Activity of avian retroviral protease expressed in Escherichia coli.

0022-538X/88/082696-05$02.00/0

Copyright © 1988, AmericanSocietyforMicrobiology

Activity of

Avian Retroviral Protease

Expressed

in Escherichia

coli

MOSHE KOTLER,t RICHARD A. KATZ, ANDANNA MARIE SKALKA*

Institute for CancerResearch, Fox Chase Cancer Center, 7701 BurholmeAvenue, Philadelphia, Pennsylvania 19111

Received5January 1988/Accepted22April1988

The3' end of the aviansarcomaleukosis virus (ASLV) gag geneencodes a 124-amino-acid protease (PR)

responsibleforprocessingthegag andpolpolyproteinprecursorsinto the mature virion structuralproteinsand the reverse transcriptase. Herewereportthesynthesisof the mature ASLV PR and anucleocapsid(NC)-PR gag precursorfragmentinEscherichia coli.E. coli extractscontainingmature PRcorrectlycleaved asynthetic decapeptide homologoustoaknown ASLVcleavagesite.Also,the NC-PRprecursorfragment appearedtobe correctly processed to produceNC and PR in the bacterial cells. Thiscleavagewasblocked byamutationin the putative active site of PR. These resultsstronglysupportthehypothesisthat PR is involved incleavingitself from the gag precursor.

The retroviralgag,pol, and env genes encode precursor

polypeptides, which are cleaved to produce the mature

forms oftheproteins found in virions (6).The

processing

of gag and pol precursors is directed by a virus-encoded

protease(PR) (7, 8, 18, 26-28) thatbelongs tothe

family

of

aspartic proteases (20, 22, 23). (We have used the recently

proposed nomenclature for retroviral proteins [14]: PR

[aviansarcomaleukosis virus {ASLV}p15]andnucleocapsid protein [NC] [ASLV ppl2].) The PR-directed processing presumably occursjust before or after budding (6). PR is

essential forinfectivity, although viruses thatcontain muta-tionsin PR areabletoassemble intomorphologically imma-ture particles (4, 8, 9, 16, 29, 30).

Unlike the mammalian retroviruses in which PR is

en-coded in the pol gene and thus contained in the gag-pol

precursor (4, 13, 15), the PR ofASLV is encoded in gag.

Therefore, the PRdomain is contained inboth the C

termi-nusof thePr769'9precursor andthePr18Vag-p precursor

(6). Forboth avianandmammalianretroviruses, mature PR

isreleasedfromprecursorproteins by proteolysisduringthe

morphogenesis ofviralparticles. Since 20 to 50 times more

of thegag thanthegag-pol precursor isproduced,the bulk of mature ASLV PRis formedas aconsequenceofcleavage

betweenPRandtheadjacentNC(ppl2), locatedupstream in

thegag precursor (21). In contrast, release of PR in

mam-malian viruses, such asMoloneymurine leukemia virus and

humanimmunodeficiencyvirus,requires cleavage at both N

andC termini ofthe PR in thegag-polprecursor (13, 15). It is not known exactly how PR is released from these precursors. Atleasttwomechanisms are possible. (i)

Cellu-lar proteases may be

responsible

for the initiation of the process, resulting in the release of a few mature PR

mole-cules. The releasedPRmightthen complete the processing,

producing more PR and the other mature viral proteins as well.

(ii)

PR might cleave itself from the precursor via either aninter-orintramolecularmechanism. Similar mechanisms ofactivationareknown for a number of aspartic

proteases,

in which an inactive zymogen is converted into an active enzyme by autocatalysis. Oneexample is the autocleavage ofpepsinogen to pepsin (22).

Inthisreport, we describe the expression in Escherichia

coli of the predicted ASLV PR-coding region. A protein

*Correspondingauthor.

tPermanent address: Department of Molecular Genetics, the HebrewUniversity, Hadassah Medical School, Jerusalem, Israel.

structurally andimmunologically indistinguishablefrom PR was produced, and a proteolytic activity analogous to the viral PR was detected in E. coli extracts. These results confirm the location of the ASLV PR-coding region. In

addition, we show that anNC-PR gag fragment

undergoes

processingin E. coli, resultingin theformation of amature PRthat appears tobe identical tovirion PR. Mutation ofa highly conservedaspartic acid residue that is thought to be partof thePR active site eliminates proteolytic activity. This

demonstratesthatthe ASLV PR is responsible for

releasing

itself from the NC-PR gag precursor

fragment

in E. coli.

MATERIALS ANDMETHODS

Bacterialcells. E.coliMC1061(3)wasused as a hostfor all expression vectors. The cells contained the plasmid pRK248cIts, which directs expression of a temperature-sensitive bacteriophage lambda repressor protein (5).

Constructionof thebacterialexpressionclones.The bacte-rial expression clones were constructed by using the

pEV-vfrexpression vectors which supply a lambda phage promot-er-operator region (PLOL), a ribosome-binding site, and a translational initiation codon (5). The pANC-PR clone was constructed by replacement of a pEV-vrf2 BamHI-PvuII

fragment with a viral 817-base-pair BamHI-HpaI fragment

(Fig. 1) derived from an infectious clone of the avian sarcoma virus PrC strain, pATV-8 (11). The viral DNA fragment contains the C-terminal two-thirds of the

NC-coding region, all of the PR-coding region including the translational terminationcodon for Pr76 gag, and the 5' end of thepol gene. The construction of pNC (formerly called

pRKP12) was described previously (10). The pNC-PR plas-mid wasconstructed by substitution ofaca. 1-kilobase-pair

pNC-derived PstI-BamHIfragmentinto pANC-PR(Fig. 1). Thisfragmentcontained thePLOLregion andthe ribosome-binding site ATG region properly aligned for expression of thenative Nterminus of NC (10). The pPR expression clone was constructed from pANC-PR by using oligonucleotide-directed, site-specificmutagenesis (10, 19) to delete the NC region and to precisely align the ATG codon in the vector with the first codon of the mature PR, a strategy used

previouslyto construct pNC (10). Theoligonucleotide used for deletion mutagenesis was 5'-AGGAGGAATTAAT ATGTTAGCGATGACAATGG-3'. A mutation was intro-ducedinto theputativeactive site region of PR (20, 23) by using site-directed oligonucleotide mutagenesis (19). The construct coding for the mutant PR [pNC-PR(S-I)] was

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BamH I

1844 1917

^A a ..

pANC-PR pNC-PR pNC-PR(S) pNC-PR(S-I)

gag

I

p1

2110 2472

--a_. _

Hpi I

ZO2734

_CA

I

NC v PR v RT I _

P OL ATG

i ~~~~TAA

PLLAT

TM

a...M. , ...I * ____

PLOL

_-i.

ATG TM

i _

Asp-m.419

PL0L

_,

ATG

_i

pPR

pNC r--- -- * ________.

PLOL ATG

PstI

TM

i

TM

i

I

1917

FIG. 1. Maps of PR, NC-PR, and NC E. coliplasmidexpression clones. All clones are derivatives of pEV-vrf vectors (5). A partial map of theviral DNA clone pATV-8 (11), from which PR and NC sequences were derived is shown at the top; the flankingcapsid (CA) andreverse transcriptase (RT) regions are indicated. Nucleotide positions indicated correspond to the avian sarcoma virus sequence described by Schwartzetal. (21). Symbols: ---, pBR322-derived vectorsequences; _,bacteriophage lambdapromoter-operator region (PLOL)(not drawntoscale). The initiation codon provided by the expression vector is indicated (ATG). The position of thetermination codon forthe PR-coding regionisindicated (TAA). The site of the Asp-*Ile mutationintroduced into the putative active site of PR isshown. Numbers indicate nucleotide positions.

provided by V. M. Vogt. It was derived by site-directed

mutagenesisattheGAC codon that encodes aspartic acid 37, which converted this codon to an ATA (isoleucine). In this construct, a portionof NC-PR sequences are derived from the Schmidt-Ruppin strain of avian sarcoma virus. A wild-typeversion of this clone was also constructed [pNC-PR(S)]. Analysis of NC and PRexpressed in E. coli. The ASLV NC and PRexpressed in E. coli were detected by

immunoblot-ting with rabbit sera directed against NC (10) and PR.

Anti-PR antibody wasprepared by immunization of rabbits with purified viral PR provided by J. Leis. Expression in cellscontainingthevarious vectors was induced by shifting

cultures from 30 to 42°C for 2 to 4 h, and virus-related proteins were detected by immunoblotting as described

elsewhere (1, 10, 24).

Preparation of bacterial extracts. Induced and uninduced E.coli cultures (1.75 ml)werepelletedandsuspendedin 400

,Iu of 1%toluene-10 mMTrishydrochloride (pH8.0)-S5mM

mercaptoethanol-0.1

mMEDTA-0.1%Triton X-100.

Bacte-rial cellsuspensionswerevigorously agitated by vortexing (3

min)andwereincubated for20 min at37°C. Unbrokencells andcell debriswerepelleted by centrifugation. Supernatants

were freeze-dried and dissolved in 50

RI

of water, from which 2

RI

was assayed.

Synthesis and cleavage ofdecapeptidesubstrates. The

syn-thesis and

purification

ofthe

decapeptides

Thr-Phe-Gln-Ala-Tyr-Pro-Leu-Arg-Glu-Ala (wild

type) and Thr-Phe-Gln-Ala-Ala-Pro-Leu-Arg-Glu-Ala

(variant)

are described elsewhere (M. Kotler, R. A. Katz, W.

Danho,

J.

Leis,

and A. M. Skalka, Proc. Natl. Acad. Sci. USA, in press). ASLV PR cleaves thewild-type

peptide

betweenTyrand Pro

(Kotler

et al., inpress).

Proteolytic

reactionswerecarriedoutin 10

,ul

of0.1 Msodium citrate buffer

(pH 5.5) containing

1.1 mMof

decapeptide substrate and 2

pul

of bacterial extract. The

reactions were incubated at 37°C for 10 h. The

cleavage

productswereanalyzed bya

thin-layer

electrophoresis

assay

(Kotler et al., in press).

Samples

of4

pul

were

spotted

on

cellulose plates (Art 5577, 20

by

20 cm; E. M.

Reagents,

Federal Republic of Germany). Plates were subjected to 45 mA for 1 h in pyridine-acetic acid-acetone-distilled water (volume ratio, 1:2:8:40). They were then air dried and

sprayed first with 1% triethylamine (Pierce Chemical Co.,

Rockford,

Ill.)

inacetoneand thenwithfluorescamine (Hoff-mann-La Roche Inc., Nutley, N.J.)at0.1 mgof acetone per ml. Photographswere taken under UV light.

RESULTS

Expression of PR in bacteria. ASLV PR is an abundant

virionprotein formed byproteolytic processing;itis

derived

from the Cterminus ofthe Pr769'9 precursor. Forexpression

of the mature PR inE. coli,a translationalinitiation codon

provided by theexpression vector wasintroduced immedi-ately

preceding

the PR

reading

frame

(Fig.

1, PR). No

alterationwasnecessaryatthe3'end, since the termination

[image:2.612.132.499.65.253.2]

codon ofPRcanalsofunctioninbacteria(Fig. 1). Thesame vectorwas also used forexpression ofa viral NC-PR gag precursorfragment derived fromSR-A aviansarcomavirus

[pNC-PR(S)]

or PR-C avian sarcomavirus

(pNC-PR)

(Fig.

1). A truncated version of the

pNC-PR

clone which is

missingtheN-terminalone-thirdof NCwasalsoconstructed (pANC-PR).

E.colitransformed withpPRwasgrowntomid-log

phase

at30°C. Expressionfrom the PL promoterwastheninduced byshiftingtheculturesto

42°C.

Cellswereharvested 3to4 h

postinduction,

and

proteins

were

analyzed by

polyacryl-amidegel

electrophoresis

and

immunoblotting,

with antisera

directed

against

the

purified

ASLV PR and NC. A

protein

that comigrated with viral PRwas

produced

in the induced but not in the uninduced cells

(Fig. 2A,

lanes 1 and

2).

Neither the viral PRnorthe

bacterially

produced

PRreacted with anti-NC serum

(Fig.

2B, lanes 1 and 2), as

expected.

FromCoomassie blue

staining,

it canbe estimatedthat PR represents 5 to

10%

of the total

protein

in inducedbacteria (datanotshown).

Synthesisandcleavage ofNC-PR inbacterial cells. Immu-noblot

analysis

ofthe

proteins

produced

after induction of

.mn.mm- m I I

I

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A

-ANC-PR

7 -PNC-PR

1 2 3 4 5 6 7 8

B

a: a: <0 cr

> EL z z

-L a Ca

Zn U I U I U I U I

43-25- .. ..:.:_...§-PR

1ANC-PR

25-12 ..=t-NC-

-

6-12 345 678

FIG. 2. Immunoblot analysis ofPRand NC productsexpressedinE. coli.E. colilysateswerefractionatedona15% polyacrylamide-sodiumdodecylsulfate gel,and virus-relatedproteinsweredetectedbyusingrabbit antiserum directedagainstASLV PR(A)orNC(B)and 1251I-labeledprotein Gasdescribed elsewhere (1).LysateswerepreparedfromE.colicontaining pPR (lanes1 and2),pANC-PR (lanes3 and 4),pNC-PR(lanes5and 6), andpNC (lanes7 and8).Lanes1, 3, 5, and7,lysatesfromuninduced(U)cultures;lanes2, 4, 6, 8, lysatesfrom cultures induced(I)for3 h at42°C. Molecularsize markersareindicatedinkilodaltons. Viral NCand PRmarkersareshown. The anti-PR serum (A) cross-reacts withanumberofE.coliproteins,as seeninlysates from uninduced cultures(lanes 1, 3, 5,and7).Theanti-NC serum (B)cross-reacts with an E.coli protein ofca. 35kilodaltons.

thepNC-PR vectoridentifiedaproteinof themasspredicted for the NC-PR precursor fragment (ca. 23 kilodaltons); it reacted withboth anti-PR and anti-NC (Fig. 2AandB,lanes 5and 6). Two smaller proteins which had migration coeffi-cientscorrespondingtothose ofproteins produced by pNC andpPR and those ofthe analogous purified viral proteins werealsoidentified. One of these two proteins reacted with anti-NC, while the other reacted with anti-PR, indicating that the NC-PR precursor underwent cleavage at (or near) the siterecognized duringvirusmaturation(Fig. 2A and B,

lanes5 and6). Analysis ofproteins produced after induction ofpz&NC-PR gave similar results (Fig. 2AandB, lanes 3 and 4). As expected, both the precursor fragment and the

NC-related productwere shorter than those produced by pNC-PR, while the PR product was unaffected. This result

con-firmed the identities of the small anti-NC and anti-PR

reactive bands.

One explanation for the production of small,

discrete-sized products is that the NC-PR and ANC-PRprecursors werecleaved specifically by PR. To test this hypothesis, we used a mutated clone in which the codon for the highly conserved Asp residue(amino acid 37 in PR) located in the

putativeactive site of PR (20) was changed to an Ile codon

[pNC-PR(S-I), Fig. 1]. Expression of proteins from this

plasmid was assayed by immunoblotting with the anti-NC serum. The results showed that the NC cleavage product wasproduced from the wild-type construct, pNC-PR(S), but notfromthe mutant,pNC-PR(S-I) (Fig. 3, compare lanes 6

and 8). Thus, we conclude that bacterially produced PR

activity is responsible for specific cleavage of the NC-PR gag precursor fragment. The protein which contained the Asp toIle mutation migrates more rapidly than the wild-type NC-PR protein (Fig. 3, lane 6; see arrows), indicating that this change may have a significant effect on the protein

conformation.

Detection of PR activity in cell extracts. To confirm that an active PR was produced in E. coli, proteolytic activities

encodedbyallsix plasmids described in Fig. 1 were tested in cell extracts by using a synthetic peptide cleavage assay

(Kotler et al., in press). We have previously shown that

decapeptideswhich are composed of a known ASLV

prote-olytic processing site in pol (between the ot and pp32PoI

domains) are precisely cleaved by ASLV PR in vitro. A change inone of the amino acids which flanks thecleavage

site(e.g.,TyrtoAla) abolishes thiscleavage(Kotleretal., in press).

Extractsprepared from induced cells which express proc-essed PR [from plasmids pPR, pNC-PR, pANC-PR, and pNC-PR(S)] contained activities which cleaved the suscep-tible peptide. Extracts from cells which express pNC or

pNC-PR(S-I), which donotproduce processedPR, showed

00

0- C

a-> z z z

z

0-~

u

43- l Q

25

-18- .. .!...

14/12-f;,

NC

6-FIG. 3. Immunoblotanalysisof NC-PRproteins expressedin E. ccli.Analysis was carriedoutas described in thelegend toFig. 2, except that cultureswereinduced for 4 h.NC-relatedproteinswere

detectedbyincubation with rabbitanti-NC,followedby "'5I-abeled

protein G. Lysates were prepared from cells containing pNC-PR (lanes3 and4), pNC-PR(S-1) (lanes5and6),andpNC-PR(S) (lanes 7 and 8). Lanes 4, 6, and 8 contain lysates from cells that were

induced forexpression (I); lanes 3, 5 and 7 contain lysates from uninduced(U)cells. Lanes1and 2 contain authentic viralproteins.

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[image:3.612.141.469.65.217.2] [image:3.612.357.511.405.630.2]

no cleavage (Fig. 4A). As a control, a peptide containing a single amino acid change at the cleavage site (Tyrto Ala) was incubated with the same E. coli extracts. No cleavage was seen even after a 10-h incubation. Byusingthis sensitive peptide assay, low levels of PR activity could be detectedin some extracts prepared from uninduced cells which con-tained plasmids with the wild-type PRsequence,althoughno PR-related proteins could be detected by immunoblotting (Fig. 2 and 3). We presume that this is duetolowexpression (leakiness) of the respective plasmids under uninduced con-ditions. Since the peptides are quite stable incrudeextracts, the reaction mixtures can be incubated for very longperiods of time, thus further increasing the sensitivityofthe assay.

DISCUSSION

In this report, we describe expression of a retroviral protease in bacteria. This enzyme isactiveinbacterial cells as well as in cell extracts. Amutationintheputative active site of PR (Asp to Ile) in the pNC-PR construct resultedina failure to release NC and PR. Thisresultsuggests thatPRis responsible for releasing itself from the gag precursor. However, the experiments with the mutated PR cannot exclude limited participation ofcellular proteases. For ex-ample, it might be imagined that limited proteolysis by a cellular protease could release some maturePR molecules, and these might then be responsibleforadditionalprocessing

A

cc

cc z

-LZ <

a

IU IU IU

--l

CD) C

z z _aD-z

IU IU IUPRS

B

c:

0-IU

P2-

S-

P1-

B-FIG. 4. DetectionofPR-specificactivity in bacterialextractsby

using a thin-layer electrophoresis assay. Synthetic decapeptides

containingTyr-Pro(wildtype)(A)orAla-Pro(mutant)(B)cleavage

siteswereincubatedwithbacterialextractspreparedfrom induced

(lanes I)anduninduced(lanesU)cultures. Asacontrol,theTyr-Pro

peptide was subjected to purified viral PR (lane PR) or was incu-batedwithnoPR(laneS).Theextractswerepreparedfromcultures containingthe indicated clones. After incubation ofpeptides with

extracts,themixtureswerefractionated by thin-layer

electrophore-sis(seeMaterialsandMethods). The mobilitiesofpeptidesubstrates

(S), products (P1andP2),andanunidentified spotassociatedwith thebacterialextract(B)areindicated. Theoriginis indicated(0).P1 isapentapeptidecleavageproductcorrespondingtotheN terminus

of the decapeptide substrate. P2 (dark spot) corresponds to a

pentapeptidederivedfrom theC terminus.Productswerelocatedby

stainingwithfluorescamine,followed byvisualizationwith UVlight

(see MaterialsandMethods). The P2spotappears dark becauseof theN-terminal Pro whichproduceschromophores that absorbUV light.Detailsofthemethodologyand characterization of the

prod-uctsidentified bythethin-layerelectrophoresis assayareprovided

elsewhere(Kotleretal., inpress).

which would release more active PR molecules. If the

processing is due entirely to PR

activity,

it would be of interesttodeterminewhether thefirstmaturemoleculesare released as aconsequence of inter- or intramolecular inter-actions. Introduction of mutationsintotheNC-PR

cleavage

site may

provide

some

insight

into this

question.

Our

finding

that the PR-NC gag precursor

fragment

is cleaved

intracellularly

is consistent with earlier reports of

proteolytic

processing

ofthe ASLV gag precursor inE.

coli

(17) and human

immunodeficiency

virusgag-polprecursors in yeastcells(13). In contrast, the ASLV

Pr76fia

precursor

synthesized in vitro

by

using

a rabbit

reticulocyte

system does not

undergo

processing

unlessPRis added

(28). Also,

Pr769' is not

processed

in mammalian cells infected

by

avian sarcoma virus

(25),

and these cells are unable to support

assembly

of infectious

particles

(2,

12).

There are

several

possible

explanations

for these differences.

(i) Avian,

bacterial,

and yeast

cells,

but not mammalian

cells,

might

containaprotease which is abletoactivatePR

by

releasing

it. The active PR molecules

might

then

complete

the

proc-essing as mentioned above.

(ii)

Mammalian cells may

con-tain aninhibitor of ASLV PR which prevents

autocleavage

or

subsequent

enzymatic

activity.

(iii)

The releaseofPRmay

require

dimerization ofthe precursor

(20)

or an intermolec-ularreactionwhichoccurs

only

whena

high

concentration of

precursor is present, as in

bacteria,

yeast cells, and the

permissive

avian cells.Thresholdconcentrationsmaynotbe

achieved in vitro or in mammalian cells.

(iv)

The

Pr769'9

may be folded in a

specific

structure which prevents

auto-cleavage

until the time ofvirion

budding.

Subsequent

acti-vation of the protease

might

occur in virions

produced

in avian cells but not in mammalian cells. Precursor

proteins

producedinbacteria

might

notfold in themanner

required

to prevent

cleavage.

Some ofthesemodels may be testable

by

using

methodsdescribed inthis paper.

The bacterial

expression

systemsdescribed here will allow usto

produce

large

amountsofnormal andalteredASLV PR

proteins.

These should be usefulfor

studying

the

biochemi-cal

properties

ofPRandfor

providing

material for

biophys-ical

analyses.

The gagprecursor

fragment (NC-PR)

will bea

useful substrate to test the effect of various

cleavage

site mutations. Since

cleavage

occurs in

bacteria,

it should be

possible

to

study

the effect of such mutations without

purification

ofthe PR or substrates.

ACKNOWLEDGMENTS

Wewishtothank W. Danhoforthe

synthesis

of

peptides,

J. Leis for supplying purified viral PR, F. Erlitz forthe

synthesis

ofthe oligonucleotides, R. Crowl for

pEV-vrf

DNA,and D. Markham for adviceon extract

preparation.

We

gratefully acknowledge

V.

Vogt,

K. Jones, J. Kulkosky, and P. Tsichlis for critical reviews ofthe manuscript. We also thank V.

Vogt

and G. Schatz for

providing

mutantconstructs and for

helpful

and

stimulating

discussions. This work was

supported

by

Public Health Service grants CA-06927and RR-05539 fromtheNational Institutes ofHealth, agrant

from the Pew Charitable Trust, and an

appropriation

from the Commonwealth of

Pennsylvania.

Partial support for R.A.K. was

providedby the W. W. Smith CharitableTrust. LITERATURE CITED

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