Biologic and molecular characterization of producer and nonproducer clones from HUT-78 cells infected with a patient HIV isolate

Mary

Ann

Liebert,

Inc., Publishers

Biologic

and Molecular

Characterization

of Producer

and

_Nonproducer

Clones from HUT-78 Cells

Infected with

a

Patient

HIV

Isolate

MAURIZIO

FEDERICO,1

FAUSTO

_TITTI,1

STEFANO

_BUTTÓ,1

ANGELA

ORECCHIA,1

FRANCESCA

CARLINI,1

BRUNELLA

TADDEO,1

BEATRICE

MACCHI,2

NICOLA

_MAGGIANO,3

PAOLA

_VERANI,1

and

GIOVANNI B.

ROSSI1

ABSTRACT

HUT-78 cells

were

infected with

a reverse

transcriptase (RT)-positive

supernatant

of

a

cul-ture

of

_peripheral

blood

_lymphocytes

_(PBL)

from

an

AIDS

patient

and then cloned. Of

these

_clones,

two

have

been isolated and

characterized.

Clone DIO is

_persistently

_and

pro-ductively

infected

with

an

HIV variant. The clone

F12,

in

spite

of

the presence of

an

inte-grated

full-length

HIV

_provirus,

does

not

release virus

_particles

in the medium.

DIO and F12 clones

_{substantially}

differ in

terms

of

_protein

_pattern;

that

_is,

_{DIO is}

super-imposable

to

infected HUT-78

cells,

whereas F12 exhibits

a

decreased

uncleaved

p55

_gag

precursor and the presence of uncleaved

«pi60

and of

a

unique pl9,

although they

do

not

show

_qualitative

or

quantitative

differences in viral RNA

synthesis.

Restriction

_patterns

of

F12

_proviral

DNA

do

not

show

_{major genomic}

deletions. These

results

indicate that

F12

clone cells carry

an

HIV genome with minor mutations that

probably

affect the

correct

production

of viral

_proteins

at a

posttranscriptional

level.

In

addition,

the F12 clone is

resistant

to

_{high-multiplicity superinfection}

with HIV-1

or

HIV-2.

INTRODUCTION

A

peculiar feature of human immunodeficiency virus

(HIV),

the

etiologic

_agent

of the

acquired

immunodeficiency syndrome

(AIDS)

and associated clinical

_disorders,

is the

_biologic

and

_genetic

heterogeneity

among different isolates.

'Laboratory

of

_Virology,

Istituto

_Superiore

di

Sanità, Rome,

Italy.

department

of

_Experimental

Medicine, 2nd

_University

ofRome, Tor

_Vergata,

Rome,

Italy.

'Department

of

_Pathology,

Université Cattolica del S. Cuore, Rome,

Italy.

Since 1985 wehave

performed

a

large

number of HIV isolations from

patients

withAIDS or

AIDS-re-lated

_complex.1

_Preliminary

characterization

_suggested

_{that isolates vary}both interms of

_biologic

behavior

(i.e.,

differential

_ability

to

_productively

infect human

_cells)

and of structural

_components

_(i.e.,

_viral pro-teins and restriction

_patterns

of

_proviral

_DNA).

These results are in

_agreement

with data from different

laboratories. Evanset

al.2

foundthatvarious

CD4+

human cellsare

differently

sensitivetothe infection

_by

thesame HIV

isolate;

at thesame

time,

different isolates may or_may notinfect the same cell

_type.

Also

genomic variability

of HIV

_isolates,

_analyzed

via DNA restriction

_patterns3-7

or

by comparing

the

se-quences of differentcloned

HIVs8

has been

_reported.

Taken

_together,

_{these data demonstrate that HIV isolates vary}

_remarkably

_{in nucleotide sequence} and

consequently

in

_{protein composition.}

Themostvariable sites are located in theenv_{gene, which codes for}

the

_{envelope proteins,}

andmore

_precisely

ina

region coding

fortheextracellular

portion

of HIV

gpl20.9-10

Biologically,

the

_{genomic variability}

of

_HIV,

also

_typical

of other

_{lentiviruses,}

such as the

_equine

infec-tious anemia virus

_(EIAV),

can

_play

a role in

fighting

the host immune defense mechanisms

during

the courseof

infection.1112

The

_great

_variability

ofHIV_{poses the}

_question

of whetherit is

_possible

todetect in the same

_subject

the

simultaneous presence of two or more

genotypically

distinctHIV

variants,

derived either

by

an in vivo

superinfection

with different HIVs or

by

the de novo_{formation of}HIV _variants

_during

_thecourse _ofHIV

infection. This

_possibility

was _raisedwhen the presence _oftwo

complete

_{genomic equivalents}

was

recog-nized

_{by HIV-specific}

probes

both intheDNA of H9/HTLV-IIIB cells

_(originally

infectedwith amixture

of HIV

_isolates)13

and in the DNA of cells infected witha

single

HIV

isolate.1>3'5

More

_{recently, Koyanagi}

et

al.14

have isolated two related but

_{genotypically}

distinct variants of HIV from two different sources

(brain

tissueand

_{cerebrospinal}

_fluid)

of thesame

patient.

In

addition,

Von Bliesenet

al.15

have isolated and

molecularly

cloneddifferent

_genetic

variants of HIV from

_peripheral

blood

_lymphocytes

_(PBL)

ofa

single

patient.

Inour

laboratory

we have infected HUT-78 cultures witha reverse

transcriptase (RT)-positive

superna-tantof PBL fromanAIDS

patient.

A

subsequent

cellular

cloning by

the

limiting

dilution method_{gave rise}

to several

clones,

two of which are characterized here. One clone releases HIV

virions,

and the other

exhibits HIV markers but does notrelease

_{RT-containing}

virions orviral

proteins

and cannotbe

superin-fected

_by

other HIVisolates.

MATERIALS

AND

METHODS

Cell cultures and

_cloning

Separation,

stimulation,

and cultivation ofPBLfrom AIDS donorswere described

elsewhere.1

Allcell

cultures

_(uninfected

_HUT-78,

_DIO,

and F12

clones,

HTLV-IIIB-infected

HUT-78)

were grown in RPMI-1640 medium

(Flow

Laboratories, Scotland)

_containing

20% inactivated fetal calfserum

(FCS)

and

split biweekly.

Cell

cloning

was

_performed

by

the

limiting

dilution method in96-well

plates.

HIV

_infection

and detection

HUT-78

_cells,

_previously

treatedwith

_polybrene

(1

p-g/ml; Sigma,

St.

Louis, MO),

were infected

by

an

ultracentrifuged

(200,000

x _gfor 30

_minutes)

_RT-positive

supernatant

(about 10,000

cpm per

106

cells).

Virus was absorbed for 1 hour at

37°C

with occasional

_shaking,

and then cellswere

resuspended

in

com-plete

medium and

_split

at the concentration of 3 x

105

_{per ml twice}a week. The presenceofHIV in the

supernatant

was _monitored

by

reverse

transcriptase

_assay as

described,1

and/or

by antigen

_capture

_assay

(Cellular

Products

_{Inc., Buffalo, NY).}

Electron

_{microscopy analysis}

Cellswere

prefixed

in

suspension

with 2%

_{glutaraldehyde}

in 0.1 M

_phosphate

buffer,

_pH

7.4,

for 1 hour

1% osmium tetroxide for 45

_minutes,

_dehydrated

in

graded

ethanol,

and embedded in

_Epon-812.

Thin sections werestained with

uranyl

acetateand leadcitrateand examined ina

Philips

EM400transmission

electron

_microscope.

Indirect

_{immunofluorescence}

_assay

_(IFA)

The _presence of

_cytoplasmic

HIV-related

_antigens

was assessed

by

IFA as

described.16

_Briefly,

cells werewashed twicewith and

_resuspended

in PBS at

106

_{per ml.} Cell

_suspension

₍₂₀

_pi)

was

_spotted

ontoa

slide,

air

_dried,

andfixedincoldacetonefor 10minutes atroom

_temperature.

Patient'sserumdiluted 1:20

(20

p,l)

was

_applied

onthe fixed cells and incubated for 45 minutes _atroom

_temperature.

The slides were

then washed for 1 hour in

_PBS,

and 20

_p,l

of 1:20 rabbit antihuman

_IgG

_conjugated

with fluorescein

isothiocyanate

(FITC,

_Cappel

Lab., Cochranville,

PA)

was added and _{incubated at} 37°C for 60_minutes.

The slides werewashed

_extensively

in PBS before

_microscope

examination underultraviolet

_(UV)

illumi-nation. Uninfected HUT-78 cells were usedas

_negative

control.

To assess _{the presence of} CD4 membrane

_antigen

and of

_HIV-specific

_membrane

_antigens,

we

per-formed the membrane indirect immunofluorescence as

described.17

_Briefly,

5 x

105

cells were washed

three timesin PBS

_plus

5%

_FCS,

the

_pellet

was

dried,

_and 10

_pj

_of

appropriately

diluted_anti-CD4

mono-clonal orof 1:20 diluted

patient's

serum wasadded andincubated for 20minutes. Cells werethen washed three times in PBS

_plus

5%

_FCS,

and 10

_pJ

of either 1:20 rabbit antimouse

_F(ab')2

or 1:20 rabbit

anti-human

_{IgG, conjugated}

with

FITC,

wasaddedatroom

_temperature.

Appropriate

cellnumbers were

_spotted

ontoa

slide,

_air

dried,

and_fixedin _{ethanol-acetic acid}

(9:1)

_for20 minutesat

—

20°C,

andthen

_rehydrated

and washed

three

times in PBS before UV

_microscope

examination.

Radioimmunoprecipitation

assay

(RIPA)

HIV-infected and uninfected HUT-78 cells were seeded at 1.5 x

106

_{per ml and} labeled with 100

p.Ci/ml

of

_{[35S]cysteine}

_{(NEN-DuPont, Boston, MA,}

_{specific activity}

1008

_Ci/mmol)

for 6 hours after 2 hours of starvationin

_{cysteine-free}

medium. For

32P

_labeling,

HTLV-IIIB-infected HUT-78 and F12cells

were seeded at

106

_perml in

_{phosphate-free}

medium and afteran

overnight

incubation labeled with 200

p-Ci/ml

of

_{[32P]orthophosphoric}

acid

(NEN-DuPont,

specific activity

3000

_Ci/mmol)

for 6 hours. Thecell

pellet

was

_resuspended

_in

_{radioimmunoprecipitation}

_(RIPA)

_buffer

₍₅₀

mM Tris

_pH

_7.4,

_{0.5% Triton}

X-100,

100 mM

NaCl,

and 100U/ml of

_aprotinin).

The

_postnuclear

_supernatant

_containing

about4 X

106

cpm was

_{exposed overnight}

to

_{protein-A Sepharose}

_(Pharmacia,

Fine

Chemicals, AB,

Uppsala,

Sweden)

previously

incubated with a

positive

reference human serum. After three washes in RIPA buffer and one

wash in 10 mM

_Tris-HCl,

_pH

7.4,

and 0.1 M

_NaCl,

the

_pellet

was

resuspended

in 50 u,l

_{double-strength}

SDS-PAGE

_sample

buffer

₍₁₀

mM

_Tris-HCl,

_pH

_8.0,

2%

_SDS,

and 2%

_{ß-mercaptoethanol),}

boiled 5

minutes,

andrun on adiscontinuous 12% or on a 12-20% linear

gradient

_{SDS-polyacrilamide gel,}

both

with 3.5%

_{stacking gel.}

The

_gel

was

fixed, stained,

and

_{autoradiographed.}

_The

_{experimental procedure}

for

pulse-chase experiments

was identical as forthe

RIPA,

_except

that after 6 hours_of

_labeling

the cells_were

washed three times withPBS and

_resuspended

at 1.5 x

106

_perml incold

_complete

medium. Cellswere

then

collected

and

_processed

as in RIPA_at

_{0, 3,}

6,

_{and 9}hours _{after the removal of}

[35S]cysteine.

Western

blot

_(WB)

_analysis

Postnuclearcell

_lysates

₍₂₀₀

_u,g)

werefractionated

by electrophoresis

on a

polyacrylamide-SDS

slab

_gel.

Proteinswere transferredto a_{nitrocellulose sheet}

(0.22

_p,m;

_{Bio-Rad, Richmond,}

_VA)

_via

electrophoretic

blotting,

washed,

and blocked with nonfat

_dry

milkto minimize

_{nonspecific binding.}

Nitrocellulose

_strips

werereactedwith 1:100 dilution ofa

positive

humanserum. After the

washing cycles,

bound viral

proteins

werevisualized

_through

a

peroxidase-labeled

_goat

antihuman

IgG (Bio-Rad).

Nucleic acid

_analysis

DNA.

_{High-molecular-weight}

DNA was extractedfrom cell

clones,

and 15 p,g was

digested

with the

appropriate

restriction enzymes

according

to the manufacturer's recommendations. The DNA was then

subjected

to

electrophoresis

in 0.8% agarose slab

_gel

and blotted

_through

a

_high

electric field

(Transblot,

Bio-Rad)

ontoGene Screen-Plus filters

(NEN-DuPont).

Hybridization

was

performed

at 42°C in a

_shaking

_water bath for 24 _hours in 50%

formamide,

1 M

NaCl,

5%

_{dextran-sulfate,}

1%

_SDS,

and 100

_p.g/ml

of sonicated salmon _sperm DNA. Filters were then

washedtwice for 30minutes in

_{double-strength}

standardsalinecitrate

_(SSC)

atroom

_temperature,

twicein

double-strength

SSC and0.1% SDS at

_42°C,

and

_finally

twicein SSC diluted0.1 x atroom

_temperature.

The

_probes

were nick translatedat a

specific

activity

of about2 x

108

cpm/p,g

DNA

_using

a

32P-labeled

deoxicytidine triphosphate

(NEN-DuPont;

specific activity

3000

Ci/mmol).

Probes utilized were

the

full

genomic length

(less

180

_bp

at

5'-LTR)

BH10

_{probe (gift}

of Dr. R.C.

Gallo, Bethesda, MD),

a 3.8 kb

gag-pol,

and a 1.9 kb env

probe (gift

ofHoffmann-La

Roche, Basel,

Switzerland)

corresponding

to the

most conserved sequences

overlapping

the HIV exones. Filters were

finally exposed

_to Kodak SO-282

films for 24-72 hours inan holder with

intensifying

screens.

RNA. Total RNA was extracted

by

the

guanidine-isothiocyanate method18;

the

poly-A+

RNA was

separated

by oligo-dT

cellulose

_(Pharmacia)

_{chromatography.}

_Poly-A+

RNA

(3-5

p,g)

was

subjected

to

formaldehyde-formamide denaturing gel electrophoresis

and then blotted and

_hybridized

asdescribed for

theDNA

_procedures.

RESULTS

Isolation and

_biologic

characterization

_of

D10 and F12 clones

HUT-78 cellswere_infectedwithan

RT-positive

_supernatant

of PBL fromanAIDS

patient.

HIV-infected

cultures weremaintained

_{by adding weekly}

fresh HUT-78cells until cell

viability

dramatically

decreased.

Fresh HUT-78 cells were then infected with a 100-foldconcentrate

_supernatant

of the former culture and after 16 _passages were cloned

_by

the

limiting

dilution method. Several HIV-infected cell clones were

obtained,

two ofwhich were

expanded

and characterized. The first clone

(D10)

is

chronically

HIV

in-fected,

releases HIVvirionsat

_high

levels

(Table 1),

and

_{replicates indefinitely}

withoutcocultivationwith uninfected cells. The second

_(F12),

albeit

_persistently

HIV infected

(Table 1),

doesnotrelease infectious viral

_particles

in the

_supernatant,

asconfirmed

by

the unsuccessful infection of

HIV-susceptible

cells

(PBL,

H9,

or

HUT-78)

witha500-fold concentratedF12

_supernatant.

The_{presence of}noninfectious aberrant HIV

particles

in F12 clonewasruledout

_by

electron

_{microscopic analysis.}

Nomatureor

budding particles

were

noticed in

F12,

whereasmature

_type

C

_particles

were

clearly distinguishable

in D10 cells

(data

not

shown).

Table 1. BiologicCharacterizationofD10andF12 HIV-Infected CellClones

Cell membraneIFAb

(%)

Cytoplasmic IFA,b

-RT1 HIV

antigen

Syncytia

anti-HIV

_(%)

anti-CD4 anti-HP/ HUT-78 - — 98.0 — HUT-78/HTLV-HIB + + + 93.6 — NDd D10 + + + 91.0 — 73.4 F12 -92.3 — 1.6 "Mean valuesatconfluence:HUT-78/HTLV-IIIB, 3 x 105

_cpm/ml;

_D10,5 x 105

_cpm/ml;

HUT-78 and

F12,

below the

_background.

bValuesofa

representative experiment.

c_{Abnormalamountsof cellulardebris in F12}

supernatantsmaycause afaint

positivity, just

above the

background.

Toassessthe

possible

release of viral

proteins,

F12cellswerelabeled_with

[35S]cysteine,

the

_supernatant

was concentrated 500-fold via _{ultrafiltration}

(Centricon,

Amicon, Danvers, MA; cutoff, 10,000 daltons)

and

_{analyzed by}

RIPA in

_parallel

with a similar

preparation

from a D10 _{clone culture.} Even

_loading

a

10-fold excess of cpm, no viral

_products

were

_recognized

in F12

_supernatant,

_{whereas the}

typical

HIV

antigens

were

_remarkably

_present

in D10

_supernatant

(data

not

_shown).

The

_growth

rateof thetwoclones is

_similar,

asboth reachsaturationatabout2 x

106

_{cells per}ml_after4

days

ofculture. Viral infectionand

_production

wereassessed

by

RT_assay,

by

the

antigen

capture

assay, and

_{by cytoplasmic}

IFA

_performed

witha

_polyclonal

anti-HIVserum. Table 1 shows that the

_percentage

of

IFA-positive

cells is

_{equally high}

_(80-90%)

in

F12, D10,

and control

_positive

cells

_{(HUT-78/HTLV-IIIB).}

Thetwoclones were also

analyzed

for the_presence of

HIV-specific antigens

on the

plasma

membrane.

A

_{partially purified}

human anti-HIV serum

recognized

73.4% D10 cells and

only

1.6% F12 cells. These

data as well as those from RIPA

experiments

_{(see later)}

indicate the presence of

HIV-specific gpl20

antigens

onthe

plasma

membrane ofthe D10 cellclonebut notonthat of the F12clone.

Finally,

the presence of CD4

_receptor

sites was tested

using

a monoclonal anti-CD4+

antibody.

As

already

described for otherHIV-infected cell

lines,19

neither D10 norF12 clones exhibitany CD4

mem-brane

_antigen.

Allthese features are

invariably

conservedeven after 12 monthsofculture.

Attempts

to rescueHIV

from

F12 clone cells

F12clone cells havebeen treated withoutsuccesswithawide

_spectrum

ofdosesof

_{fluoro-, bromo-,}

and

iododeoxyuridine

for

_possible

rescue of the

integrated provirus

(data

not

_shown).

Further,

wetriedto

_{complement possible}

defects of F12

_proviral

_genome

_{by superinfecting}

these cells with different HIV-1 or HIV-2 isolates. Even with a

_{high multiplicity}

of

HIVs,

_every

_attempt

_met with

failure.

Transcription

directed

_by

the HIVLTRs was showntobe frans-activated

_{by superinfection}

with

_herpes

simplex

virus

_type

1

_(HSV-1).20

F12 clone cells were thus infected with

_{1, 4,}

or 10

_{plaque-forming}

units

JLL

JLUL 1XL

^p ^^^^. ^^^^_

FIG. 1.

_{Radioimmunoprecipitation}

of

_{[35S]cysteine-labeled}

cellular

_lysates

of HTLV-IIIB-infected HUT-78 cells

(c

+

₎

and D10 and F12clones. About4 x 106_{cpm per condition}wasincubated with human

_negative

(

—

)

or

HIV-posi-tive

(

+

₎

serumand

_processed

asdescribed. Thearrowsindicatethe

major

viral

antigens

detectable in the RIPAtest.

gp160*

gp120»

p55* gp4l» p24*

p19

p17

389

(PFU)

percellof

HSV-1,

butnoHIV

_production

wasdetectable under_any

conditions,

evenifthe

superin-fecting

HSV-1 did

_replicate

in F12 as well as in HUT-78 control cells

(data

not

_shown).

Protein

_pattern

_analysis

The

_productively

infectedDIO clone shows a viral

protein

_pattern

resembling

that of

HUT-78/HTLV-HIB cells

_except

for the lower band of the

_p24-25

doublet and the

_{electrophoretic mobility}

of

_gpl20,

possibly

dueto adifferent

_pattern

of

glycosylation (Fig.

1).

In contrast, in the F12 cell clone there

is

a consistent reduction in

p55

_{gag precursor,} which is

poorly

cleaved with the

consequent

almost total absence of

p24

and

pl7

gag

proteins.

Further,

only

gpl60

pre-cursor is

_present

but there is a lack of

_gpl20

and

_{gp41, easily}

visible in HUT-78/HTLV-IIIB and in DIO

cells. Pulse-chase

_experiments

didnotshowany

cleavage

of

_gpl60

in F12 clone cellseven9 hours after the

removal of radioactive label

_(data

not

_shown).

In DIO and F12

_cells,

_p64

and

_{p38 proteins}

_(the

reverse

_{transcriptase}

and the viral

_{endonuclease,}

respec-tively)

are _visible

_by

WB

_analysis

while

being

undetectable

_by

RIPA. In F12 cells there _is a

prominent

band ofa

protein

withan

Mr

of 19

kDa,

also visible

by

WB

analysis (Fig.

2),

thatmaybe eitheran_aberrant

product

of thegag geneor_the

_product

_{of the}rev HIV exone. Todefine more

exactly

the natureof this

protein species,

we have labeled HTLV-IIIB-infected HUT-78 and F12 cells with

[32P]orthophosphoric

acid

_(Fig.

3).

The failuretoreveal substantial differencesin the RIPA of

32P-labeled

_proteins

between the

positive

control and the F12

_clone,

that

_is,

theabsence of any 19 kDa

signal,

suggests

that this

_protein

does not

_belong

tothe

_{phosphorylated}

_gag

_{antigen family. Conversely,}

the

_species

most

_represented

is the

_p27

nef

protein,

which is

_present

at

_comparable

levels in both F12 andHTLV-IIIB-infected HUT-78 cells. The

_possibility

ofa double infection

by

HTLV-I and HIV-1 in the F12 clone

(a

pl9 protein

is a core

protein

of

HTLV-I)

has been ruledout

_by

(1)

utilizing cytoplasmic

IFAwith monoclonal antibodies

_against

the HTLV-I

_pi9

core

protein,

and

(2)

hybridizing

F12DNA withan HTLV-I

probe

of full

genomic length

(data

not

shown).

F12

P38-.«

P24- _g P17- •• + - + -+

-FIG. 2. Westernblot

_analysis

of uninfected HUT-78

(C

-), DIO,and F12 cell

_lysates.

The nitrocellulose

_strips

were

incubated with human

_negative

₍

—

)or

HIV-positive

(+

₎

serum. On the left are indicated the

position

of themore

FIG. 3.

_{Radioimmunoprecipitation}

of

_{[32P]orthophosphoric}

acid-labeled cellular

_lysates

of HTLV-IIIB-infected HUT-78cells(c+

₎

and F12 clone. About3 x 105_(lanesAand

B),

5 x 105

_(lanes

C andD),or106

(lanes

F, G,and

I)

cpm per condition was incubated with an

HIV-positive

(

+) or

negative

(lanesE and H, 5 x 105

_cpm)

serum and

processed

as described. On the leftare indicated the

_major

viral

_antigens

detectable in the RIPA test

_(the

_p55

_gag

precursorand the

_p27

nef

protein);

onthe

right

are indicated the

MT

of the standards.

DAM

_analysis

The

_pattern

_{analysis (Fig.}

₄₎

ofHIV

_proviral

DNA restricted

_by

_{the SstI enzyme, which cleaves in the} LTR

_regions,13

shows that both DIO and F12 clones have an HIV genome ofa

length

_{similar tothat of}

HTLV-IIIB-infected HUT-78

_cells,

thus

_ruling

outthat the lack of HIV release in F12cellsis dueto

_major

genomic

deletions. As

_already

demonstrated for HTLV-IIIB-infected H9

_cells,13

the _presence of two

coexisting

HIV genomesinDIO and F12 cells is demonstrated

_by

the SstI and EcoRI

_patterns.

Most restriction enzymes

(six

of

_eight

forthe DIOcloneand six ofnine for the F12

clone,

notall shown in

_Fig.

4)

generate

different

_cleavage

_patterns

with

respect

to HUT-78/HTLV-IIIB control cells. In

con-trast, the

_{heterogeneity}

is less

pronounced

between thetwoclones

_(e.g.,

EcoRI)

andbetween each clone and the infected

_parenteral

cell

_population,

from which

_they

derive

_(data

not

_shown).

The Xbal _enzyme does not

_{recognize proviral}

sites in HTLV-IIIB-infected HUT-78 and in DIO cells. Thattwo additional bandsof

_high

molecular

_weight

are

_{generated only}

in F12 clone may be

_interpreted

asthe appearance ofan

Xbal-specific

internal site inoneof thetwo

_coexisting

_{HIV genomes.}

Finally,

as

reported

for the H9/HTLV-IIIB

cells,3,10

the restriction

_pattern

of the two clones did not

change noticeably

even after 12_{months of culture}

_(data

_not

_shown).

RNA

_analysis

Several studies of

_HIV-specific

RNAs21,22

_reported

the _{presence in HIV-infected cells} of three

_major

bandsat

9.3, 4.3,

and 1.8-2

kb,

_{representing, respectively,}

the

_{genomic length transcript,}

the

_env-specific

mRNA,

anda

family

of minormessengers

_coding

forthe

regulatory

HIV

_proteins.

An additional

transcript

was

occasionally

detectedat5

kb.23

The

_{hybridization}

ofintracellular viral

_poly-A

+ _{RNAwith the}

full-length genomic

BH10

_probe

reveals the same three

major

bands in both

positive

control

cells,

in

DIO,

and F12 clones

(Fig.

5).

The evident

overproduction

of 1.8-2 kb viral RNA in the F12 clone

_hybridized

withaBH10

_probe

can

_{hypothetically}

fit with the

_{overexpression}

ofa 19 kb

regulatory

protein

in the F12 clone

(Fig.

1).

Hybridization

ofthe sameRNAs with the

gag-pol

or env

subgenomic probes

fails to show any

quantitative

(as

shown also

by

c+ dtO c+ _dlO c-f dtO c+ <flo c+ dlO c• .; : 4Êt *fr c+

fl2

c+ «2 c+ _ft2 c*

(12

<3- 2.3-2

SstI

_pcoRl

_Hind

III Xbal

BamHI

FIG. 4. _{Restriction enzyme}

_analysis

ofHIV DNA

_integrated

inDIO_{and F12 genomes. Eachpattern} was

compared

with that of HTLV-IIIB-infected HUT-78 cells

(c

+_);

_(c-),

uninfected HUT-78 cells. At the bottomareindicated the restriction enzymes

employed;

onthe leftare indicatedthe kilobase

pairs (kbp)

of the \ DNA markercut

_by

Hindlll.

hybridization

with the

_{glucose-6'-phosphodehydrogenase}

_probe)

or

qualitative

differences among

DlO,

F12,

and HUT-78/HTLV-IIIB cells

_(Fig.

_5).

DISCUSSION

The

_cloning

ofHUT-78 cellsinfected withan

_RT-positive

_supernatant

of PBL_fromanAIDS

patient

led

totheisolation oftwoclones

_harboring

differentHIV

_variants,

one

(DlO)

persistently

infected

by

a

replica-tion-competent

HIV,

the other

(F12)

exhibiting

an

integrated

HIV genome _unable to

_generate

infectious viral

_particles.

The isolation of such clones

_emphasizes

the

_possibility

of

_recovering

more than one HIV

variant from PBL of the same AIDS

_patient.

More

_{interestingly,}

fromthe same virus isolation assay we

have detected adefectiveHIV variant

_that,

once

_integrated

in the host genome, is able to

_protect

the cells from HIV

_{superinfection through}

a

_phenomenon

of

homologous

viral interference.

The simultaneous _presence of more than one variant in cell

populations

infected with HIV was first

reported by Wong-Staal

etal.

,3

whodescribedthe coexistence ofatleasttwodifferent

_genotypes

in 2 of18 AIDS isolates. This evidencecamefromrestriction

_pattern

analysis

of

integrated

viralDNA

using

enzymes

BH10 o a u. 9.2* 4.8» 1.8_*r 92» GAG POL o m o

S

£ M*^^ ^^^^ JIB

9.2*

4.8*

ENV i O CM ' T- T-Ü Q LL ^^^m Hft ^^^m.

-kit

FIG. 5. Northern blot

_analysis

ofviral RNA: 3-5(Jig

poly-A+

RNAofDIO, F12,andHTLV-IIIB-infectedHUT-78 (c+₎cellswere

hybridized

withthenick-translated full

_{genomic length}

BH10

_probe,

with

_{subgenomic gag-pol}

or env

probes,

both

_representing

themost_{conserved sequences of}the

_respective

exones. Thearrows ontheleft indicate the

kbp

of theHIV RNA

major signals.

The

_possibility

that

_proviral

genomes

present

in DlOand F12 clones

_originated

fromin vitro mutations cannot be ruled out, sincesomany culture passages werenecessary toestablish theclones.

_However,

this

hypothesis

is

_strongly

in contrast to the in vitro

great

genomic stability

of

_{integrated proviral}

HIV,

as

already

shown

_{comparing proviral}

restriction

_patterns

after

_{3,10 9,3}

or even 12months

(our

results forDlO

and F12

_clones)

of culture.

The

_nonproducer

HIV-infectedF12 clone may have

originated

fromanHIV-defective

particle originally

present

in the AIDS

patient

and released from the

patient's

PBL. This virus could have infected CD4+

HUT-78

cells,

_taking

advantage

of the presence ofa

coinfecting replication-competent

HIV. Such viral

complementation

could inturn leadto release in the

_supernatant

of reinfected fresh HUT-78 cells of viral

particles

with normal

_{nucleocapside}

and

_envelope

structurebut witha_{genome unable}tocode for infectious HIV virions.

Both DlO- and F12-infected clones may

represent

a cell

_{population originally harboring}

HIV variants

with limited

_cytopathic

effects that could conferaselective

_advantage

with

_respect

tocellsinfected

_by

more

cytopathic

HIV variants. The

_nonproducer

F12 cell clone resembles the DlO clone in terms of cellular

growth,

percentage

of cells

_positive

for

_cytoplasmic

HIV

_antigens,

and down

_regulation

of CD4

_receptor

sites and viralRNA

_transcripts.

In

_addition,

the F12 clone is

_by

restriction

_pattern

much more related to DlO thantothe HUT-78/HTLV-IHB cells.

The

_inability

of the F12clone torelease infectious

_particles

canbe duetoboth viral and cellularfactors.

Among

the

_latter,

Joneset

al.24

showed that the

_activity

ofacellular

transcription

factor

(Spl)

isnecessary for thecorrect

_{transcription}

ofHIV sequences. The absenceof

_Spl

factor leadstoa 10-fold_reductionin_the

in vitroHIV

_{transcriptional efficiency.}

Our resultsarenotin

agreement

withthe

_hypothesis

ofa

Spl

defect

in F12clone becauseofthe

_comparable

presence of viral

_transcripts

in

DIO,

Fl

_2,

and HTLV-IIIB-infected HUT-78 cells

_(Fig.

_5).

The most

_important

difference between DlOandF12 clones is atthe level of viral

_protein

_pattern.

The very low

_positivity

of F12cells

_by

themembrane IFA withananti-HIV antiserum fits well with the absence

of

_{cleavage products}

of

_gpl60.

The lack of

_{gpl60 cleavage}

is not due to a defect in the

viral-specific

protease,

which

_plays

arole

only

in the_gag

p55

cleavage25

and may beduetoa mutation in_the

_cleavage

site.

F12

_pl9

can

_hardly

be considered an aberrant

_product

ofoneofthe three structural genes, since

(1)

the

env

gpl60

is

substantially

uncleaved,

(2)

the_gag

p55

_precursor is

poorly expressed,

and

(3)

the

p64

viral

polymerase

as well as the

p34

endonuclease are well translated in both F12 and DlO clones

(Fig.

2).

In

addition,

the absence ofa

_signal

atthe 19-20kDa level inthe

_{phosphorylated protein}

pattern

of the F12 clone does not

_support

the

_hypothesis

of

_{pl9 being}

an aberrant gag

product (Fig.

3).

Experiments

in

progress

utilizing

anti-rev

polyclonal

andmonoclonalantibodieswill establish whether theF12

pl9 protein

is the

_product

of therevgene.

Sodroskiet

al.26

_assigned

an

aníí-repression

functiontotherev

protein

to counterthe effects of

_cw-acting

negative regulatory

sequences

present

in viral mRNAs

_encoding

HIV structural

_{proteins. Conversely,}

Feinberg

et

al.22

_hypothesized

arole of therev_{gene in}

regulating

the

splicing

of viral mRNAs.

Regardless

of the mechanism of

_action,

allthese authors

_assigned

totherev_gene a

_{positive regulatory}

_{function ofgag}

and

_pol

_{protein synthesis}

atthe

_{posttranscriptional}

level.

It is thus difficult to correlate the

_{overexpression}

ofa

_putative

rev

_{pl9 protein}

_{with reduced gag}

_p55

production

unless we

_hypothesize

_{the presence of} a mutated _defective rev.

_{Alternatively,}

_{the increased}

presence of

_pl9

may accountfor this

_block,

ifone

hypothesizes

a_{bimodal effect of}rev

_{protein by}

which

low and

_high

amountswould induce

_opposite

effects.

Further,

the p 19

_{overproduction}

may bea_responseto

some_{undefined block related}toeither cellularorviral

_regulatory

_genes.

Anaccurate

_study

of the sequences and of the

_secondary

structureofviral mRNA willalso be useful to

exactly

understand both the

_mechanism(s)

of translation inhibition of gag

specific

messengers and the

origin

ofthe

_uncleavage

ofenv

gpl60.

ACKNOWLEDGMENTS

This workwas

supported

in

_part

by

_grants

from AIDS

Project

1987-1988 ofthe

Ministry

of

Health,

Rome,

from

_Gruppo

di

_Virologia

87.01640.04

_Consiglio

Nazionale delle

_{Ricerche, Rome,}

and from the Associazione Italiana_perle Ricerche sul Cancro.

WeareindebtedtoMs. A. Guderzofor excellent secretarial assistance.

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