Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro.

Vol. 65, No. 1 JOURNALOFVIROLOGY, Jan. 1991, p.225-231

0022-538X/91/010225-07$02.00/0

CopyrightC) 1991, AmericanSociety forMicrobiology

Evolution of Human Immunodeficiency Virus

Type

1

nef and

Long

Terminal

Repeat Sequences

over

4 Years In

Vivo and

In

Vitro

SYLVIE DELASSUS, REMI CHEYNIER, AND SIMON WAIN-HOBSON*

Laboratoire deRetrovirologie Moleculaire, Institut Pasteur,

28 Rue de Dr.

Roux,

75724 Paris Cedex

15,

France

Received 2 August 1990/Accepted 19 October 1990

The evolutionofan851-bpsegmentof the human immunodeficiency virustype 1(HIV-1)genomeencoding

thenefopen reading frame and U3/R elements of the long terminal repeat has been followedover a 4-year

period in vivo andinvitro. The population of viralsequencesatanygiven timewasestablished by sequencing

clonedpolymerase chain reaction products. Thesamples studied werederivedfromthesamemanfor whom

adetailed analysis of thetatgene waspreviouslydescribed (A. Meyerhans, R. Cheynier, J. Albert, M.Seth,

S. Kwok, J.Sninsky, L. Morfeldt-Manson, B. Asjo, and S.Wain-Hobson, Cell 58:901-910, 1989).Once again

invitro culture resulted in the selection ofminorforms. Overa4-year period in vivo, there was noobvious

selection for,oroutgrowthof,anyparticularneforU3/Rsequence. Fewdefective nefproteinsequenceswere

observed, whicharguesagainst nef actingas a negative regulatoryfactor. Althoughnofunctionally defective

promoter/trans-activation-responsive elementswereidentified, the transactivation efficiencies varied between

0.2 and 2 times that of the control. Thesequenceencodingthemostefficient trans-activation-responsiveregion

didnotoutgrowothers. Theextremegeneticheterogeneity of thedifferentsamplesofthe locus,either in vivo

orinvitro, indicates that there isnosuchthingas asingle, distinct HIVsequence.It is suggested thatdifferent

HIV-1loci evolveindependently, recombination being responsible for their uncoupling.

Theplasticity ofthe humanimmunodeficiency virus(HIV)

genome hasbeenamplydescribed. Itisdueto amultitude of

phenomenaencompassing viral polymerase miscopying, du-plication, deletion, recombination,andhypermutation of the viral genome. These events, while rendering the task of

molecular biologists particularly arduous, are probably an

advantageto thevirusinits continual efforttoadapttolocal

environmentsorrespond toselectionpressures. Inorderto

describe such complexity inherent in all RNA viruses, the

conceptofaquasispecieshas beendeveloped(11,12, 34). In

brief, a quasispecies may be defined as a population of distinct but relatedviral genomes. The 10-kb sizeofthe HIV

type 1(HIV-1) provirus effectively prohibitsaccurate

anal-ysis of populations of complete sequences. A sequence

analysis of cloned DNAfragments, derived by polymerase chain

amplification

of the HIV-1 provirus, is perhaps as

closeascan be got to a

description

ofaHIV-1 quasispecies

atthe nucleotide sequence level (28).

In a

previous

longitudinal

study

of the HIV-1 tat gene

sequencesin vivoandin

vitro,

itwasshown that the onedid

notreflect the other(28). There was no selection for

more-efficient tat genes with time despite the suggestion that

viruses isolated from asymptomatic seropositives, which

grow poorly, could be complemented by a cell line

perma-nently expressing tat(2). These isolates are referred to as

slow/low isolates (3). Bycontrast,isolates from patientswith

AIDS

replicate

well, thegrowth ofthe isolates being

unaf-fectedbypassageto atat-producing cellline. These viruses

are called

rapid/high

isolates (3). These observations

sug-gested that perhaps there was selection, coincident with

disease,

of isolates with increased trans-activation

effi-ciencies. Asit happened no selection atthe level of the tat gene wasdescribed (28).

* Correspondingauthor.

225

Theviral longterminal repeat (LTR) carries the sequences

essential for transcription, reverse transcription, and

inte-gration. A particular feature is the

trans-activation-respon-sive (TAR) sequence in the Rregion, which is a target for

tat-dependent trans-activation of provirus expression (29,

31). The tat, as well as host proteins, binds TAR RNA,

resulting in efficient transcription and a positive feedback

loop (10, 38). Thus, it was possible that the differences

betweenviruses inearlyand latestagediseasemight bedue

to subtle differences in the TARregion.

HIV-1is endowed with at leastninegenes. The ninth and

most 3' gene, called nef, is a uniquefeatureofthe primate

lentiviruses,asopposedtothe otheranimal lentiviruses (30).

It wasoriginally thought to be anegative regulatory factor

(14, 18, 27, 36), perhaps interacting with the negative

regu-latory elements in the U3 region ofthe LTR (1), hence the

mnemonic nef(16). The role of nef

is,

however, now no

clearer than when it wasfirst identified (19, 25). While it is

notessential toviral growth, its conservation inall primate lentiviruses arguesforan important role.

Sincethe U3 elementofthe LTRoverlapswith the 3' half

of the HIV-1

nef

open

reading frame,

it was decided to

amplify the entire nef open reading frame and LTR

se-quences. Apart from

addressing

the problem of possible

functional differences within the LTR and TARregions, it

wouldalsopermitthe mostextensive(approximately 10%of theprovirus)high-resolution studyof the evolution ofHIV-1

quasispecies coincident with disease

progression.

The data

provided here show that, as in the tatgene

analysis,

there

was noselection of more efficient TAR elements.Inaddition

no completely defective LTR sequences were identified.

Thus, differences between the so-called slow/lowand

high/

fast viruses mustbe encoded elsewhere within the genome.

Little evolution of

nef

orLTRsequenceswasnotedfrom the

asymptomaticto thedisease stage.

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226 DELASSUS ET AL.

I

env | I

F

rev

tat

|nef I

Oligonucleotides Amplification -_ Sequencing Subcloning

FIG. 1. HIV-1nef-U31R locus amplified.Theregionof the HIV-1

genomestudied is shownexpanded.The locations and orientations

of the oligonucleotides used for amplification, screening and

se-quencing of M13 recombinants, and subcloning of the promoter/ TARregionsaremarkedAmplification, Sequencing, and Subclon-ing,respectively.

MATERIALS AND METHODS

Bloodand culturesamples. Freshperipheral blood

mono-nuclearcells fromaHIV-1-infectedman wereanalyzedover

a4-year period. Samples Li, L2, L3, and L6were takenin

June 1985, March 1986, June 1986, and June 1989,

respec-tively. Viral isolates Vi, V2, V3, and V6 were derived by

coculture using seronegative donor

phytohemagglutinin-stimulated blasts. SamplesLi,L2, L3, Vi, V2, and V3 have

already been described (28). In June 1989, HIV-1 was

isolated from a sixth sample (L6) by cocultivation with

HIV-1 seronegative donor peripheral blood mononuclear

cells. Unlike all other isolates from this patient, the virus

derivedfrom L6, i.e., V6, wastypically arapid/high isolate (3) and induced large syncytia. It alsogrew well on estab-lishedcell lines.The DNA usedtocharacterize V6wasfrom first-passage virus.

PCR. The nef-U3/R amplification primers used for poly-merase chain reaction (PCR) were located in highly

con-served regions flanking these sequences. The DNA plus

strand primer NL1 (5'-CCAGCATGCAGTAGCTGAGGG

GACAGATAG) and DNA minus strand primer NL2

(5'-CCAGTCGACCAGAGTCACACAACAGACGGG) mapped

topositions 8278to8300 and 131to109,respectively,onthe

HIV-1Brusequence (37)(Fig. 1). The primers carried SphI

and SalI restriction sites (underlined), respectively. DNA

amplification conditions have been previously described

(28). Approximately 1 ,ug of total DNAwas used.

Denatur-ation, annealing, and elongation temperatures and times

were 94°C and 30 s, 55°C and 30 s, and 72°C and 1 min,

respectively.

Cloning and sequencing. PCR material was purified on a

low-melting-point agarose gel, phosphorylated, and

blunt-end ligated into dephosphorylated, SmaI-cleaved, M13mp8

replicative-form DNA (Amersham). Aftertransformation of

Escherichia coli TG1, plaqueswerescreenedin situ by using

amixture of three oligonucleotides, Si, S2, and S3, all of which were located in highly conserved regions within the

nef-U31R sequence. The sequences were Si, 5'-AGTC

CCCAGCGGAAAGT; S2, 5'-GGAAGTAGCCTTGCGCG;

andS3,5'-GCTGCTGTATTGCTACT, which mappedtothe

minusstrandat9047to9031,8753to8737,and8538to8522,

respectively (Fig. 1). Twenty M13 recombinants fromeach

sample were sequenced by the dideoxy chain termination

method (33) using M13 universal primer and the three

oligonucleotides, Si, S2, and S3.

Polyacrylamide gel blotting. After separation on a 3.5%

acrylamide-TBE (89mMTris-borate, 2 mM EDTA) gel,the

PCR-amplified products weredenaturedinsituby using0.2 MNaOH-0.6 MNaCl for30 minfollowedby30 min in 7%

formaldehyde. The DNAwasthen transferredtoa

nitrocel-lulose filterbyastandard method. The filterwas hybridized

withanequimolar proportion of5' 32P-labeled oligonucleo-tides Si, S2, and S3.

Construction ofexpressionvectorandsubcloning. Thelarge

HindIII-SspI fragment of the pBC12/PL/SEAP vector (5),

which encodes the entire human secreted alkaline

phos-phatase (SEAP) cDNA, was cloned into the HindIII and

SspI sites ofpUC18. This vector,

pUC/SEAP,

contains all

the pUC18 polylinker sites 5' tothe SEAP gene, and was

constructed so as to delete the simian virus 40 early

en-hancerfromthe original plasmid. The enhancer, promoter,

and TAR sequencesofHIV-1were amplifiedfrom 10to50

ngofrecombinantM13 DNAunderstandardconditions.The

amplificationprimerswere NL2andNL3

(5'-GAGAGGTC

GACCGGAGTATTACAAAGACTGCTGA, positions 8987 to9010)(Fig. 1). TheSall restrictionsite used insubcloning

ofthe HIV-1

promoter/TAR

fragments is underlined. After

10 cycles, amplified DNA from 22 M13 recombinants and

from pBRU-2(kindlyprovided by Keith Peden)wascleaved

by HindIIl and Sall and ligated into pUC/SEAP

through

the same sites (HindIll cleaved within the Rregion, 3' to

the TAR region). The sequences ofthe resulting series of

23 constructions, named pTAR/SEAPO to pTAR/SEAP22,

were all checked by double-stranded plasmid sequencing

(20) using the M13 reverse sequencing primer. No

differ-ences between the subclone and the original recombinant

M13 sequenceswere identified.

Transfection and SEAP assays. SW480 cells(human colon

carcinoma cells) were cotransfected by

pTAR/SEAP

and

pSV2tat Bru (28) by the calcium phosphate method (8),

whereas Jurkat-tat cells (31) were transfected by pTAR/

SEAPplasmids byusingthe DEAE-dextranprocedure(15). Sixty-hour posttransfection culture supernatants were

clearedbycentrifugation (15,000 x g)andheated for10min

at65°Cto inactivate any endogenous alkalinephosphatase.

SEAPactivities weredeterminedon50 ,ulof supernatantas

previously described (5). The valuesgivenarethemeansof

at least two independent transfections ofSW480 cells and

wereconfirmedby transfection oftheJurkat-tat cell line(see

Fig. 3).

Nucleotide sequence accessionnumbers. TheGenBank

ac-cession numbers for the HIV-1 nef and LTR sequences

presented hereare M58193 to M58283.

RESULTS

What is a HIV-1 sequence? Twenty recombinant M13

clones were sequenced for each sample (i.e.,

Li,

L3, L6,

V2, V3, andV6). None of the 60 sequences from

Li,

L3, and

L6 were identical over the 851-bp locus analyzed. The

maximum nucleic acid sequencedivergence noted between

anypairwas3.1%.Of the three in vitroquasispecies, V3was

the most homogeneous and V6 was the least. A major

species representing 35%of all sequences could beidentified

within V3. Nonetheless,evenwithin V3 upto 2.4% nucleic

acid sequence divergence was noted between any pair. As

was observed previously (28) the invivo quasispecies

(Li,

L3, andL6)were morecomplex than the in vitro

quasispe-cies (V2, V3, andV6).

J. VIROL.

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[image:2.612.66.296.70.220.2]

EVOLUTION OF HIV-1 nefAND LTR SEQUENCES 227

PCR analysis of the nef-U3IR region from a molecular clone (pNL4.3; see reference 30 for sequence) indicated that there were no hot spots for the enzyme within this region. A study of 20 clones indicated that 1 clone in 4 carried a single base substitution per 851-bp sequence due to Taq polymer-ase errors (data not shown). This value was concordant with those derived from analyses of 300-bp segments of the HIV-1

tat and env genes (17, 28). Since the minimum number of substitutions within any quasispecies, using any sequence as a reference, was always greater than 35, the ratio of natural

to artifactual substitutions would be 7:1 [i.e., 35:(20/4)] or

more.

A comparison of thenef-U31R quasispecies in vivo and in

vitro (i.e., comparisons of L3 with V3 and L6 with V6)

showed that there were no common nucleotide sequences, indicating once again that the in vitro culture of HIV-1, whether from early (L3 and V3) or late (L6 and V6) stagesin

disease, leads to the selection of low-abundance forms. These in vitro forms are presumably in the original periph-eral blood mononuclear cell quasispecies. However, the resolution of the analysis (1 clone of 20) must have prohib-ited their identification.

What is a nef sequence? The nef protein sequences are shown in Fig. 2 for each of the three lymphocyte samples, Li, L3, and L6. Only two sequences, L1.14 and L1.16, were present at 10% of their original quasispecies. All other sequences were present at 5%, the resolution of the study.

Of the 60 sequences only 4 were common tothe Li and L3

quasispecies. All the L6 sequences were unique. None of the

V3 or V6 nef protein sequences could be found in the

parental L3 or L6 samples, respectively. Thus,nosingle nef

protein sequence could possiblyhavereflected the

complex-ity of nef sequences present in any sample.

The mutations were not randomly distributed but were

clustered in the amino and carboxy termini. Apart from a

few substitutions, the regionbetweenaminoacids 40and 130

was highly conserved. Overall the internal sequence

varia-tion among nef proteins was between 1 and 4.5%. While

there does not appear to be any obvious selection for a

particular nef sequence, some progression may be noted at

specific positions. Thus, threonine15 ispresentin 30% of all

Li sequences, in 45% of L3 sequences, and in 95% of L6

sequences.

Sequences L3.14 and L6.01 appeared particularly

diver-gent at their amino acid termini. These substitutions maybe

explained by G--A hypermutation of RNA genome during

DNA synthesis by the HIV-1 reverse transcriptase (36a).

There were fewcases ofobviously defectivenef sequences.

L6.01 encoded a mutated initiator methionine codon

(ATG-*ATA) aswell as an in-phase stop codon atposition

57. Sequences L1.13 and L1.21 also encoded in-phase stop

codons. Finally, clones L1.09 and L3.21 carrieddeletionsof

154 and 68 residues within their nef sequences. These

deletions, 462 and 204 nucleotides, respectively, were

mul-tiples of 3 andwere considered authentic for the following

reasons. (i) Polyacrylamide gel blot analysis of the original

PCR-amplified materialbyusing oligonucleotide probes

cor-responding to sequences within the deletions showed that

there were unclonedPCR products lackingthese sequences

(data not shown). (ii) No sequence homology around the

deletions that argued against a PCRartifactcould be found.

(iii) In the positivecontrol (amplificationof the same region

from a molecular clone) no similarly deleted products could

be identified. In all the loci that have been studied in the

laboratory by PCR no deletions have ever been found.

Occasionally a deletion was found at the 5' or 3' end of a

sequence. Invariably the neighboring amplification

primer

was alsodeleted, whichargues morefora

cloning

artifact.

Functionalanalysis of the HIV promoterregion.The

essen-tialtranscriptional control sequences of the HIV-1 LTR map

to the noncoding region between bases 636 and 801 in the

locus amplified. If just this region was

analyzed

at the

nucleicacid sequence level a verydifferent

picture

wouldbe

obtained. Thus, the Li and L3 quasispecies were

complex

while that for L6 was

relatively

simple, amajor form

being

present at 65%. Again there were substantial differences

among the Li, L3, and L6 subsets. While a number of

sequences were common to Li and L3, none of the L6

sequences could be found in either Li or L3

(data

not

shown).

Whenalltheenhancer/promotersequences from the L and Vquasispecies weretaken together, mostofthe mutations

mappedtotheTARregion, notably in the baseof the stem.

All but twoofthemutationswerepoint mutations.As

usual,

transitionsgreatly outnumberedtransversions. The few

mu-tations in the upstreamregion

invariably

mapped

just

outside

ofthe NFKB and Spl sites

(Fig.

3).

Twenty-twoclones wereanalyzed atthe functional level

afterthe TARregion wasPCR

amplified

and subclonedinto the pUC/SEAP expression vector. The mutants and their

relative trans-activation efficiencies with respect to HIV-1

Bru tat and LTR are givenin

Fig.

3.Therelative

degrees

of

transactivation variedfrom0.2to2 times thatof theHIV-1

Bru tat and LTR control.These differenceswere notdueto

experimental errors. Those

pTAR/SEAP

plasmids

which

yielded a reduced relative transactivation

efficiency

were

tested at least three times

by

using

two different

plasmid

preparations. In these

experiments

thebasallevelof SEAP activity for each

plasmid

was the same. These levelswere

comparable with thatof theHIV-1 BruLTRreference. The

relative transactivation efficiencies varied

by

+0.05. All the

clones

representing

morethan5% ofa

quasispecies

showed

relative transactivation ratios

comparable

with that ofthe

reference (i.e., between 0.7 and

1.3).

Clones 7 and 12 encodeda G-*A substitution at

position

20 at the baseof the

bulge

which reducedthetransactivation

efficiency byafactorof5.

However,

deletion ofa

single

base

in the stem atposition 42

(clone

17)

hardly

affected

transac-tivation. TwoC->T

substitutions,

oneinthe

bulge

andonein the loop, didnotinfluence transactivation. A smaller

region

at thebase ofthestemmay be

important,

for substitution at

position

7 or53 either decreasedor increased the

efficiency

oftransactivation.

Interestingly,

clone

13,

which

proved

to

be the most

efficiently

transactivated sequence and which

was derived fromthe L3

quasispecies,

was notidentified in

the L6quasispecies whatsoever.

DISCUSSION

After

having

assimilated all the sequence data it is very

difficultto

speak

ofa

single,

distinct HIV-1 sequence either

atthe nucleic oraminoacid level

(11, 12,

34).

The 851 bases

sequenced,

approximately

9% of the genome, were not

among the most variable

regions

of the HIV-1 genome.

Extrapolating

from this

data,

it is

possible

to

estimate,

at

least in this individual case, that any two

complete

viral

genomes in vivomay differ

by

upwards

of 10to20 bases. It

may not be

assumed, however,

that the HIV-1 reverse

transcriptase

misincorporation

rate is of the same

order,

because the

precise

relationship

between sequences and the

numberofcycles

separating

them is unknown.

Given the number of

unique

sequences involved it is not

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230 DELASSUS ET AL.

surprisingthat any twoquasispecies aredistinct. Howeverit

is not possible to conclude that the differences are

signifi-cant. The sampling of 20 genomes, all present at low

frequencies,from a largepool wouldinvariably give different populations with perhaps different structures. These data

caution against using a single molecular clone to represent

HIV-1. Only a few sequential changes at particular sites,

such asThr-15--lAla-15, may possibly have any meaning. If

the four such sites are considered, then there was little

evolution of the nef protein sequence over the 4 years

coveredin this study.

Onceagain thein vitro data did not reflect the in vivo data.

Comparing either the nef or the LTR sequences, the

rela-tivelyabundantforms recovered in vitro represented minor

formsin vivo. In the case of the L3 and V3 and L6 and V6

pairs there were no common nucleic acid sequences (data notshown). The minimum number of differences was 1 to 2

nucleotides. This extends our previous study based on a

314-basesegment encoding the first exon of the tat gene (28).

Perhapsbecause the segment was smaller it was possible to

findtheminorform in the in vivo quasispecies. The new data

from thenef-U3/R region indicated that in vitro coculture of

HIV-1resultedin the isolation of very minor forms. V6 was

isolatedfrom the patient during late-stage disease

(CD4+

=

10/mm3) and was typically a rapid/high syncytium-inducing

isolate. Despitethe fact that patients with AIDS have little or no cell-mediated immunity (21), the virus isolated still did notreflectthe major species in vivo. Once again these data

caution against the extrapolation of in vitro data to a

descriptionof HIV-1-associated pathogenesis.

Thedistortionof the population of genomes in culture may

be due to statistical factors, methods, or the nature of the

lymphocytes.

Recentlyit has been shown that, in the context

of viruscoculture, seronegative donor cells secrete a soluble

factor capable of inhibiting HIV-1 replication (7). Another

nonnegligible factor may be the presence of substantial

proportions of HNK-1 suppressor cells, particularly in

pa-tientswithAIDS(23). Clearly a detailed study of the factors

influencingtheisolationofHIV-1, perhaps by using a genetic

fingerprinting analysisas in this study, is urgently called for. Among all the 120 nef protein sequences within this study

only a few were obviously defective. Thus, sequences

L1.15, L1.21, and L6.01 encoded in-phase stop codons;

L6.01 encodeda mutatedstart codon; and L1.09 and L3.21

carried large deletions. The L1.09 and L3.21 proviruses

should be completely defective for replication, since the

polypurine tract and 5' inverted repeat were also deleted.

Though nef was, at one time, described as a negative

regulatory element, no obviously defective nef protein

se-quences

predominated.

These protein sequences in vitro or

in vivo did not present any amino acid substitutions that

have not hitherto been observed (30). However, the

distri-bution ofamino acid substitutionsamong the Li,L3,and L6

(Fig. 2) or V2, V3, and V6 sets of sequences (data not

shown)appeared nonrandomand was essentially confined to

the amino- and carboxy-terminal regions. This would, if

anything, suggest thata

nef

gene product is selected both in

vitroand in vivo andthatthe central region between residues

40 and 130 encodes the most important functional domain.

These data are not irreconcilable with the observation that

nefmutant virusesareviable(14, 27, 36). The contribution of

neftothe HIV life cyclemay simply be subtle and

uname-nable to analysisinshort-term experiments.

That none of the LTR sequences were defective for

transcription is understandable. A functionally defective LTRwouldbe incapable of producing virus and cannot be

complemented. Itisinteresting to note that,while therewas

only a 10-fold difference in relative efficiencies of

transacti-vation, the correspondingtatgene products varied

by

more

than 100-fold in the same assay. This may simply reflect the

fact that adefectivetat maybecomplemented intranswhile

an LTR-defective genome may not. Clone

pTAR/SEAP13

(representing 5%ofL3 sequences) was twice as efficient as

any of its homologs, as well as the Bru

tat-U31R

pair, at

directing transcription. It is intriguing that this genome did

not outgrow its siblings. A number of possibilities present

themselves. However, a coupleof trivial explanationscould

be (i) that the lymphocyte(s) harboring the sequence as a

latentprovirus was neveractivated by antigenor(ii)that the

genome harbored a defect in some other gene, thus

prohib-iting viral replication. In the other clones, themutations that

greatly reduced the transactivation efficiency were

concor-dant with the deletions and substitutions that have already

been described for the

Spl

sites (24) and the TAR region

(29). In the initial study no selection for more-efficient tat

gene products wasobserved. The same conclusion can now

be drawn from the analyses presented hereof the

promoter/

TAR region. Taken together, these data indicate that the

apparent emergence of rapid/high syncytium-inducing

iso-lates (3, 9, 35) with declining CD4 cellnumbersis not due to

modulation of the transactivating system. Thus, differences

between theso-calledslow/lowandhigh/fast virusesmustbe encoded elsewhere within the genome.

Acomparison ofthe sequences fromthe V6 quasispecies showed that for both nef and U3/R there were two major

forms (36 and18% for nef and 45 and36% for U3/R). Despite

these comparable frequencies the sequences encoding the

major nef sequence did not at the same time encode the major U3/R sequence. Even when single point mutations were eliminated, thus simplifying the analysis, the same

conclusion held. This suggests that the nef andU3/R

quasis-pecies were evolving independently of each other. Since

they are located in cis, the only explanation for their being

uncoupled would be efficient recombination between ge-nomes.

The efficiency of recombination has been estimated to be of the order of 20 to 50%per cycle (4, 6, 22, 26). In view of this it is indeed likely that HIV gene sequences may be uncoupled throughout the genome. Consequently the HIV genome would resemble that of a segmented virus despite being continuous. This would once again emphasize the plasticity of the HIV and allow individual genes to evolve independently.

In conclusion these data greatly extend the notion, already described for a few molecular clones (13, 32), that the phenotype of any given clone may differ from that of another. As a consequence the biological properties of a given isolate will be made up of the sum of the properties of a myriad of subtly different genomes. In addition the ex-treme heterogeneity of the HIV-1 quasispecies either in vivo or in vitro cautions against extrapolating from sets of data that are toolimited.

ACKNOWLEDGMENTS

We thank Bryan Cullen for the gift of the plasmid

pBC12/PL/

SEAP, and Birgitta

Asjo,

John Sninsky, and Andreas Meyerhans for providing viral isolates, chromosomal DNA, and valuable help in PCR.

Sylvie Delassus and

Remi

Cheynier were supported by

l'tcole

Polytechnique and la Fondation pour la Recherche

Medicale,

re-spectively. This work was supported by grants from Institut Pasteur andl'AgenceNationale de Recherches sur le SIDA.

J.VIROL.

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