A specific inhibitor of cysteine proteases impairs a Vif-dependent modification of human immunodeficiency virus type 1 Env protein.

0022-538X/91/031325-07$02.00/0

Copyright © 1991, American Society for Microbiology

A

Specific Inhibitor of Cysteine

Proteases

Impairs

a

Vif-Dependent

Modification of

Human

Immunodeficiency

Virus

Type

1

Env

Protein

BRUNO GUY,'* MICHEL GEIST,1 KARIN DOTT,' DANIELE SPEHNER,"12 MARIE-PAULE KIENY,1 ANDJEAN-PIERRE LECOCQ'

Transgene S.A., 11 rue de

Molsheim,l

and Institut National de la Sante

etde laRechercheMedicale

U74,2

67000

Strasbourg,

France Received 22 August 1990/Accepted 3 December1990

The Vif protein of human immunodeficiency virus type 1 (HIV-1) regulates viral infectivity. Virions produced in cell culture after transfection by a Vif-negative molecular clone show a dramatic decrease in

infectivity forsusceptible CD4+ celllines, although theVifprotein doesnotappeartobeaconstituent of the

viral particle. Theexactmechanism by which Vif affects HIV-1 infectivity issofarunknown. We reportthe existence ofstructuralhomologiesbetweenVif and afamily of cysteineproteasesandpresentevidence which suggeststhatoneof the targetsof Vif is the Envproteinandmoreprecisely thecytoplasmic domain of gp4l. Vifwasfoundtomodifyboth the processing and conformation of the Env protein. Ethyl(25, 35)-3[(5)-3-methyl-1-(3-methylbutylcarbamoyl)]oxirane-2-carboxylate, aspecific inhibitor of cysteineproteases,inhibitstheeffect ofVif,asdoesthemutation of Cys-114toLeu inVif. Furthermore, Cys-114 of Vif produced in Escherichiacoli, interacts directlywithtrans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane. These observationssuggestthat

acysteineprotease activityis associated withVif and that this activity playsa rolein Env maturation.

TheVifprotein belongs toa group of human

immunode-ficiency virus (HIV) regulatory proteins whose functions have not yet been elucidated. The vif gene is located

betweenthepolandenvgenes (32)onthe HIVgenome,and

related open reading frames have been reported in other lentiviruses such as HIV type 2(HIV-2) (7), simian immu-nodeficiency virus (SIV) (3), visna virus (26), and feline immunodeficiency virus (FIV) (29). However, thesequence

homology between these genes is poor. Vif from HIV-1

encodes a 23-kDa protein which is produced both in vitro

after infectionofsusceptible CD4+ human cell lines and in vivo during thecourseof the disease. Human HIV-positive seraspecifically immunoprecipitatethevifgene product(13).

Viffunction isessential for the full infectivity of the HIV-1 virion (14) since infectious Vif-negative cDNA clones of HIV-1generateviralparticlesaftertransfection,thatare2to 3 orders ofmagnitude less infectious than normal particles (4, 28). Vif is thought to act at a posttranslational level;

however, the exactmechanismhasnot yetbeenelucidated (4). In this report, we have investigated the biological activity of Vif and itspossible interactions with other HIV proteins, particularly Env, usingin vitro-producedVif from Escherichia coli and recombinant vaccinia viruses (VVs) expressingVif in mammalian cells. We have observed that expressionof Vif in BHK-21 cells is linkedtoamodification

of the C terminus ofgp4lenv and that this modification is inhibited by trans-epoxysuccinyl-L-leucylamido-(4-guanidi-no)butane (E64), a specific inhibitor ofcysteine proteases. Amino acid sequence analysis and the effects of point mutations in Vif suggest that Vif could be acysteine prote-ase.

MATERIALS AND METHODS

Construction ofplasmidsand recombinantviruses. The vif

gene contained in M13TG185 (20) (fragment

BglIl-EcoRI)

*Correspondingauthor.

wasinserted under the control of the bacteriophage lambda

promoterpL into pTG959, generating pTG4190. By using oligonucleotide TG2540 (5' GCAGAGTCTGAAAAGAGC TCAAAGTAATACAG 3'), Cys-114 was changed to Leu. Thecorrespondinggene wassubcloned into pTG959,

gener-ating pTG4193, or transferred to the VV genome (12), generatingVVTG4185. By usingoligonucleotideTG2658(5' GCTTGATATTCAAGCTTAGGGCTAACT 3'), Cys-133

was changedto Leu. The mutatedgene was subcloned into

pTG959, generating pTG5106. By using oligonucleotides TG2540 and TG2658, the vifprotein was mutated at both cysteines. The corresponding expression plasmid is pTG5115.

Expressionof Vif in E. coliandlabelinginthepresenceof E64. After transformation of the TGE901 E. coli strain (containing a thermosensitive c1857 repressor of bacterio-phage lambda) by pTG4190andinductionat42°Cfor4h,the cells werepelleted andsonicated. The sameprocedure was

used for the Vif with mutationsatCys-114, Cys-133,orboth (pTG4193,pTG5106, andpTG5115,respectively).Acontrol plasmidwastransformed intoTGE901 cells,and the culture

was induced by the same procedure. The insoluble and solubleproductswere analyzedon asodiumdodecylsulfate (SDS)-polyacrylamide gel stained with Coomassie blue or

transferred onto nitrocellulose for Western blot (immuno-blot) analysis, using monoclonal antibodies against Vif. In

experiments involving E64, 40 ,lI of soluble extract in phosphate-buffered saline wasincubatedin thepresence or

absence of 10,uME64 (Sigma)for 30minat37°Cand labeled with 10 ,uM 14C-labeled iodoacetamide (Amersham) for another30minat37°C.Theextractswereloadedon anSDS polyacrylamide gel. After migration, thegel was fixed and

autoradiographied. All experiments have been reproduced fiveto seventimes depending onthe mutants.

Analysis ofproteins expressed in BHK-21 cells. A total of

106 BHK-21 cellswerecoinfectedwithrecombinantVVs. 15

PFUpercellwasused for controlVV, VVTG1160(20) (Vif),

orVVTG4185 (mutated Vif), and 10PFU per cell wasused

1325

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forVVTG9-1

(12) (Env)

orVVTG1131

(mutated Env)

(11).

After 1 h of

adsorption

at

20°C,

cells were labeled for 4 h

with

[35S]methionine (Amersham)

and

immunoprecipitated

withahuman

polyclonal

serum or arabbit

polyclonal

serum raised

against

the last 15 residues of

gp4l (Neosystem,

Strasbourg, France).

Immunoprecipitation

was carried out

at5 h

postinfection.

Themoredramatic effect of VifonEnv

expression

wasobserved 4 and 5 h after infection

(results

not

shown).

The difference between the control VV- and

VVTG1160-infected cells decreased after 6 h

postinfection,

indicating

that a VV

protein

produced

at the late

phase

of

infection could

possibly complement

for the lack of Vif. All

experiments

have been

performed

at least five times with

different

preparations

of recombinantVV.Thetitersof virus used in each

experiment

were determinedtwice.

For Westernblot

(immunoblot)

analysis,

extractsof

unla-beled cells were loaded 5 h after infection

directly

onto a

15% SDS

polyacrylamide gel

and transferred

by

standard

protocols

onto nitrocellulose. Mouse monoclonal

antibody

directed

against partially purified

E.

coli-produced

Vifwas

produced

atTransgene (17a).

As

needed, ethyl(25,

35)-3[(5)-3-methyl-1-(3-methylbutyl-carbamoyl)]oxirane-2-carboxylate (E64-D)

1at10or100

jiM

(synthesized by Neosystem)

wasadded tothe medium with

[35S]methionine.

A

nonspecific

effect on

protein synthesis

wasobservedatdoses

higher

than 100,uM

(data

not

shown).

All

experiments

have been

performed

morethan five times.

Westernblot

analysis

of HIV viral

proteins.

Westernblots

were carried out

following

the instructions and

using

the reagents of the Pasteur

Diagnostics

New Lav blot I kit. When the

peptide

was

used,

it was

preadsorbed

with the rabbitserum 0.5 h at

4°C

and remained present

throughout

the

experiment (1

jg/ml).

The

experiments

were

performed

with

antigen samples

on

strips

derived from thesamegeland

were

reproduced

with different batches.

RESULTS

Homologies with cysteine proteases. Our initial efforts to

discover a

putative activity

for Vif involved a computer search for

homologies

between Vif and amino acid se-quencesin

protein

data bases. Because the Vif

proteins

of HIV-1andHIV-2/SIVare

only slightly

homologoustoeach other

(about

30%

homology

between HIV-1 andHIV-2),we

focused on small conserved

regions.

A domain rich in

tryptophan,

located downstream of His-48 of

HIV-lBRU

and

spanning

about 30 amino acids, was found to share some

homology

witha domain of the

cysteine

proteasecathepsin

B

(Fig. 1);

therefore this

family

of proteases was examined

more

closely (18). Cysteine

andhistidine residuesarecritical

for the

activity

of

cysteine

proteases so we looked for

conservation oftheseaminoacids in ViffromHIV-1, HIV-2,

SIV,

FIV and visna virus (3, 7, 26, 29, 32). Localized but

significant

similaritieswerenoticed aroundtworesidues(His

and

Cys)

involved in the active site of thiol proteases (18)

(Fig.

1). Although the Vifproteins of the different

lentivi-ruses showlittle sequence similarity, thehomology of these

putative

active sites is intriguing. FIV Vif displays a

se-quencemotifparticularly closetothethiol protease

consen-sus sequence. Both Cys and His are located in secondary structures

predicted

by Chou and Fassman (DNA STAR program;

IBM)

to be similarto those ofcysteine proteases

(data

not

shown).

It is however noteworthy that the His

domain of the Vif

proteins

of lentiviruses is positioned

upstream of the Cys domain, unlike known thiol proteases

(18).

Expression of the Vif protein. We therefore sought to determine whether Vifpossesses biochemical activities as-sociated with thiol proteases. Thevif genewasexpressed in E. coli by using plasmid pTG4190 (Fig. 2A). Since we suspected thatCys-114 might be involved in the active site of Vif, we mutated this Cys codon into Leu to generate pTG4193. For controls,we mutated ina similarmannerthe otherCys codon (133) inpTG5106 and both Cys codons(114 and 133) in pTG5115. After introduction into E. coli and inductionat42°C, these plasmids directed theproduction of large amounts of Vif protein (about 30% of the insoluble proteins) of which only a small percentage remained soluble (Fig. 2B). Two species of Vifprotein were visualized after Coomassie blue staining of the SDS polyacrylamide gel; the larger one comigrated with Vif expressed in mammalian cells. We have shownby protein microsequencing that the larger species corresponds to a Vif protein initiated at the first methionine (nucleotide 4587), while the smallerspecies is derived from initiation at a GTG valine codon atposition 10(not shown).

The vif gene was also expressed in mammalian cells by using recombinant VV VVTG1160 (20) encoding HIV-1 Vif and VVTG4185encoding the mutated vifgene corresponding to pTG4193 (protein with Cys-114--->Leu). Each of the recombinantproteinswasrecognizedby antibodies directed against a Vif protein produced as a fusion protein in E. coli (unpublished results).

InteractionofVif with E64. E. coli-producedVifwasthen examinedby labeling inhibition foraninteraction with E64, a specific thiol protease inhibitor (1). Soluble extractsfrom E. coli containing Vif were labeled with radioactive

iodoac-etamide,whichreactswithcysteine residues. Vif, which was

expected to be labeled on two cysteines (114 and 133)

(pTG4190), migratedas afuzzy band after gel

electrophore-sis (Fig. 2B, panel c, lanes 2). Preincubation with E64 (10

p.M) prior to labeling with iodoacetamide modified the apparent migrationofVif, indicatingthat E64 could bind to at least one of the two cysteines. To clarify this point,

iodoacetamide labeling was performed on the mutant Vif

proteins. E64 abolished thelabeling ofCys-114 (pTG5106), while it had no effect on the labeling of Cys-133 (pTG4193)

(Fig.2B,panelc,lanes 4 and3). As expected, the Vif protein

devoid ofcysteines (pTG5115)was notlabeledwith

iodoac-etamide. Although a large number of E. coli proteins were

labeled with iodoacetamide in ourpreparations, labeling of Vifwastheonly modification induced by E64, indicating that thiseffectwasspecific(data not shown). Incubation prior to

labeling with inhibitors of serine proteases such as

phenyl-methylsulfonyl fluoride and soybean trypsin inhibitor (STI) didnotmodifyViflabeling. We also performed Western blot

analysis of native and mutated Vif proteins following

incu-bationwith E64. Under theseconditions, we observed a shift in the electrophoretic migration of native Vif (data not

shown). This indicates that E64interacts directly with Cys-114and suggests that theCys-114 residue in HIV-1 Vif is in a domain which is structurally related to the active site of thiol proteases.

Interaction between Vif and gp4le"v. We then sought to determine the putative substrate of Vif. Immunofluores-cence and immunogold techniques (2) localized Vif, in recombinant VV-infected cells to the Golgi apparatus and on the surface of vesicles (results not presented). A similar

localization, although inside related vesicles, was obtained with a recombinant VV expressing Env and we surmised that Vif and thecytoplasmicdomainofEnvmight interact in vivo. To test this possibility, we investigated whether the

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S D S A I

T AG V E M E S I

N L M C L

A F G A V

R 1 VIF HIV1 - BRU

R 2 VIF HIV2 ROD/SIV mac

F

IR

IE ITIF S T TIG IAIF S A VIV

A L

F S A IhA

L A A IhG

y y

3 Q VISNA 4 VIF (?) FIV 5 Cathepsin B 6 Cathepsin H 7 Papain 8 Actinidin

9 Calcium protease

W W

E S

PIHIP

RII

Y V P

R G Q

W S F H H

HI

V M

GIHI

H K - K T R A-V L y I

K V NIHIA -IV

A V DIH K V D V K G

H H A -A -A -I v y 3 9 - YI R L v I A

II

A A 7

V H I

V I F

M F V I G Y I G W

V G Y

V G Y V G Y

8 7 5 13 5 5 5 3

I T Y W G

L

H T G E

I

Q

A

Y W

N L

-

T P

E

Y

QEA Y W

E

N -

T

S G

L

RM

Y I Y I ---

S

V -P Y

W

L

V - -

A

L - L Y W I V - -K

-V

D- Y

W

I

V - -K

N D G Y I L I -- -K

Q

L I RI-- R

C

* *

W W W H W W W C C

5 11 21 48 71 81 89 114 133 192

VIF

HIV13Ru

FIG. 1. Analysisof thesequencescorrespondingtotheputative active site of Vif andrelatedgenesofHIV-lBRU, HIV-2ROJSIVmac, FIV,

andvisnavirus,comparedwith theactivesiteofthiolproteases.Asterisks indicatetheCysand Hisconserved residuespresumably important

for Vifactivity. Gaps have beenintroduced to align all the related proteins. The single-letter code is used. Residues considered to be

homologousareboxed.Residuesaregroupedasfollows:hydrophobic (L,I,V, F,Y,andW), hydrophilicorneutral(P, G, T,S,andA),basic

(K and R), andacidic (DandE). Asparginine (N) andglutamine (Q) residues are boxedwith acidic residues. (a) Sequences surrounding

Cys-114 (HIV-lBRU), -116 (HIV-2ROD/SIVmac), -222(visna virus), and-187(FIV) compared with those surroundingthe active Cys ofthiol

proteases. (b) Sequences surrounding His-48 (HIV-lBRU), -44

(HIV-2RODJSIVmac),

-57 (visna virus), and -78 (FIV) compared with those

surroundingtheactiveHisof thiolproteases. (c)Conservedresidues inHIV-lBRU Vifproteincomparedwith HIV-2ROD and SIV

Env protein was modified in BHK cells coinfected with

recombinant VVsexpressing Vif andEnv (VVTG9-1) (12). Thetotalamount of Envprotein present inthe cellpellet afterimmunoprecipitation wassimilarin thepresence(V)or

absenceofVif (W);however, theamountofgpl20 released into the medium of the infected cells markedly increased whenEnvwascoexpressedwithVifcomparedwith when it

was expressed with a control VV (Fig. 3a). For another

control, coinfection of VVTG1160 (Vif) and recombinant

VVsexpressingp55gag,

p259ag

or

p189a9

proteindid notlead

to detectable differences in the pattern of expression or

release of theseproteins (datanot shown). Addition of 1 to

100,uMof E64-D (25)(a cell-permeablederivativeofE64)to

cellsexpressingbothVifandEnv decreasedthe sheddingof

L I L I T I

H L Y H S T

P W S

a

ADQ A D V

MGY

GE I A Q I

S PV

IT

PIV

V D I

TD

I

Y F DC F YF P C F

LQrc

w

C GDHCW

C G SC W

C G S CW

C G S CW

C G G CW

LG DCW

D P K K W R D Q G Si

K N Q G A K N Q G S K S Q G E C - Q G A

b

H

*

R D W

K G W

K Q W

N P W

IN

SW

6

6

6 H -N

-I E

W

I

W

L D L

SQ

Y

V D

W

SN W

T

GW

T

T W

V E

W

1 2 3 4 5 6 7 8 9 IN S WIG

-;lN

S

WIG

-IN S

WID

-N P W|G Q

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E.coli

Vaccinia

A

pTG

VV

TG

c c

114 133

L

X c

114 133

L

C

X

LI

I

I

1 14 133

L

L

L.

_-

I --1

b

1 14 133

C - +

_10

.._WM

1 2

4190

4193

1160

4185

5106

5115

_ + - 4

._ ._-Nw ._,

3 4

4

14

a0

B

p

S

a

10.

v

- V V.Virus

FIG. 2. (A)Schematic representationof thevifgenesinserted into E. coliplasmids and thegenome.The vifgenefromHIV-1BRUwas

expressed in E. coli and in mammaliancells,usingrecombinant VV. Thevifgenewasexpressedin E.coliby usingpTG4190.Thevifgene

mutatedsothatCys-114waschangedtoLeu in VifproteinwasexpressedbypTG4193,whereas inpTG5106,Cys-133waschangedtoLeu. InpTG5115,bothCys werechangedtoLeu. Thenative andCys-114-+ Leumutated Vifgeneshave beenexpressedfromrecombinant VV

VVTG1160 andVVTG4185, respectively. (B) Expression of the vifgene in E. coliandlabelingwithiodoacetamide. (a)Coomassie blue stainingofthe insoluble(P)andsoluble(S)fractions ofacontrol culture of E.coliexpressingVif(V).The arrowheadspointto the twospecies of Vifobtained intheextracts(initiationatthe first Met andatVal-10).(b)Westernblotanalysisof the soluble fractions of E. coli cultures

expressingVif(V) and of theVVexpressing Vif(VVTG1160)(V.Virus).Themutated Vifproteinswererecognizedto thesame extentas

parental Vif(datanotshown) byamonoclonal antiserum.(c)Labelingof the E.colisolubleproductswithiodoacetamidefollowingincubation

without (-)orwith (+) 10 ,uM E64(Sigma). Lane 1, control (pTG959 without foreign insert);lane2,native Vif(pTG4190);lane3,Vifwith Leu-114(pTG4193); lane 4, Vif with Leu-133 (pTG5106);lane5,Vif mutatedatbothcysteines(pTG5115).Thefigureshowsonlythe smaller

species of Vif; the larger speciescomigrated withabackgroundE.coliproteinlabeled with iodoacetamide. Theupperbandcorrespondsto

anE. coliprotein whose quantity variesdepending ontheexperiment.

gpl20 into the medium (data not shown). Furthermore, expression of Vif mutated at Cys-114 (VVTG4185) had no

effect on Env shedding. When VVTG9-1 was replaced by

recombinant VVTG1131, which expresses an Env gene

whose sequencecoding forthe cytoplasmic and transmem-branedomainswas substitutedby theequivalent sequences

from therabies virus glycoprotein (11),noeffect of Vifwas

observed (Fig. 3b), suggesting that the cytoplasmic tail of gp4l is the target of Vif activity. SDS-polyacrylamide gel electrophoresis withhigh salt concentration in the samples revealed that coexpression of Vif and Env results in a

slightly increased mobility of gp4lenV, possibly correspond-ingtoalossofapproximately 1kDa (datanotshown). This difference in migration was also observed after complete

deglycosylation of gp4l by endoglycosidase F (data not shown). Becausesuchaposttranslational modification could

be a consequence of a proteolytic cleavage of gp4l, we

therefore examined the extreme C terminus of the Env

precursorpolypeptide.

Antibodies were raised in rabbits against the last 15

residues of the gp4l ofHIV-lBRU (32) and used to immu-noprecipitate the [35S]methionine-labeled gpl60 precursor

and gp4l synthesized in VVTG9-1-infected cells. Similar amountsof thegpl60precursor(Fig. 3c) were

immunopre-cipitated from cells coinfected with VVTG1160 (Vif) and VVTG9-1 (Env) or with control VV and VVTG9-1. In contrast, the amount ofgp4l immunoprecipitated was

sig-nificantly decreased when Vifwas coexpressed with Env,

suggesting that the C-terminal end of gp4l had been

re-moved. In cells coinfected with VVTG9-1 and the virus encodingVifmutatedat Cys-114, gp4lenv was

immunopre-cipitated as well as in the singly infected sample. The addition of E64-D(1 to 10,uM) restored thelevel of immu-noprecipitated gp4l tothe control level. No difference was

observed in the controls in thepresenceorabsence of E64-D (datanotshown). Furthermore,theincreasedrecognitionof gp4lin thepresenceof E64-Dwasnotduetoaninhibition of

lysosomal thiol proteases, as no corresponding increase in 5

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P S b P S C

;

*~~~~~~~~-

GP

160

i,

4

GP120

w4

vw vw

WV WV a

i

"'

Ili -- GP

160

L.-Fw

*.

_{b_}

+*o

_s,

_{ts_}

ho__.

_.A

_mb._

VIF*- Ur

v w

1

2

3

4

5

FIG. 3. (a) Immunoprecipitationbyahuman HIV-positiveserumofEnvproteins expressed inBHK-21cellscoinfected with recombinant VVTG9-1 expressing the nativeEnvprotein ofHIV-lBRU andacontrol VV(W)orrecombinantVVTG9-1 and VVTG1160 expressing the Vif

protein of HIV-lBRU(V).P,Pellet;S,supernatant.The positions ofgp160,gp120, and gp4lareshown. At the bottom of panelaisaWestern blotof thesamecellextractsusingamonoclonal antibody directed against the Vif protein. (b) Immunoprecipitationof Env proteins expressed by VVTG1131 (rabies virus glycoprotein cytoplasmicdomain). The protein correspondingtogpl60comigrateswithgp120as aresultofthe reduced size of its cytoplasmic domain.(c) Immunoprecipitationbyarabbit polyclonal serumdirected againstthe C terminusof gp4lenv proteinsexpressed in BHK-21cellscoinfectedwith VVTG9-1 and VVTG1160 (nativeVif)(lane 2),controlVV(lane3),VVTG4185(mutated

Cys-114) (lane 4),orVVTG1160 (native Vif) inthepresenceof 10,uME64-D(lane 5). Lane 1, Control VV alone.

gpl60wasobserved.Moreover,as noincreasein theamount

of gpl20 was noticed (not shown), it seems that E-64

specifically affects gp4l recognition by the rabbit antiserum. Whenthe same immunoprecipitations wereperformed with

ahumanpolyclonalserum,gp4lwasrecognizedtothesame

extent in the presence or absence ofVif. Taken together,

theseobservations arein agreement witha modification of

theCterminusof gp4l by Vif and itssensitivitytoinhibition by E64. Thismodification ismostlikelyaproteolytic

cleav-age, Cys-114 being part of the active site of the putative protease.

IfVifentails cleavage of Env during HIV-1 infection, the Cterminusofgp4l shouldbe absentfrom HIV virions. We therefore examined the immunoreactivity of the serum

di-rected against the 15 C-terminal residues of Env, with virion-associated HIV proteins present on a commercially

available Westernblot (Fig. 4). For controls, we used two

mouse monoclonal antibodies directed against gp4l

1 2 3 4 5 6 7 8

160K*

-GP120 *

GP41

0- a

FIG. 4. Western blot of HIV viralproteins presenton a

commer-cial HIV Western blot(NewLavblotI;Diagnostic Pasteur).Lane1, Monoclonal antibody (Mab) 41-1 (Genetic System) diluted 1/500; lane2, Mab MATGO025 directed againstgpl20 (Transgene) diluted 1/500; lane 3, Mab MATGO023 directedagainst gp4l (Transgene)

diluted 1/2,000; lane 4, Mab MATG0023, 1/500; lane 5, rabbit 1

serum, directed against the last 15 residues ofgp4l (Neosystem)

diluted1/500,preincubatedwith 1 ,ugof thecorresponding peptide (Neosystem)perml;lane6,rabbit 1polyclonalserumdiluted1/500; lane 7, rabbit 2polyclonalserum(Neosystem)directedagainstthe last 15 residues of gp4l, preincubated with the corresponding peptide;lane8, sameserumwithoutpeptidediluted 1/100.

(MATGO023 obtained after immunization with purified gpl60 synthesized from VV-infected cells [Transgene]) and gp41-1 (Genetic Systems) raised against the native HIV Env

pro-tein, whichis tetrameric inthe virions.

Aprotein migrating withamolecularmassof 160 kDa has

previously been shownto be astable tetramer of gp4l (19) and thiswas confirmed bytheabsence ofrecognition ofthe

160-kDa species by amonoclonal antibody directed against

gpl20(MATGO025) (Fig. 4, lane 2). The anti-peptide serum

(lanes 6 and 8) reacted predominantly with the tetramer, whereas the control antibodies strongly reacted with the monomeric gp4l in addition to the tetramer (Fig. 4). A monoclonal antibody directed against native HIV Env

pro-tein (lane 1, gp41-1) reacted only with the multimer and is

presumed to recognize a conformational epitope. As the

gp4l monomer present on the blots is most likely derived from aless stable multimer (19), these results indicate that

onlythe stablestructureresistanttoSDS-polyacrylamide gel electrophoresis possess the C-terminal epitope. Vif could thus be involved in this modification of Env through a

proteolytic cleavage, resultinginaless stable multimer.

DISCUSSION

Inconclusion,ourresults indicate that theproduct of the

vif

gene of HIV-1 (and presumably of other lentiviruses)

regulates theprocessing and conformation of the Env

pro-tein. Moreover, we suggest that this regulation is likely to involveathiol proteaseactivity, althoughwehavenotbeen ableto directlydemonstrate the activityof Vif inaninvitro

assay. Delayed Env transport during HIV infection in the

absence of Vif or an abnormal Env conformation could explain the lack ofinfectivityofthevirions expressedfrom

aVif-negative provirus. Since directbinding ofEnv to the CD4 moleculeat the surface oftarget cells isnecessary for infection,virusparticles lackingEnvordisplayingan incom-pletely processedEnvwould notbeable toefficientlyinfect cells. Thispossibilitymaybecorrelatedwith theobservation that Env is notcorrectly processed in CHO cells whenthe

C-terminal domain ofgp4l (8) is present. However, it has

been demonstratedthat Env could be expressed and proc-essed in HeLacells in the absence of Vif(30), indicatingthat

cellular factors or other viral proteins whose activity

de-pendsonthecelltypemayalsoparticipateinsuchaprocess. a

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[image:5.612.91.516.76.194.2] [image:5.612.95.257.495.616.2]

Data concerning the processing of the Env protein in Vif-negative virions are presently lacking, but the fact that the

general

pattern of synthesis HIV proteins is unmodified in

cellstransfected by Vif-negative infectious clones (4) seems tobeinagreementwith a Vif-induced conformationalchange of HIV structural proteins.

Interestingly, it was demonstrated that an HIV mutated

provirus

(X10-1) (5), whose Env C terminus is deleted but

replaced

by different amino acids (KRRRRWVFQSHL

RYL), although replication competent after transfection, is

markedly less cytopathic and has a significantly delayed

kinetics of viral expression when virions produced after

X10-1 transfection are used to infect susceptible cell lines. This studyunderlinedthe critical importance of the natureof the gp4l C terminus. Cleavage of the cytoplasmic tail of transmembraneglycoproteins has already been observed for other viruses. In particular, the cytoplasmic domain was

foundtoregulatethe kinetics of transport of the glycoprotein

(G)

of vesicular stomatitis virus (VSV) (21). Partial or

complete deletion of this domain, as well as the addition of

"poison"

sequences, blocked the VSV G protein inside the

cell or significantly slowed its transport to the cell surface

(21).

This domain ofthe G protein of VSV is susceptible to

proteolytic cleavage, and its accessibility to proteases is

increased in the late steps of intracellular transport (15). However, nocorrelation has been established between

pro-teolytic

cleavageand the efficiency of transport or assembly

ofthe Gprotein. Cleavage also occurs at the C terminus of thetransmembrane proteinof the Moloney murine leukemia retrovirus before virusbudding (peptide R corresponding to the last 16 residues) (24). Examination of the protein

se-quence around this cleavage site suggests that the viral

protease responsible for Gag cleavage is involved in Env

cleavage. Incontrast,the C terminus of HIV-1 Env does not contain aconsensus sequence for the HIV (aspartic) prote-asewhich could thus imply another protease.

In ourmodel,truncationofgp4l, as observed in many SIV and HIV-2 viruses isolated in vitro, could result in

Vif-independent virions, depending on the localization of the stop codon andthe sequenceof the resulting cytoplasmic tail

of

gp4lenv.

Recent data supporting this have shown that

mutationswithin the vifgene of SIVAGM, which synthesizes atruncatedgp4l(gp32), have no effect on virus infectivity in

vitro (23), in contrast to the situation observed with HIV-1

(4, 28). On the other hand, an HIV-2 isolate with a mutated

Vif displayed a decreased kinetics of infection, although

possessing

atruncated gp4l(22). Interestingly, the

localiza-tionof the stop codonin SIVAGM (23) is different from that in HIV-2 and most other SIV clones (3, 6). The remaining

cytoplasmic

tail oftheseisolatesis thus different and would

orwouldnotrequire furthercleavage by Vif. Analysis of the

C terminus ofalltransmembrane proteins in HIV-1, HIV-2, and SIV clones reveals a highly conserved RIRQGL

se-quence (residues 852 to 857 in HIV-lBRU) (32). This

se-quence is located just before the end ofgp4l and is down-streamofaputative

membrane-associated

amphipathic helix

(31). Cleavage could occur after a basic residue (R) or between the Q and G residues as observed for the 3C

proteaseofpoliovirus (9). Interestingly,a related conserved

sequence, RXRQGX, where X is a hydrophobic residue

(residues

712 to 717 in HIV-lBRU) (32), is located just

downstream of the

transmembrane

domain of gp41. The nature andthelocalizationofthis latter site would thus be

similar to the C-terminal site RIRQG, in most SIV and HIV-2 isolates (3, 6), but at adifferent position in SIVAGM

(23).

Nevertheless, truncation of

gp4l

is counterselected in

vivo and the virus could in any case revert to a Vif-dependent situation(10). Interestingly, anSIV isolate which is nonpathogenic in vivo has been described; it

presumably

has a defective Vif according to our

hypothesis,

as the

putatively active Cys has been replaced by a leucine as a

result ofaframeshift mutation (6). Ofcourse, we are aware

that this clone could be less

pathogenic

for several other

reasons.

In preliminary experiments, we have tried to

directly

assaythe

proteolytic

activity

ofthe Vif

protein

purified

from insoluble E. coliextracts. No specific activity was detected by using the solubilized Vif

protein

and the C-terminal peptidecontaining theRIRQG sequence(last 15

residues)

of

gp4l as asubstrate, possibly due tothe denaturation ofthe

Vifprotein expressed in bacteria ortothe fact that Vifacts indirectly on theC terminus of gp4l through anothercellular

protease. Alternatively, the conformation of the substrate

couldbecriticalfor Vifactivity. Experimentsarein progress to express Vif in different systems, in order to

perform

assays under other conditions.

The possible relationship between Envprocessing by Vif and virus infectivity is unclear. One can only speculate on thispoint, takingintoconsideration ourobservationthat the processing, transport, and stability of Env seem to be regulatedbyVif. Since Envrecognizes theCD4 molecule at thecell surface and in a second step enters into the cellvia apH-independent membranefusion involving gp4l

(27),

the

conformation of the gpl2O-gp4l complex must be critical. Vif-negative viruses might thus bind to

CD4+

cells but not enter these cellsif the hydrophobic N terminus of thegp4l

were unable to fuse with the cell membrane.

In addition to the effect of Vif on Env, Vif could have other cellular or viral targets and this point remains to be investigated. Finally, the function of Vif could beimportant with respect to AIDS therapy. Encouraging results have been obtained with inhibitors of the HIV aspartic protease (16, 17), and specificinhibitors ofVif may also bebeneficial

indecreasing the viral load in the infected individual.

ACKNOWLEDGMENTS

We are indebted to H. Kolbe for helpful discussions; M. Mon-signy and his team for invaluable help; A. Balland for help inprotein purification; M. Girard, P. Chambon, and P. Kourilsky for their interest in this work; R. Lathe and R. Drillien for critical readingof the manuscript; and E. Chambon for typing the manuscript.

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