0.5 mM

MnCl2

and then applied to a 1-ml column of

DEAE-cellulose.

Thecolumnwaswashed

exten-sivelywith0.1MNaClto removeunbound and

weaklyboundmaterials. Under these

conditions,

all thereversetranscriptasemolecules existedas

acomplexwithpoly(G)andtherefore remained

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558 NOTES

boundtotheDEAE-cellulose column. When the NaCl concentration in the bufferwasraised to 0.3 M, the complex betweenreverse

transcrip-tase and poly(G) was broken and the reverse

transcriptase emerged from the column as a sharp activitypeak, leaving poly(G)still bound tothe column (Fig. 3). The recovery of enzyme

activity fromthe columnwasgreater than 70%.

When

'25I-labeled

envelope glycoprotein

(gp7O)

orthemajorstructuralprotein (p30)of R-MuLV

wasmixedwithpoly(G)andappliedto a

DEAE-cellulosecolumn under the conditions described

above,therewas noretention of the radiolabeled

protein on the column, indicating that these

proteins did not form stable

complexes

with

poly(G). Further, when

'25I-labeled

gp7O of

R-MuLVwasmixed with anucleic acid-free virus extract and the mixture was incubated with

poly(G) and

chromatographed

on

DEAE-cellu-lose, thelabeled gp7Owasrecovered in the

un-retardedflow-throughfractions and thereverse

transcriptasewasadsorbedtothe column (data

notshown).

The simple method outlined above for the

purificationofreverse

transcriptase

is

essentially

asingle-step

procedure,

and therefore is fast and

convenient. It was of interesttodetermine the

purityof the enzyme obtained

by

this

procedure.

Forthis purpose, the

purified

reverse

transcrip-tasefrom theDEAE-cellulosestepwas

radiola-u I-4

U)

4

I-U) U

z 4E

uL

LU LU

cc

FIG. 3. Chroma of poly(G) and R DEAE-cellulose.A NaClfrom 3.5x10 threefoldwithbuff

with50 4Mpoly(G, wasthenapplied t< -equilibrated with

with buffer B con transcriptasewas taining 0.3M Na( lected and sample scriptase activity plate.

beled with 125I usingthe chloramine-T method (1), and the labeled protein was analyzed by

electrophoresis on a polyacrylamide gel in the presence of sodium dodecyl sulfate. The radio-activityprofile obtained on thegelis shown in

Fig. 4. The enzyme moleculemigrated withan apparent molecularweight of about 70,000 and

was substantially free ofother contaminating

proteins.

Theprocedure describedabove,therefore, in-volves the selectivecomplexingofreverse

tran-scriptase in a crude viral extract with poly(G)

and capturing thecomplex, and only the

com-plex,onDEAE-cellulose (Fig. 3). Since thefree

viral proteins, includingfree reverse transcrip-tase, donotbindtoDEAE-celluloseatthesalt

concentrations employed, the retention of

re-verse transcriptase is solely dependent on the

E

GELSLICENUMBER

E 0.1 M NaCI FIG. 4. Electrophoreticprofile ofiodinatedreverse transcriptase on asodiumdodecyl sulfate-polyacryl-amide

gel.

A

sample of

the reverse transcriptase, 0.3 M NaCI purifiedasdescribedinFig. 3, was labeled with125i

by themethodof Greenwoodetal. (1). A 100-,ulportion

of

thereaction mixture contained0.5to2,ug

of

the 5 15 35 40 45 enzyme,50 mM sodiumphosphate (pH7.5),30,ug of

chloramine-T,250mM

NaCl,

0.1%TritonX-100,and

VOLUME(MI) 0.5mCiofNa'25. After1 min at room temperature, ,tography of a preformedcomplex the reaction was terminated by the addition of 50 ,ig

t-MuLV reverse transcriptase on of sodium metabisulfite and 20

til

of5MNaCl. The Inucleic acid-free extract in 0.3 M iodinatedprotein was thenseparated from unincor-12particlesofR-MuLVwas diluted porated 1251 on a BioGelP-10column equilibrated 'er B and incubated in ice for 5min with1MNaCl, 10 mM sodiumphosphate buffer (pH )and 0.5 mMMnCl2.Thecomplex 7.5), 10%, glycerol, and 0.2 mM phenylmethylsulfonyl

oa1-mlcolumn ofDEAE-cellulose fluoride. The labeled protein was subjected to

electro-bufferB. After extensive washing phoresis on aI

010

polyacrylamide gel in the presence itaining 0.1 MNaCl, the reverse of sodium dodecyl sulfate bythe method ofLaemmli

eluted with 10 ml of buffer B con- (3). The gel was divided into1-mmslices, and their Cl. Fractions of 0.65 ml were col- radioactivity was determined in a gamma counter. es were assayed for reverse tran- The standard molecular weight markers run in par-with (dT)-15 '(A), as primer-tem- ellel gels were: P, phosphorylase; B, bovine serum

albumin; 0,ovalbumin; and C, chrymotrypsinogen.

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complex formation. For thisreason, the size of

the DEAE-cellulose colunm is

independent

of

theamount of viral

proteins

in the extract,but

isdeterminedbythe amountof

poly(G)

present.

Routinely,aslittleas 1ml ofDEAE-cellulose is

enoughtoretain50mlof50,uMpoly(G),making

thisprocedure

extremely

attractivefor

achieving

atremendous concentrationofreverse

transcrip-taseduring this step. Unlike mostother

proce-dures,thisprocedure

effectively

eliminates the

needtohandlereversetranscriptaseatlow

pro-tein

concentrations,

because the

only

steps

in-volved are (i) the

preparation

ofnucleic

acid-freeextractsfrom virus concentrates, whichdoes

not expose reverse transcriptasetolowprotein

concentrations,

and

(ii)

the

chromatography

of the

complex

of reverse

transcriptase-poly(G),

which yields the freeenzymeinaconcentrated

form.

LITERATURE CITED

1. Greenwood,F.C.,W. M.Hunter,and J. S. Glover. 1963. The preparation of"1'I-labeledgrowthhormone

ofhigh specific activity. Biochem. J. 89:114-123. 2. Kacian, D. I., K. F. Watson, A. Burny, and S.

Spie-gelman.1971.Purificationof the DNA polymerase of avian myeloblastosis virus. Biochim. Biophys. Acta 246:365-383.

3. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685.

4. Lewis, B.J., J. W.Abrell, R. G. Smith, and R. C. Gallo. 1974. DNA polymerases in human lymphoblastic cellsinfected with simian sarcoma virus. Biochim. Bio-phys. Acta349:148-160.

5. Pogell, B. M., and M. G.Srngadrharan.1971. Specific elution with substrate. MethodsEnzymol. 22:379-385. 6. Sarngadharan, M. G., V. S. Kalyanaraman, and R.

C.Gabo. 1978. Inhibition by RNA of RNase H activity associated with reverse transcriptase inRauscher mu-rineleukemia virus cores. J.Virol. 27:568-575. 7. Sarngadharan, M. G., M.Robert-Guroff, and R.C.

Gallo.1978.DNApolymerasesof normal and neoplas-ticmammalian cells. Biochim.Biophys. Acta 516:419-487.

8. Sarngadharan,M. G.,A. Watanabe, and B. M. Pogell. 1970.Purification of rabbit liver fructose 1,6-diphospha-taseby substrate elution. J. Biol. Chem. 245:1926-1929. 9. Waters, L C., and W.-K. Yang. 1974. Comparative biochemical propertiesof RNA-directed DNA polym-erases fromRauscher murineleukemia virus and avian myeloblastosisvirus. Cancer Res. 34:2585-2593.

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