Complementation studies with Rous sarcoma virus gag and gag-pol polyprotein mutants.

0022-538X/92/063873-06$02.00/0

Copyright

X 1992, American

Society

forMicrobiology

Complementation Studies with

Rous

Sarcoma Virus

gag

and

gag-pol

Polyprotein Mutants

SUZANNEOERTLE,t NEILBOWLES,* AND PIERRE-FRAN0OIS SPAHR DepartmentofMolecular Biology, UniversityofGeneva,

30quaiEmnest-Ansennet, 1211 Geneva 4, Switzerland Received 31 October1991/Accepted 17 March 1992

Avian retroviruses (with the notable exception of spleen necrosis virus)expresstheirprotease (PR) both in theirgagandtheirgag-pol polyprotein precursors,incontrast tootherretroviruses, notably, the mammalian retroviruses, in whichPR is encoded inthe gag-pol polyproteinorin a separatereadingframeas a gag-pro

product.Theconsequenceis that the avian PR is expressed in stoichiometric rather than catalyticamounts.To

investigate the significance of the particular genome organization of the avian retrovirus prototype Rous

sarcoma virus, we developed an assay that measures complementation between the gag and the gag-pol

polyproteins by expressing them from twodifferent plasmids in transfected cells. By using this assay, we

showed that the protease PR from the gag-pol polyprotein is capable of autocatalytic self-cleavage and -activationwhen coexpressed withaprotease-deficientgagprotein and that the PR domain hasa rolein viral

particle assembly. Furthermore, this complementation assay can be used to investigate the role of thegag

domain in thegag-pol polyproteinbydetermingwhether itcanrescueadefect in thegagpolyprotein. Wereport

here the results ofsuchanexperiment,which studieda mutation in the N terminus of thegaggene.

The retroviral genome encodes at least three genes: 5' gag-pol-env 3'. Theenvgenecodesfor theenvelope protein ofthevirion. Expression of thegag geneyieldsapolyprotein precursor (Pr76Wag for Rous sarcoma virus [RSV]) which migratestotheplasmamembrane of the host cell, where it is cleavedduringvirus assemblyandbuddingtoyieldthefive maturegagproteins. Thepolgeneisalways expressedas a gag-pol fusion polyproteinprecursor (Pr1809ag-Po forRSV) by translational suppression of the gag gene termination codon at a frequency of about 5%. Thegag-pol precursor incorporated into theviralparticleduring budding (ataratio of 1:19 withrespect tothegagprecursor) is then processed toyieldtheenzyme reverse transcriptase, which is entirely codedby thepolgene(forreviews,see references 2, 9, and 15). Thus, thegaggeneisexpressedbothas aseparateentity and as a fusion product with thepolgene. This raises the questionof therole,ifany,of thegagportionofthegag-pol fusion protein. It has been shown that the NH-terminal portion ofgagis sufficienttoallowrelease of fusionproteins from cells but thatsequencesextendingasfarasthecapsid (CA) proteinarerequiredforformation of viruslikeparticles (6), suggesting thatgagis necessaryto transportpolto the membrane.Analysisof virusmutantsexpressingthegag-pol fusion protein in the absence of thegagprecursor has been

performed with Moloney murine leukemia virus, spleen necrosisvirus, human immunodeficiency virus type 1, and RSV (1, 3,4, 12, 14, 17). In allcases, itwasfound that the gag-pol fusion protein failed to induce formation of viral particles (unless rescued by coexpression of the gag-en-coded protein). However, the stability of the

gag-pol-en-codedpolyproteinswithin the cellvaried, being processedin spleen necrosis virus and human immunodeficiency virus type 1 but stable in Moloney murine leukemia virus and

RSV.

*Correspondingauthor.

tPresent address:Departmentof CellularBiology, Universityof

Geneva,1211 Geneva4,Switzerland.

The structure and expression ofthe genome of RSV are unique among retroviruses in that the protease PR is en-coded in both thegagand thegag-pol polyprotein precur-sors,with theconsequence that thisenzymeisexpressedin stoichiometric rather thancatalyticamounts.This raisesthe question of whether the PR ofRSVgag-pol polyprotein is active(asis thecasefor the PRs of otherretroviruses,which only express PR as part of gag-pol). To investigate the significance of the particular genome organization and expression ofRSV, we developed an assaythat measures complementationbetween thegagandgag-pol polyproteins by expressingthem in chickenembryofibroblasts (Gs- and Chf-; Spafas, Norwich, Conn.)fromtwodifferentplasmids derivedfrompAPrC (a nonpermutedcopyofprovirusRSV PragueC[10]).By usingthisassay, weshowed thatprotease

PRfrom thegag-pol polyproteincan beactivated autocata-lytically when complemented by a protease-deficient gag protein. Furthermore, this complementation assay can be usedtodetermine whetheranydefect in thegaggenecanbe rescued by thegag portion of thegag-pol gene, and we

reporttheresults of suchanexperimentthat usedamutation in the N terminus of thegag gene.

Expressionof thegag-polfusionproteinwithoutgagprotein doesnotinduce virionformationorproteolyticprocessing. A mutant expressing a gag-pol fusion protein but no gag

protein was constructed (Fig. 1) by deleting the first two

nucleotides of thegag gene termination codon, thus over-coming the translation arrest and constitutively expressing thepolgene in the same readingframe asgag (5). In this

mutant,U180,thegag-polfusionproteindiffers from that of

the wildtypebydeletion of the firstamino acid (isoleucine) of thejunction peptide.TheU180mutantproviralDNAwas transfectedinto cultures of chicken embryofibroblasts,and

the viralproteins,eitherincorporatedintoparticlesreleased

into the culture mediaorsynthesizedinthe transfectedcells, were analyzed by immunoblotting using anti-CAserum as already described (10). As shown in Fig. 2A and 3A (lane U180), themutant U180synthesized astable fusionprotein in the transfected cellsbut,inthe absence of thegagprotein 3873

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A ENV SRC E-T

B

C

WT

MA|plO CA NC

PRI

alpha p32

MA

210

CA NC PR r69

Pr76 9-aP

BBRYMMMMQfi,!1-.-1 I PrlBO9 9~~~pO

U1807

U76 1OMI3EFW'l

CM5

U76pl5.1 WWEZZ .

U18Op15.1 MinU76 MinUI80

MA. matrix associated protein

E plO.protein of unknown function

_ CA.major structural component of the capsid

M NC.nucleocapsid protein EZ3 PR. protease

ED PR, protease mutated in its active site

FIG. 1. Structuresof gag andgag-polpolyprotein mutants. (A) Complete genome of RSV PrC as a linearprovirus. The regions

encoding thegag,pol, env, andsrc proteins are shown in boxes.

LTR, long terminal repeat. (B) Enlargement of the gag andpol genes. MA, matrix-associated protein; plO, protein of unknown

function; CA,capsidprotein; NC,nucleocapsidprotein; PR,

prote-ase; alpha, 58-kDa componentofreverse transcriptase; p32, inte-grase. (C) Schematicrepresentationof themutants(symbolsfor the

codingregionsareshownatthebottom).The wildtypesynthesizes

both gag precursor Pr769ag and gag-pol precursor

Pr180QagPo.

MutantU180was constructedbydeletingthe firsttwonucleotides of the stop codon of the gag geneby site-directedmutagenesis (8,

16) using the oligodeoxynucleotide 5' GGCCCTCCCTAAATT TGTCAAGCGG 3'; it differs from the wild-type (WT) gag-pol polyprotein bythe absence of the first amino acid(isoleucine)of the

gag-poljunctionpeptide.Mutant U76 hasalreadybeendescribed: it

synthesizes only the Pr76gag polyprotein and produces

noninfec-tious viral particles (11). Double mutants MinU180 and MinU76

were obtained by changing the initiation codon of the gag (or gag-pol) polyproteinfrom AUGtoTTCby site-directed mutagene-sis (8, 16) with the oligodeoxynucleotide 5' TATGACGGCTTC

GAAGCTTGATCCACC 3'.CM5,a mutantlackingthePRdomain,

has already been described (11). Double mutants U180p15-1 and U76p15-1 were constructed by exchanging the 2,393-bp EcoRI restriction fragment containing the protease active site mutation p15-1(a changefromAsptoArgatthe activecenterof PR[11])with the samerestrictionfragment ofthe DNAproviralvectorcarrying either the U76 or the U180 mutation outside of this restriction fragment.Allof the mutationswere confirmedbyDNAsequencing (19).

Pr76gag, therewas noviral particle formation. In

addition,

noCAantigenwasdetected in the culturemedium,

showing

that thegag-pol fusion proteinitselfwas notreleasedornot

stable enough to be

immunoprecipitated (Fig.

3B).

Similar

results have beenobtainedwithamutant

expressing only

the first 85 amino acids of the

pol

genefusedtothe gag gene

(1).

Our results thus indicate that the

phenotype

of the shorter fusion mutantis notdueto the absence of the remainder of thepolgene.These dataare

compatible

withthose

published

after our work was

completed,

showing

no

particle

forma-tion when

gag-pol

is

expressed

alone

(3,

14).

The

gag-pol

fusion mutant can be rescued in trans

by

coexpression

of the

gag

polyprotein.

We have

already

de-scribed a mutant U76

expressing

only

the gag

polyprotein

precursor

(11).

This mutant, upon

transfection,

yields

fully

processed

viral

particles

as

efficiently

asthe wild

type.

To

rescue

gag-pol

fusion mutant

U180,

we cotransfected the

two

plasmids

(U180

and

U76)

at different U180-U76 ratios

from 1:10 to 4:1 and

analyzed

the intracellular and viral

proteins

and

production

of viral

particles

by

immunoblotting

and the presence of the

pol

gene

product

in the

particles by

thereverse

transcriptase

assay

(10).

The results

presented

in

Fig.

3CandDshow thatatall

input

ratios of U180toU76 up

to

1:1,

viral

particles

wereformedwithan

efficiency

similar

tothatof the

wild-type

virusortransfectionwithmutantU76 alone

(some

transfection

efficiency-related

variation was

observed).

At a U180-U76

input

ratio of

4:1,

efficiency

of virus

particle

formationwas

markedly

impaired

(<5%

of the

wild-type

level).

Results of thereverse

transcriptase

assayof these

coexpression

experiments

(Table

1)

showed that while all

particles

contained this

enzymatic

activity,

its level increased when the

input

ratio of U180toU176increased. At

high

U180-U76

input ratios,

particles

that contain more

reverse

transcriptase (and

therefore more

gag-pol

polypro-tein)

than the

wild-type

virions were formed. Itwas

only

at

aU180-U76

input

ratio of 1:4 that thereverse

transcriptase

activity

of the

particles

reached the level found in the wild type

and, therefore,

the

stoichiometry

of gag and

gag-pol

incorporated

into

particles

was

probably

the same as in

wild-type

particles.

Examination of the intracellular

proteins

(Fig.

2B and

C)

shows that at

increasing

concentrationsof

input

U180

DNA,

the amountof

gag-pol

precursor

synthe-sized increased

concordantly.

However,

the

efficiency

of

U180

transcription-translation

appeared

to be lower than

that of

U76,

although

there wasvariationbetween different

transfection

experiments

and different

plasmid

preparations

(compare

Fig. 2A, B,

and

C).

AtU180-U76 ratios which gave

particles

withreverse

transcriptase

activities closetothat of

wild-type

particles,

the relative

proportions

of

gag-pol

and gag precursors in thecell

lysate

weresimilar.

However,

the

efficiency

of

particle

formation with gag and

gag-pol present

at

wild-type

levels was also

dependent

on the

requirement

thata

single

cell express both

plasmids.

The resultsof these

complementation

experiments

demonstrate that

(i)

the

gag-polfusion

polyprotein

canbe

packaged

into

particles

when

the gag

polyprotein

is

provided

intrans

(in

agreement

with

otherreports

[3, 14]),

which

implies

aninteraction between

thetwo

polyproteins

during

the processof

particle assembly,

and

(ii) particles

which containmore

gag-pol

fusion

polypro-tein than the wildtype canbeformed. The

inability

of

gag-poltobe released fromcells andtoform virus

particles

when

expressed

alone can be

explained

in two ways.

(i)

The

presence of gagis necessarytotransport

gag-pol

tothe cell

membrane,

or

(ii)

the

gag-pol

molecule is

sterically

inflex-ible and cannot condense into a

capsid

structure unless

"spaced" by

the presence ofgag molecules.Similar

results,

showing

that

gag-pol

can be releasedfrom acell and form

particles only

whenrescued

by

gag

expression,

were

pub-lished after our studieswere

completed

(3, 14).

The

protease

from the

gag-pol

polyprotein

can be activated

,5il CM5

.1-.1 .1 .1 If .1 .1

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Ao

D

CD

PrI80-" '* _ M

B U76+

U76+U180 a MinU180

r ~ ~-- + I

!8 16 15 13 0 E 25 25

uJ 2 4 5 7 IC) 6 12 3

am m

m

4m

-m

w M

m

-m

C

U76 U 180

6 10 4 4 10 6

3:

14> w

CA- - _ _ i. - 4-

-FIG. 2. Analysisof intracellular viral proteins from cells transfected with the viral mutants DNAs. (A) Proteins of celllysatesfrom two

petri disheswere immunoprecipitated with a polyclonal antibody against CA, followed by protein A-Sepharose adsorption. The eluted

proteinswereresolved by polyacrylamide gel electrophoresis followed by immunoblotting with anti-CA serum and'251-labelledprotein A, as

previouslydescribed (10). Single transfections were performed with 15pLgof plasmid DNA per plate. Forcotransfection (U76 plus U180), the

plasmidDNAinputwas30p.gofU76 and 10pLgof U180. (B and C) Analysis was performed as described for panel A. Cotransfections were

performed either with theamountsofplasmidDNAindicatedabovethe lanes (per plate) or with 25 ,ug of plasmid-encoding gag sequences

plus10 1Lgof plasmid-encodinggag-pol sequences. C, control; WT, wild type.

autocatalytically

by coexpression with a gag polyprotein. To investigate the potential of the protease encoded in the gag-polpolyprotein ofRSV,weused ourcomplementation assay tomimic a mammalian retroviral system: we cotrans-fectedmutantU180 with mutantCM5. In this mutant, which hasalready been described (11), codon 1 of the protease PR isreplaced bya stopcodon. Ittherefore synthesizes only a truncated gag precursor without PR (analogous to that of mammalianretroviruses) andnogag-pol protein(Fig. 1) but

formsnonprocessedviralparticlesat alowerefficiencythan the wild type (Fig. 3B in reference 11). Cotransfection of

CM5 and U180 resulted in virus particle production less

efficientthan that of the wild type(Fig. 4A): they contained aproteinof about 63 kDa(the translation product ofCM5),

a small amount of Pr18h99P° (made visible by overexpo-sure of the autoradiograph), and a significant amount of

matureCA (the amount ofprocessing was variable between transfections, but CA was always demonstrable). Further-more, theparticles produced had a relative level ofreverse transcriptase activity comparable to that of the wild-type virus, albeit slightly reduced (Table 2). That the amountof CA in the progenyparticlesis greater thanunprocessed Pr63 suggestseither thatCA is derived from gag (and gag-pol) or that the ratio ofgag-pol to gag is much higher than in wild-type particles and that CA is derived only from gag-pol. However, the relativereversetranscriptase activity is simi-lar to(even slightly lowerthan) that of the wild-type virus,

suggesting that the amount ofgag-pol incorporated is not

significantly higher (assuming partial activation of reverse

transcriptase). Increasing the proportion of U180 to CM5

hadaneffect similartothatproduced bycomplementation of U180byU76:particleswereformed withasimilarefficiency

J~~~~~~- IfU76-UiBO

co i8 16 15 13 10 ,_

.) 3 u 2 4 S 7 10 3

D U76+LJ180

16 10 4

u 4 10 10 B

BSA* __

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CAe

FIG. 3. Analysisofgagproteinsofviralparticles produced bycells after transfection. (A) Culture mediumwasharvested 48 to 60h

posttransfection,theparticulatematerialwascollectedbyhigh-speed centrifugation,and gagproteincontentwasanalyzed by

immunoblot-ting usinganti-CAantibodyaspreviouslydescribed(10).Transfectionswerecarried outasdescribed in thelegendtoFig.2A.(B)Themedium at48to60 hposttransfectionwasimmunoprecipitated, and theelutedproteinswereelectrophoresed asdescribedin thelegendtoFig.2A.

(CandD) Analysisof viralproteinswasperformedasdescribedforpanelA. Thenumbers abovethe lanesareratios of U76 to U180plasmid

DNAs inmicrograms. C, control; WT,wild type;BSA, bovineserumalbumin.

A

co

U , D D

Z --n

J j-,.

Pr76 ewm qmmmw. _-M.N!Illll

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TABLE 1. Reversetranscriptase activities ofparticles produced bycomplementation of U76 and U180'

Reverse

Complementation transcriptase

activity(%)

Wildtype... 100%

U76 +U180 at: 18/2... 38

16/4... 128

15/5... 286

13/7... 494

10/10... 728

4/16... >1,000 "The culture medium from two 100-mm-diameter petri disheswas har-vested at 48 to 60 hposttransfection,the particulate material wascollected by high-speed centrifugation, and reverse transcriptase was quantitated by exogenoustemplate assay, aspreviouslydescribed(10), normalizedtothe CA proteincontentofwild-typeviralparticles,andexpressedas apercentage of thewild-type particlesafter subtraction of thenegativecontrol. at a U180-CM5 input ratioof 1:1, butat 4:1particle forma-tion was greatly reduced(data not shown). However,aswith U180-U76 cotransfection, the relative reverse transcriptase activity of the particles increased andwasdetectableeven at the highest input DNA ratio. These data support the idea that theparticles, formedataU180-CM5input ratio of 2:5, shown inFig.4Adidnotincorporatemoregag-pol than gag during virus assembly and that CA was derived, at least partially, from gag. Analysis of the intracellular viral proteins revealed the presenceofPrM8gQag-PoIandPrO3gag at levels similartothose of thewild-type virus (Fig. 2B, Prl8O andPr76), suggesting that the lowerefficiencyof virusparticlerelease is duetothe absence of the PR domain in gag. A similar reduction in virus particle assembly has been reported for a protease-deficient gag mutant (14). These results show (i) that RSV PR canactivate itself from the gag-polpolyprotein (as is the casefor mammalianretroviruses), sincewehave shown that anunprocessed gag-polpolyprotein does not exhibit reverse transcriptase activity (11); (ii) that the PR encoded in the

A

_o~ n CD a CD U CD CD w+ = CO CO F i+ + MI r- r-- C-Uu D EDD Pr180' P0I w1

B

CD_co aD tD in cl- r-I D E . TABLE 2. Reversetranscriptase activities of viralparticles incomplementationexperiments' Reverse Complementation transcriptase activity (%) Wildtype... 100

CM5 + U180... 60

U76p15.1 + U180... 30

U76 + U180p15.1... 150

U76p15.1 + U180p.15.1... 0

MinU76 + U180... >500 U76 + MinU180... 140

U76 + U180... 150

a Fordetails, seethe footnote to Table 1. Thenumber ofparticleswas

normalized by quantitation ofmatureCAprotein and/or unprocessedgag protein.

RSV gag-pol polyprotein can probably act in trans to process the PR-deletedgagpolyprotein; (iii) thatrescue of the U180 phenotype by coexpression ofagag polyprotein can occur in the absence of the PR domain of the latter protein, although the PR domain hasarole in efficientrescue andvirusassembly; and (iv) that the PR from RSVdoes not requireafree carboxyl endfor autocatalyticprocessing, as has been suggested recently (1). Why the PR of RSV is presentalso in thegagpolyprotein isnotunderstood. Oneof the reasons might be quantitative, since it has been shown that RSV PRintrinsically has a lower specific activity than nonavian retroviral proteases(7); thus, more enzyme may be needed. This would becompatible with the partial process-ing observed inourcomplementation experiments. Itshould be noted that inputratios of U180 to CM5 lower thanthose used in the experiments presented in Fig. 4A reduced processing and resulted in lessmatureCA (datanotshown). Another explanation for the presence of PR in the gag polyprotein is the role ofthe PR, directly or indirectly, in RSV RNA packaging (11). Finally, it appears possible that PRplaysastructural role inthe assembly of the RSV virion, possibly through interaction ofPRdomains.

Another approach to investigate the roleofthePRof the

C

Cn

aD

-D

(0

0- H- U

-.

ao

c co

2 a

LO UN

D CL

. r

D

U76 U76

MinUI80 U180 25 25 25 25

U 6 12 6 12 3:

Pr76909'> 4

Pr63T- w

CA - -ow

MA P

_od

FIG. 4. Analysis of gag proteins of viral particles released by cells after transfection orcotransfection. The analysis was preformed as described in the legend to Fig. 3A, except that for B and D an anti-MA antibody was used in addition to an anti-CA antibody.Transfections wereperformedas described in the legend to Fig. 2B and C. C, control; WT, wild type.

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gag-pol

polyprotein

isto useactive-site mutantsin

comple-mentation

experiments.

Two double mutants were

con-structed: a U76 mutant in which the active-center aspartic

acid of PR has been mutated to arginine, destroying its

proteolytic activity (U76p15.1

[Fig. 1andreference11]) and a U180mutant withthe same additional mutation in its PR

(U180p15.1 [Fig. 1]).

Transfection of cells by mutant

U76p15.1

alone

yielded

viral particles as efficiently as the wild type withanunprocessedgag precursorprotein,

show-ing

that the PR was

fully

inactivated (data not shown).

Cotransfection of cells with mutants

U76p15.1

and U180

produced

viral

particles

asefficientlyasthe wild type, but by

comparison

with the

coexpression

of U180 and

CM5,

there

weremuchmoreunprocessedgagandgag-polpolyproteins

and much lessmatureCA

(made

visible

by

overexposureof

the

autoradiograph) (Fig.

4B andTable

2).

Twofeatures of

retroviruses may offer an

explanation:

the facts that the active PR is adimer and that manymore molecules of gag thangag-polare

incorporated

into virus

particles.

Assuming

that

only

PRdimers

homogeneous

forafunctional active site are

active,

the numberof such active molecules will be small

by comparison

withthe wild typeorCM5-U180

complemen-tation

(in

which there are fewer

copies

of PR but all are

active).

The resultsof theconverse

complementation

exper-iment withmutantsU76 and

U180p15.1

supportthis

hypoth-esis

(Fig.

4Aand Table

2).

In this case, the active protease wasinexcessand thePRofthe gagpolyproteinwasableto cleave the

protease-deficient

gag-pol

polyprotein

com-pletely, resulting

in reverse

transcriptase activity

similar to that observed in the

complementation

between U76 and U180. It was observed in another

study (3)

that some

mutationsin the PR active site of U180 affected the

ability

of PR to activate the

RT, probably through

an effect on the precursorstructure: ourmutation of

aspartic

acidto

aspar-agine appeared

to have no such effect. The control

experi-mentin which U76

p15.1

andU180

p15.1

werecoexpressed

yielded

viral

particles containing

unprocessed polyproteins

(Fig.

4C).

Thus,

it appears that the PR present ingag-pol

may be

dispensable

for

processing

of gag and gag-pol

polyproteins although

it is

intrinsically capable

of

autocata-lytic self-cleavage

and precursor

processing.

The data

presented

here are,atleast in part,contradictory

to two studies

published

while our studies were

being

completed

and submitted

(3, 14).

The datafrom theanalysis

of

point

mutations of the active site of the PR of gag are similar to our data. In one report

(3),

no CA but some RT

activity

was detected in the released

particles,

and in the othernoCAorRTwasdetected

(14). However,

inour

study

of active-site

point

mutants very lowlevels of CA and RT

were detected. The

analysis

of gag PR deletionmutantsby

these groups

yielded

results

essentially

identicaltothe

point

mutation studies.

Thus,

thesetwogroupsconcluded that the embedded PR is inactive. In contrast, ourdata from

com-plementation

between CM5 and U180 showed

significant

levels of

polyprotein processing and, thus,

that the

embed-ded PR can be active. An

explanation

for the difference betweenourdata and the

study

of Craven etal.

(3)

maybe the nature of the protease deletion construct. With our

construct CM5

(in

which a stop codon

replaces

the first codon of

PR), production

of virus

particles

was

significantly

reduced when the construct was

expressed

alone or with U180

(reference

9and

Fig. 4A), suggesting

arolefor the PR domain of gag in

particle assembly.

Thisreductionwasalso observed

by

Stewart and

Vogt

(14)

with an identical

muta-tion.

However,

in mutant 3h of Craven et al.

(3)

the first

sevenamino acids ofPRarepresent and thismutantis

fully

efficient inparticle formation, suggesting that this part of PR is the domaininvolved in efficient assembly. If these amino acidsareable tointeract with the PR domain of gag-pol, the effectonPRactivity may be similar to that observed with the active-site mutants inwhich an inactive dimer is formed.

Inthe study of Stewart and Vogt, they established a cell lineexpressing gag-pol constitutively and then introduced a plasmid encoding a protease-deficient gag. The reason for thediscrepancywith out data may result from the levels of gag andgag-pol expression, since we have shown that at

highratios ofgag-poltogagexpression particle formation is rendered muchless efficient. However, it should be stated that theirimmunoblots of cell lysates do not suggest a large difference inexpression.

Duringtheconstruction of mutant U180, a one-amino-acid deletion occurred in the linkerpeptide. It is possible that this mutation hasan effecton theactivity of the PRof gag-pol. Anearlierstudyshowed that sequences at the C terminus of the PR of gagdo affectactivity (1).However, to explain our

data,this mutation would havetoresult in increased

suscep-tibilitytoprocessingof the precursoratthisjunction, either

by PR itself or by a cellular protease (that gag-pol is not

apparently processed intracellularlyexcludesthe latter

pos-sibility).

Deletion ofthe N terminus of thegag polyprotein can be rescuedby complementationwiththegag-pol polyprotein. The complementation assay thatwedeveloped can also be used tostudytherole of the gagportionofthe gag-pol polypro-teinbyassayingfor therescue of any gag gene mutants by

wild-typegag-pol polyproteinprovided in trans. It is thought that the MAprotein located at the N terminus of the gag gene is involved in the interaction of the gag polyprotein with the plasma membrane (13). We therefore constructed double mutantMinU76(Fig. 1),which carries adeletion of the N terminus of the MA protein. This was achieved by

changingthe normal AUG initiator of the gag geneto TTC

so that translation should begin at a downstream

AUG,

probablythe second AUG in the MA sequence, resulting in

synthesis of agag polyprotein shorter by 28 amino acids.

Transfected alone, MinU76 synthesized a truncated gag

polyproteinof about 72kDa,asexpected,atabout half of the

efficiencyof Pr76 synthesized by the wild type (3a) but did notproduceanyparticles (Fig.4B).Asimilar mutant which

displays the same phenotype has already been described

(18), which is compatible with the hypothesis that the N

terminus of the MAprotein is needed for interaction of the gag

polyprotein

with the plasma membrane (since U76

produces

particles

as efficiently as the wild type). Mutant U180 was therefore used to complement mutant MinU76

(Fig. 4B). Relatively few particles were produced by

com-plementingMinU76by U180, comparedwith the

cotransfec-tion of U180 and U76(<5% attheequivalent plasmidDNA

input ratio; this is not overspill from the adjacent lane, as

equivalent results were obtained when the proteins of the

viral mutants were electrophoresed separately). The

parti-cles had reverse

transcriptase activity

about fivefold higher

than thatof theparticles produced by coexpressionof U180 and U76 when the activity was normalized to their CA

protein content (Table 2). Thus, it appears that the N

terminus of thegag-pol

polyprotein

is abletodirect the gag

polyprotein lacking

the first 28 N-terminal amino acidstothe

plasma membrane, allowing formation of viral

particles,

although this rescue is inefficient and the

proportion

of

gag-pol to gag is higher than in wild-type

particles.

The converse

experiment

was carried out

by

coexpressing

an-otherdouble mutant, MinU180

(analogous

to MinU76 [Fig.

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http://jvi.asm.org/

1]), with single mutant U76 (Fig. 3D and Table 2). In this case,the phenotype of theparticlesproducedwassimilarto that obtained bycoexpression of U180 and U76atthe levels ofviral protein synthesis (Fig. 2B), particle formation (Fig.

3D), and reverse transcriptase activity (Table 2). Control experiments in which MinU76 and Min U180 were coex-presseddid not yield anyviral particles (Fig. 3C). Thus, the N terminus of the gag-pol polyprotein is dispensable for rescue of gag-polfusion mutant U180 by the gag polypro-tein. Thedifferent efficiencies ofsynthesis andincorporation

of gag and gag-pol and the role of the Nterminus of gagas amembrane"anchor" and in the transport of the precursor proteins to the plasma membrane couldprovidean

explana-tion for the differences between these two complementa-tions. It may be hypothesized that eachgag-pol molecule interacts onlywith a small number of gag molecules during virus assembly (rather than all 19 in excess). Therefore, in the complementation between U180 and MinU76, the gag-polmolecules interactwith, and transporttothemembrane, fewergag molecules than would reach theplasma membrane inthe wild type.This results in the phenotypeobserved, i.e., fewerparticles andanincreasedproportion of gag-pol in the virions. In thecomplementation between MinU180 and U76, according to this model, there will be no limitation or hindranceof the interaction between gag andgag-polorthe

efficiency with whichgag-pol is transported to the

mem-brane, resulting in the phenotype observed. Rescue of a deletion mutantofthe N terminus of gagbywild-typegag has been reported (18).

The observation that a mutant such asU180, expressing only the gag-polpolyproteinbutproducingnoparticles,can be rescued by coexpression with a mutant such as U76, expressing only the gag polyprotein, implies that there isan interaction between the two precursors and that some re-gion(s) of the gagpolyprotein is involved in thisrescue. By using the complementation assay described here, we can exclude from this function the first 28 amino acids of theMA protein of the gag-polpolyprotein but the PR domain may have a role, since CM5 can only partially rescue mutant U180. The dataare in agreementwith those of Stewart and Vogt (14), but it should be noted that deletionmutant3h of Craven et al. (3),which has the first seven amino acids of PR, is fully competent in particle formation. Studies of

complementation between gag mutants should, therefore,

prove useful in analyzing the role of this gene in the retrovirus lifecycle.

We are indebted to 0. Donze for construction of the vector carrying the Min mutation and to P. Damay for excellent technical assistance. Wethank D. Bonnet for critical reading of the

manu-script,P.Dupraz and0.Donze for helpful discussions, D. Rifat for

oligonucleotide synthesis,and0.Jenni for photographs and figures. We gratefully acknowledge the financial support of the Swiss League againstCancer (to S.O.). This research was supported by grant 31-29983.90 fromthe Swiss Foundation.

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