Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1.

Analysis of Sequence Diversity

in

Hypervariable Regions of

the

External Glycoprotein of

Human

Immunodeficiency

Virus

Type

1

PETERSIMMONDS,lt* PETERBALFE,' CHRISTOPHER A. LUDLAM,2 JOHN0. BISHOP,13

AND ANDREW J. LEIGH BROWN'

Department ofGenetics, University of Edinburgh, EdinburghEH9

3JN,l

andDepartment of Haematology, Royal Infirmary, Edinburgh EH3 gyW,2 Scotland, and Department of Biological Sciences, University of

Maryland-Baltimore County, Baltimore, Maryland212283

Received 13July 1990/Accepted 5 September 1990

Nucleotidesequencesin threehypervariable regionsof thehumanimmunodeficiency virustype1(HIV-1)env

gene were obtained by sequencing provirus present in peripheral blood mononuclear cells of HIV-infected

individuals. Singlemoleculesoftargetsequences wereisolatedbylimiting dilution andamplifiedintwostages

by thepolymerasechain reaction, usingnestedprimers. Theproductwasdirectly sequencedtoavoiderrors introduced by Taq polymerase during the amplification process. There was extensive variation between

sequences from the same individual as well as between sequences from different individuals. Interpatient

variabilitywasmarkedly less in individualsinfectedfromacommon source.Ahighproportionofamino acid substitutions in thehypervariable regionsaltered thenumber andpositionsofpotentialN-linkedglycosylation sites.Sequences intwohypervariable regions frequently containedshort(3-to15-bp) duplicationsordeletions, and by amplifying peripheral blood mononuclear cell DNA containing 102 or 103 proviral molecules and

analyzingtheproductby high-resolution electrophoresis, the total number and abundance of distinctlength

variants within anindividual could beestimated, providing a more comprehensive analysisof the variants presentthanwouldbeobtainedby sequencingalone.Sequencesfrommanyindividualsshowedfrequentamino

acid substitutions at certain key positions for neutralizing-antibody and cytotoxic T-cell recognition in the

immunodominant loop. Theratesofsynonymousandnonsynonymous nucleotide substitution in theregionof this and flanking regions indicate that strong positive selection for amino acid change is operating in the

generation of antigenic diversity.

Understanding the nature of sequence change in the

human immunodeficiency virus type 1 (HIV-1) genome is central to current theories of viral pathogenesis and the immune response to infection. In common with other

eu-caryotic viruses with RNAgenomes, HIV-1 shows

consid-erable sequence diversity between different isolations,

par-ticularly those from geographically distinct regions, where divergence has taken place over a number of years.

Se-quence diversity is seen within isolations from the same

individualaswellasbetween HIV strains infecting different individuals (11, 29). Therate of HIV sequence variation is not uniform throughout the genome. Comparison of pub-lished sequences has shown that thegagandpolgenes are more conserved than env. Furthermore, within env the

pattern of variation is unusual (3, 34, 38). Five regions in

gpl20,VltoV5,have beendesignatedashypervariable (23). Theseweredefinedasregionswith25%orlessconservation ofaminoacids betweenanumberof published sequences.In addition to high rates of amino acid substitution, the

pub-lished sequences ofsome hypervariable regions have been

previously reportedtocontain short deletions and insertions (3, 11, 23, 34). Several actualorpotential gpl20 epitopesare located in these regions (23, 34). Antigenic variation

conse-quent to sequence differences in V3 (the immunodominant

loop)hasbeen reported(19, 36).Thissequencecorresponds

tothepredominant neutralization epitope of gpl20(28), and ithas beenargued that the observed variabilityrepresentsan

* Corresponding author.

tPresentaddress:DepartmentofMedicalMicrobiology,Medical

School, University of Edinburgh, Teviot Place, Edinburgh EH3

9AG, Scotland.

adaptive responseby HIVto evade the immune system, as

proposedforequineinfectious anemia virus and visna virus

(4, 5, 24). Whether othervariable areasofgpl20are

impor-tant in neutralizing-antibody or T-cell recognition is not known.

In thiswork,variation in thegpl20sequencesofprovirus

present in circulating peripheral blood mononuclear cells

(PBMCs) was studied. Anumber ofindividuals included in thisstudywereinfected fromacommon source(20),

allow-ing variation between patients to be assessed. The hyper-variableregionsofenvnotonlyarepolymorphicinsequence but inmanycases also differ inlength (3, 11, 23, 34). Thus, thespacingof theconservedregionsofgpl20shows consid-erable variation. Using the polymerase chain reaction

(PCR), we have investigated length variation of amplified DNA in different regions of gpl20 and have visualized distinctive patterns ofcoexisting variants in each infected individual. Thedataonlengthvariationtogetherwith exten-sive data obtained by sequencing hypervariable regions of

single isolated molecules (30) indicate that the number of variants present in eachpatientis extremely large. Despite

this heterogeneity, HIV sequences from individuals in the

hemophiliac cohort, who were infected from a common

source, wereclearlymorecloselyrelated toeach other than

to published sequences of HIV-1 and to those ofa

hemo-philiac infected in the United States. In particular, the V4

sequence of the cohort memberswasdistinctive and served

to distinguishthem from other hemophiliacs.

MATERIALSAND METHODS

Clinical samples. Blood samples were obtained from 11

hemophiliacs infected with HIV-contaminated factor VIII

5840 0022-538X/90/125840-11$02.00/0

Copyright ©1990,American Society forMicrobiology

on November 10, 2019 by guest

http://jvi.asm.org/

ANALYSIS OF SEQUENCE VARIATION IN gpl20 5841

approximately

5 years ago

(p12

to p95). For sequencing

studies, samples

were used from eight haemophiliacs who

were exposedabatch of factor VIII thatwasimplicated in infection

by

HIV-1 of 18 outof 32 recipients(20). Alleight individuals seroconverted for antibody between 3 and 10 months after

receiving

the factor VIII (31). p82 is currently

asymptomatic,

and p83 has thrombocytopenia but is other-wise

well;

all others have been classified as IVc and suffer from a range of

opportunistic

infections and constitutional symptoms of HIV infection. Samples from individuals in-fected from othersources include

p12,

whowasinfectedin theUnited StatesfromcommercialfactorVIII,and 10drug abuse-related and

pediatric seropositive

individuals (nl to

nlO)

from J. Mok

(Infectious

Diseases Unit, City Hospital,

Edinburgh, Scotland).

HIV primers. Oligonucleotides were synthesized by the OswelDNA

Service,

Departmentof

Chemistry,

University of

Edinburgh,

andwere

purified by high-performance

liquid

chromatography.

The

primers

werebasedontheconsensus

of the

following published

HIV sequences: HIVHTLVIIIB

(clones HXB2,

BH102, and BH8), HIVBRU, HIVCDC42, HIVELI, HIVMAL, HIVMN, HIVPV22, HIVRF, HIVsc,

HIVSF2,

HIVWMJ22,

and

HIVZ6.

The

primer-binding

sitesin

gpl20

were chosen to be as

highly

conserved as

possible

between

published

sequences of

geographical

variants of HIV-1. Nomore than one mismatchwith any of the North

American, Haitian,

orAfrican sequenceswaspresentin any

oneof the

primers.

The sequencesof the

primers

are

given,

with their

positions

in HIV clone HXB2indicated in paren-theses

(+,

sense; -,

antisense):

V3(a)TACAATGTACACA TGGAATT

(+, 6957),

(b)

TGGCAGTCTAGCAGAAGAAG

(+, 7009), (c)

CTGGGTCCCCTCCTGAGG (-, 7331), and

(d)

ATTACAGTAGAAAAATTCCCC (-, 7381); V4-V5 (e) TCAGGAGGGGACCCAGAAATT

(+, 7316), (f)

GGGGAA TTTTTCTACTGTAAT

(+,

7360), (g)

CTTCTCCAATTGT CCCTCATA

(-, 7665),

and

(h)

CCATAGTGCTTCCTGCT GCT

(-,

7814).

Double-PCR method. DNA fromat least 5 x

106

PBMCs

was

prepared

as described

by

Simmonds etal.

(30).

Ampli-fication of DNA

by

double PCR and

quantification by

limiting

dilutionwerecarriedout asdescribed

by

Simmonds

etal.

(30).

All DNAextractions and

amplification

reactions carried

appropriate parallel negative

controls

(blood

from

seronegative,

low-risk group blood

donors)

to detect

con-tamination at any stage in the

procedure.

In

principle,

at

least five outer

primer pairs

may be used

simultaneously

in the first

amplification

reaction.

However,

DNA sequences

amplified by

the outer V3 and V4-V5

primers overlap.

To

permit

the same

sample

to be

amplified

in the two

regions,

the

following

combinations of

primers

wereused. In the first

reaction, positive-sense,

outer V3

primer (primer

a; see

above)

was used with the

antisense,

outer V4-V5

primer

(primer

h),

to

amplify

a

858-bp

fragment;

in the second

reaction,

a

sample

of the

product

was

amplified by using

the inner V3

(primer b)

andinner V4-V5

(primer g) primers (657

bp);

in the third

reaction,

the final

amplification

used the innerV3

(b plus c)

andV4-V5

(f

and g)

primers

in separate reactions. Thismodified

procedure

is

equivalent

in

sensitiv-ity

and

yield

to separate

amplification

in two stages

by

double PCR

(data

not

shown).

Length analysis of PCR products. To

investigate length

variation in

product

DNA

amplified

in the threeenv

regions,

the final PCR reactionwascarriedoutin the presence of0.05 to 0.1

,uCi

of

[cI-355]thio-dATP

(1,000

Ci/mmol; Amersham)

and 8.3 mM each of unlabeled

deoxynucleoside

triphos-phates (dNTPs).

A

1-,ul sample

of the PCR

product

was

heated to 95°C in 50% formamide for 3 min and electro-phoresed on a denaturing polyacrylamide gel (6% acryl-amide, 0.3% N',Nbisacrylamide, 8 M urea, 0.089 M Tris, 0.089Mboric acid,0.002MEDTA, pH 8.3). Standard DNA sizemarkers (1,635, 1,018, 516, 506, 394, 344, 298, 220, 200, 154, and 142 bp; BCL)were end labeled with [_y-32P]dATP. Gels were dried and exposed overnight on X-ray film. Analysis of migrationdistances and intensity of bands was assisted by theuseofadensitometer.

DNA sequencing ofPCR products. The double-PCR pro-cedure produces sufficient DNA from single isolated mole-cules oftarget sequencetopermit direct sequencing of the PCR product (30). Unreacted dNTPs and primers were

removedfrom product DNA by using Gene-Clean (Bio 101, Inc.). Direct sequencingwasthencarried out by usingoneof theprimersusedfor the finalamplification bytheSequenase protocol (U.S. Biochemical Corp.), with the following

mod-ifications: the standard annealing mix was adjusted to 10%

dimethyl sulfoxide (40), and the sample was denatured by boilingfor 5 min andimmediatelychilled onice. The labeling reactionwas performedatroomtemperaturefor5min with 0.25 ,uM two unlabeled dNTPs and 5 ,uCi ofal-35S-labeled dATPordCTP(1,000Ci/mmol; 37).Thechoice of unlabeled and labeled dNTPs wasbased onthe nucleotides present 3'

to the priming site used. Termination reaction mixtures contained a final concentration of 10% dimethyl sulfoxide. Sequences were analyzed on a 6% polyacrylamide wedge sequencing gel.

Nucleotidesequenceaccession number.All sequences used in this study have been submitted to GenBank (accession numberM36997).

RESULTS

Amplification of HIV provirus by PCR. Two different

approaches to PCR amplification were used. In the first, a

sample of DNA large enough to contain a representative sampleofproviruswas amplified. In this case, theproduct should be representative ofthe provirus population in the

sample (see below). In the second method (30), single moleculesofproviruswereisolatedbydilution beforebeing amplified. In this case, the product of each amplification derives fromasingleprovirus.In many cases,adouble-PCR reactionusing nested primers was used. In this

method,

a

small sample of the first PCR reaction is used to prime a

second(30). Consequently, theproductsofafirst

amplifica-tion can be usedto prime more than one

amplification,

for example, one with labeled precursor for visualization by autoradiographyandasecondwithout labeled precursor for directsequencing (see below).

In published HIV-1 sequences, the length of the region amplified bythe V4-V5 primers

(spanning

V4,

C3,

and

V5;

23) varies by as much as 30 of around 300 nucleotides. Substantial size variationin this

region

is also found within the

provirus

population

presentina

single

infected individ-ual. Forexample, a sample ofDNAfrom

hemophiliac p79,

containing about 60 molecules of

provirus

(30), was

ampli-fied with the V4-V5

primers

and labeled with

[a-35S]thio-dATPduringthe second

amplification

step. When

analyzed

by gel

electrophoresis,

the

product

showeda

complex

setof size variants (Fig. 1, lane U). To confirm that these were

natural variants present in the DNA

sample

rather than artifacts of the PCR

reaction, single provirus

moleculeswere

isolated by dilution before

amplification

(30). A total of 31 replicate tubes, each containing 625

cell-equivalents

of DNA, were

amplified

in the double

PCR,

with

[a-35S]thio-VOL.64, 1990

on November 10, 2019 by guest

http://jvi.asm.org/

M U 1 2 3 4 5 6 7 8 9 10 11 12 13 N M

Wm.

394 _

344-_

220-.

FIG. 1. Size analysis of amplifiedproviral DNA in the V4 and V5 regions fromPBMCs ofanHIV-infected individual (p79). Positive

reactionsatlimiting dilution of PBMC DNA fromanHIV-infected

hemophiliac (p79)were amplified with V4-V5 primers and

electro-phoresedon apolyacrylamide gel (lanes 1 to13). Lane U, 1 ,ug of PBMC DNA from p79; lane M, product size estimated with DNA markers of sizes (in nucleotides) indicated (see Materials and Methods); lane N, 1 ,ug of PBMC DNA fromanuninfected

individ-ual.

dATPadded to the second reaction. Thirteen of the

reac-tions were positive. From the 18 negative reactions, the Poisson formula predicts that approximately 75% of the

positive reactions are due to the amplification ofa single

provirus molecule. Electrophoretic analysis of the positive reactions showed that the lengthvariants found by amplify-ing 60 provirus molecules togetherwerealso foundwhenthe

moleculeswereisolated before amplification (Fig. 1, lanes1 to 13). A single lane (lane 5) clearly contained two length variants, consistent with the prediction of the Poisson for-mula.

Todetermine the basis of the length variation, the prod-uctsof 12 of the 13 first PCRreactions (omitting reaction 5) were againamplified in the second PCR but without labeled

substrate and directly sequenced (30). The 12 nucleotide

sequencesare showninFig. 2. Thelengths of the amplified

sequences calculated from electrophoretic analysis agree

well with thelengths determined by sequencing (Fig. 2,last

two columns). Thus, the differences in electrophoretic

mo-bility reliably reflect differences inlength.

Theisolatedmolecules appearedtobearandom sampleof

the sequences visualized in the bulk amplification of 60

provirus molecules (Fig. 1, lane U). Thus, the four more

intense bands in lane U withlengths of 296, 299, 302 and 305 nucleotides were represented by one, one, six, and two

isolatedsequences,respectively. Faint bands with lengths of

284 and 293 nucleotides were represented by one isolated

sequence,and others withlengths of 287 and 281 nucleotides

were not represented. Thus, the analysis of bulk samples providesaconvenient and rapidassessmentof thespectrum

of size variation within the provirus populationpresentin a

DNAsample.

Amino acidsequencevariation in HIV-infectedindividuals.

PBMC DNA from eight other infected individuals was diluted andsequencedin theV4 andV5 regionsasdescribed

for p79, in all cases using low frequencies of positive

reactions at limiting dilutiontoavoid multiple positives. To simplify the presentation, the nucleotide sequences were

translated. Figure 3 shows alignments of such amino acid

sequences obtained from each individual. Considerable

di-versity in the V4 and V5 regions was seen within and between individuals. In many individuals, itwas necessary to insert notional gaps to preserve alignment of the

se-quences. These gaps were concentrated exclusively in the

V4andV5 regions,where therewasalsoahighrateofamino acid substitution. Eachgap was amultiple of 3 nucleotides,

maintainingthereadingframe of thesequence.Thedegree of

sequencevariationwithinanindividualvariedconsiderably,

the sequences from p77and p79 showing the most

hetero-geneity in this groupandthose from p84 showing theleast. A number of sequences showed short repeats of 3 to 6 amino acids in V4 and V5 (Fig. 3). The same repeated

sequences were found in different individuals. In V4,

se-quencescontaining (N)STWwererepeatedin p74,p77, and

p82, whilep77, p79, p83, p84, p87, andp91 showedrepeats

ofsequences TTGSN and TTESN. In V5, repeats ofNET

were found intwoindividuals, p77 and p12. The number of repeated sequences varied both between and within

sam-ples.This duplication ofsequence motifsaccountsforsome of the observedlength variation. SitesforpotentialN-linked

glycosylation sites were concentrated in the hypervariable

regions. In each individual, there was a concentration of

potential N-linked glycosylation sites in the V4 and V5 regions. The number and positions ofsuch sites were vari-able between individuals and within each patient sample (Fig. 3). Variation in the number of repeats changed the overall potentialforcarbohydrate addition in this region of

gpl20 (Fig. 3).

Lengthpolymorphism of DNAamplifiedin the V4 and V5 regions. With theexception of p82, few identicalsequences

Seq. < V4- > < C3 > <---V5 --->

<-Sequence Measured

C4 Length (bps) Length (bps)

1 51 bps+

2 51 bps+

3 51 bps +

4 51 bps+

6 51 bps +

7 51 bps+

8 51 bps +

9 51 bps+

10 51 bps +

11 51 bps +

12 51 bps +

13 51 bps +

Cons51 bps+

+ 132 bps+ + 132 bps+

+ 132 bps+

+ 132 bps+ + 132 bps+

+ 132 bps+

+ 132 bps+

132 bps+

+ 132 bps+ + 132 bps+ + 132 bps+ + 132 bps+ + 132 bps+

+41 bps =

+41 bps=

+41 bps=

+41 bps=

+41 bps=

+41 bps=

+41 bps=

+41 bps=

+41 bps =

+41 bps=

+41 bps=

+41 bps=

302 302 293 305 302 284 302 305 302 302 296 299 303.4 302.7 294.3 306.3 303.0 285.1 302.0 305.8 302.0 302.0 295.8 300.1

[image:3.612.70.289.75.232.2]

+41 bps= 305

FIG. 2. Alignment ofsequences obtained by limitingdilution of PBMC DNA from p79. Sequence labels V4, C3, V5, and C4follow

previous usage (23). Symbols: ., unsequenced; -, gap introducedto preserve alignment; ?, no majority consensus. Sequences 1 to 13 correspondtolanes 1 to13inFig. 1. Differences from theconsensusareindicatedinlowercase.

a g t 9 9 a

c t- a

ttg a 9 a

t

t 9 a

t t a

.GATACTACAGGGTCAAACACTACAGGGTCAAATAAC ACTGAACCTATCACA

t g

t g .

ta_{t a --- .}

c ac tgacc

9 9 a

---gg a a g ----a

t 9

c ac g ac

t g a

---t c

---t ac c--- ..

ggg a a--- a

---AAAAACGTGAGCAAC G?GTCCACCGAGACC

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.612.64.552.571.686.2]

< V4- > N LFNSTWI Ng N-P# P# g# p# pN p# s gn i 9s i ge i ge i ge

#---# t

#---Nd TTesN tTeS? NtEplt

11

<---- V5--->

Int ns

DGGrn

ANALYSIS OF SEQUENCE VARIATION IN gp120

#g

NE

N#--t #9- t--N- -N- -INeT - i TEt I 79-a 79-b 79-c 79-d 79-e 79-f 79-g 79-h 79-i 79-j 79-k 79-L Cons

<---V4--->

[image:4.612.77.537.92.676.2]

STwNd * i a iN# Nf a a a a gd t 9 d t d t t

#---t

ttGSnI|

tTGSN|nTEpIT

<---V5 ->

t#t mts . #v #a-#v ke-#v

#g-#v fg-#v #g

-Ng

#e-t rrkt-# .

i#tt---n

rdm#-e-N#v

e-s deNk-

-dGgkn?s??-tET

83-a N [T# ] #dt en#k .91-a N la e# pi # --#

83-b N LNj# #et knk#e .91-b. L#d P

83-c s# t t # k #e . 91-c N N eN

83-d N L# i # n -#g . 91-d Ni e N --# #

83-e N [ t --- # n -#gg91-e N#

Cons STWND TL TGSN N?gnIT DGG?egN?TE. Cons STWND tTgsN tTgSN NTEiIt DGGgsNss--NET

84-a N N N d N.87-a a# r e#ksspt

84-b N N N d #a . 87-b

eN-s

s p t

84-c N N s d . 87-c - e#ks s p t

84-d N N N - . 87-d N- N N s- kg# t

84-e N N N -Ntk . 87-e N g N t -- g#t

84-f N N N -Nt . 87-f N N N -N-te

84-g . N N N t . 87-g N N N - N-te

84-h - N N N -Nt . 87-h N N N - #-te

Cons STWND TTGSN TTGSn NTETIT DGG-ENR?eTE. Cons STWNd TteSN TTgSN NnEtIT DGG?ngnk?e?Ei

-tytwN htgN#

DltqlNSTQ-NkeEN IT

# # # --n-dt #- ---dt#n---nr#es#N DGGNSgnksndTTEt 74-a 74-b 74-c 74-d 74-e 74-f Cons 12-a 12-b 12-c 12-d 12-e Cons k t n #tg #tg n d# n d# STWNN?DTSTWNK?eESgNIT

..- k sNN #gt

SNWStSPGEpNNTTGN--IT

#r- # s-#r- # s-#r- # # s---t

Ngt # t Ngt # t DGGtEn?-TENRtTEI t- a-qn qn DGG? -# #g Ng NeT d n nET t Ei

FIG. 3. Alignment ofsequencesfromninehemophiliacsinthe V4andV5 regions. Individualsequencesfrom eachindividualareindicated

as atoz.Symbols: notsequenced;-,gapintroducedtopreservealignment; #,asparagineresiduesiteofpotentialN-linkedglycosylation;

?,nomajorityconsensusatthis position;*,stopcodon.Conserved amino acidsareshowninuppercase;nonconservedaminoacidsareshown

inlowercase. Tandem repeatedsequencesare indicated by boxes. Potentialglycosylation sites shown whetherconserved ornot.

VOL. 64,1990 5843

77-a 77-b 77-c 77-d 77-e 77-f 77-g 77-h 77-i

77-j

77-k 77-L Cons stw 82-a 82-b 82-c 82-d 82-e 82-f 82-g 82-h 82-i 82-i Cons

N#

tysN |nstw STU

- I II

I

I

i

on November 10, 2019 by guest

http://jvi.asm.org/

n p

M11 2 3 4 5 6 7 8 9 10174 M 777982 87 91 89 95

394

344

298 _

[image:5.612.60.302.76.232.2]

220

FIG. 4. Size analysis ofamplified proviral DNA in the V4 and VS regions from PBMCs ofHIV-infected individuals. Samples (1 jig) of PBMC DNA from HIV-infected nonhemophiliacs (nl to nlO) and hemophiliacs (p74 top95) wereamplified with V4-V5 primers and electrophoresed on a polyacrylamide gel. Lane M, Product size

estimated withDNAmarkers of sizes (in nucleotides) indicated (see Materials andMethods).

were isolated from the same individual (Fig. 3). No two

identicalsequences wereisolated from p12, p79, p83, p87,or

p91; p74 and p77 each hadtwoidenticalsequences,p84 had

three, and only p82 and p87 had multiple incidences oftwo

sequences. Clearly, the sequencesdetermined donot

repre-sent the full range of variation present in most of the

individuals studied. It would be possible in principle to

sequenceeach DNAsample exhaustively. However,a

sim-pler approach to the question of variation within and

be-tween individuals is to use the PCR technique described

abovetoassesstheprofile of length variation in each sample.

The length analysis can be carried out with a standard amount ofPBMC DNA, sothatmany samplescanbe dealt withrapidly.

Todemonstrate therangeoflength variation within PBMC

samples, 1-,ug samples of DNA from 18 HIV-infected indi-viduals were amplified by using the V4-V5 primers as

described above(Fig. 4). The samples comparedwerefrom

p74, p77, p79, and p82, and the number of provirus

mole-culespresentin the1-,ug samples ranged from 60(p79)to200

(p82; 30). A close concordance was observed between the

sizesof the bands measured by gelelectrophoresis and sizes determined by sequencing isolated molecules from thesame

sample of DNA(Table 1). For example, theprominentbands in the p74and p82 samples with estimated sizes of 308.0 and 316.5 bp were paralleled by a predominance ofsequences

with overall lengths of 308 and 317 bp. Similarly, the wide

rangeof band lengths observeduponamplicationof thep77

samplewasparalleled byawiderangeoflengthsamongthe

individually determinedsequences. These data show thatan

analysis of length variation serves as an approximate mea-sureof the overall sequencevariationinaprovirus

popula-tion. The sequence data(Fig. 3) show thata lengthvariant may comprise several different sequence variants. For

ex-ample,sequences74-a, -b, and-chad thesameoveralllength as 74-e and -f, although the two groups differed in the

positions of thegaps and anumber of aminoacid

substitu-tions. An impression ofthelarge range ofvariation which

canoccurwithinasamplemaybegained by consideringthe

range of size variation and the number of sequences of a

given lengthtogether. In severalcases,thismustrepresenta

tremendousamount ofvariation inthe V4-V5 region alone. Band lengths overved in the V4 and V5 regions with

samples from eight hemophiliacpatientsare summarizedin

Table 2. Data from 10drug abuse andpediatric patientsare

alsolisted forcomparison. The overallrangeoflengths and patterns were similar in the two groups. The majority of

samples contained bandsin therange of293 to 317bp, but

there was considerable variation in the diversity of bands presentinasample. Atoneextreme,n3 andp77 had 10 and 13bands,respectively, with rangesof 287to317 and 287to

326 bp. At the other extreme, n6, n7, and p95 had single bands of296, 314, and 293 bp. Therangeoflengths of theV4

and V5 regions of geographically diverse isolated HIV-1

sequence variants, from 293 (HIVSF2) to 311 (HIVCDC42),

lies within the range observed in this study and suggests

possible selectiveconstraintson thesize oftheseregions.

Sequence variation in the V3 hypervariable region. To

investigate sequence variability of the immunodominant

loop (28),sequences of individualprovirus molecules in the

[image:5.612.62.567.539.715.2]

PBMC DNAsampleswere determined with the V3 primers

TABLE 1. Comparison of size ofamplifiedDNAwithoveralllengths ofsequencesobtainedbylimiting dilutiona

p74 p77 p79 p82

Amplified No. of Amplified No.of Amplified No.of Amplified No.of

DNA sequences DNA sequences DNA sequences DNA sequences

325.1

322.5 1 at323

316.6 1 at317 316.5 8at317

313.7 2at314 313.7 1at314

311.2 311.7 1 at311

310.0 5at308 308.3 2 at308 306.9

305.1 305.1 2 at305 304.0 2 at305

301.2 302.0 301.65 6 at302 301.3

299.2 1 at299 299.9 1at299

295.5 1 at296 296.5 298.1 1at296

293.8 1 at 293

290.7

288.0 288.0 2at287

1 at284

a Prominent bandsareinbold type; all sizes aregivenin basepairs.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.574.42.540.80.279.2]

ANALYSIS OF SEQUENCE VARIATION IN gpl20 5845 TABLE 2. Distribution of length variants in theV4and V5 regions

Intensity of bandb at observed size (bp) of: Samplea

287 290 293 296 299 302 305 308 311 314 317 320 323 326

nl 3 3 1 2 1

n2 3 3 3

n3 1 2 1 3 3 1 1 2 1 1

n4 2 2 1 5 1

n5 1 1 3 3 1 2

n6 5

n7 5

n8 2 2 2 1 2 2 1 1 2 1

n9 5 2

nlO 1 1 5 1 3 1

p74 1 2 1 2 4 1

p77 1 1 2 1 2 1 2 2 1 1 2 1 1

p79C 1 1 3 2 3 2

p82 1 1 2 1 1 4

p87 2 3 3 2 1

p91 1 3 3

p89 1 2 3 3

p95 5

anltonlO,HIV-infected nonhemophiliacindividuals; p74 to p95, hemophiliacs.

bScored on a scale of 1 (weak) to 5 (strong). c Bands (intensity= 1)also at 284 and 281 bp.

(Fig. 5). Sequences bearing the same identification letter in Fig. 3 (V4and V5 sequences) and Fig. 5 (V3 sequence) were derived from thesameprovirus molecule(seeMaterialsand Methods). Sequence variation accompanied by little length variation was the dominant feature of the sequences of V3 andflankingregions(Fig. 5). The sequencediversity within a sample was no less than in the V4 and V5 regions. However, in theV3 region, each sample showed particular sequence features, oftencommon toall sequences withina sample, whichdistinguished it fromothersamples. Overthe V3 region as a whole, only 23 of 83 amino acids were conserved in all of the sequences listed in Fig. 5. The immunodominant loop sequences all differed considerably from theprototypeHIVMNand

HIVHTLVIIIB

(clone HXB2) sequences and from those of other geographical HIV-1 variants (Fig. 5). The core sequence GPGRwas well con-served between individuals, although a numberof variants existed. However, in one individual (p12), the majority of sequences wereGSGR.

The flanking regions of V3 contain a large number of potential N-linked glycosylation sites. Therearesix of these in theprototype HIVHTLVIIIB sequencein Fig. 5, and there are six potential sites for N-linked addition lying to either side of the V3 loop structure. No glycosylation sites are common to all of the sequences. By contrast, the two cysteine residues spanning the immunodominant loop are absolutely conserved (Fig. 5).

DISCUSSION

Diversityof envsequences withinHIV-infectedindividuals. Astriking feature of the proviralsequencesreportedhereis the diversity of env sequences within a number of the individuals studied. Inmany cases,there isacomplete lack of

homology

between variantsover anumber of amino acid residues (for example, the V5 region ofp79). It is clear that

avery considerable amountofsequencing would be neces-sary to describe fully the range of variation in proviral sequences within PBMCs. Thevisualization of length

vari-ants in the V4andV5 regions provides a rapid method for the partial characterization of sequence variants within a

sample. Analysis of the amplified DNA from samples con-taining large numbers of proviralsequencesshows therange and relative abundance of sequences that differ in length. The niethodprovides evidence for the existence of relatively scarce sequences that were not detected by conventional sequence analysis of arelatively small number of variants. The method does not provide comparative sequence infor-mation on the different length variants, and each length variant comprises an unknown number of distinct se-quences. However, itprovides a simple description of the provirus population whichallowslarge numbers of individ-ual samplestobecompared. Applications include the anal-ysis of sequence change over time in an individual (P. Simmonds, unpublished data) and comparisons between proviralvariants inPBMCsand viral RNAinplasma(L.-Q. Zhang, personal communication). It also allows genuine positive PCR results from patient screening to be distin-guished from those due to contamination by cloned se-quences(39).

Phylogenetic significance of amino acid changes in env. Many

hemophiliacs

treated withcommercial factor VIII in the early 1980s became infected with HIV-1. The rate of infectionwas

considerably

lower in those whoweretreated with factor VIII prepared from plasma ofvolunteer blood donors inalow-prevalenceareafor HIV-1

infection,

suchas

Scotland (21). However, somehemophiliacs treated in Ed-inburgh solely with locally produced factor VIII were in-fectedwith HIV-1in 1984(20).Asingle batch offactor VIII has been implicated in the infection of 18 hemophiliacs, including hemophiliacs p74, p77, p79, p82, p83,

p84,

p87, andp91 studiedhere. p12wasinfected intheUnited States from commercial bloodproducts.

The V4 regions of six of the eight designated cohort members(p77, p79, p83,p84, p87, and p91)arevery similar toeach

other,

and allcontaintworepeatsofa

relatively

well conserved 5-amino-acid sequence, TTGSN. p77 and p79 contain variant

proviral

sequences thatlack one ofthetwo

copies

of thissequence, whilesomevariants fromp77have

a second copyofthe

preceding

LFNSTW sequence. How-ever, p82andp74, who werealsoconsideredtobeinfected

VOL.64,1990

on November 10, 2019 by guest

http://jvi.asm.org/

77-al

kdp

# t

s#

s h --a v hat e i di # kd # vt e#- N

77-di

kdp

# t s# s h-- v hat e i di #L d # vt e#- N

77-i kdp # t s# s h-- v hat e i di #L g # n vk k N- #

77-f kdp # t s# s h --a v hat e i di N kd # vt k N- #h

77-m #n t s# s h-- v hat e i di sL ed # vt e#- #

79-m #e v # s h-- yat e t i #l e # vt k#- #

79-n #e v i N s pm-- ks yat d ii l e # vt k #-r #

87-a kdp k # g#y e s-- arrq i di k e # r vt k k#- NI

87-b kdp k # g#y e s-- arrq i di k e # r vt k e#- NI

87-c kdpk# g#y e s-- arrq i di k e # r vt k e#- #

87-d kdp k g# e s-- ar q i di k e # r vi k d#- #

87-e kd k# s# ss-- yat e i di #l e tk vte k r#- k #

87-f kd s# s s-- yat e i di #L e # r ai k k#-r # a

87-g kd s# s s-- yat e i di UL a # r ai k kf-r k #

87-hi kd s# s s-- yat e i di #L a # r ai k k#-r k #

91-a . k g# s-- atsqi di k #L ee# vt

91-b kp k # g# s-- atsqidi k #L ee# vt k#- n..

91-c . k # g# sp-- atsqi di k # ee# vt

91-d . k # gd srn-- yatd i di #L eedd vt k

91-e . k # g# s P-- yat didi #L ee dd vt ...

82-c #e v t y-- vy teq ii #e vi

e#-82-d #e v t y vy teq i i U #e vi

e#-82-e #e v# h-- vy teq i U #e vi e - a

82-f #e v i y v teq ii N #e vi

eN-82-g #e v i # y vy teq ii #e vi

e#-82-h #e v # g h-- s yatg i di #e vt k#- #

82-i #e v# # g h-- s yat g i di N #e vt k#- U

82-j #a v t h-- vy t q i di #L #e r vi g t k r

74-a #e v # rg h-- yat n i di t #d vt #-S #f

74-b #e v # rg h-- yat n i di t #d vt #-s

74-d t #e v # g# rs h --wm yat e i di Ut i vt V#- #f

74-f #e v # # rgh-- yaar i di #L t #d h vt r#-r gr

74-g #e v # s# rg h-- yat n i di t #d vt #-s t

12-b #n U sk i rs h --s y eg a dv k y tL#gt #d lvav- q p

12-c #n # sk i rsmh --s y eg a dv k y tL#gt #d Lvav- q p

12-d #s N s k rsh --s y eg t dv k y tL#gt #d L va - t p

12-e #n s#t rs h-- y t e t dveky tL#gt #d L va - k qp

MN h #e q # y k h-- y tkn i ti # #d r v k

k-RF #a q # N s tk-- viyat q i di k UL q # vt d#- ts

SF2 #e aN # s y -- h t r i di k q # e vk # UJ

HXB2 TIIVQL#TSVEI#C TRPN#NTRKRIRIQRGPGRAFVTIGKI -GNMRQAH C#ISRAKW#NTLKQIDSKLREQFGN#KTIIFKQSS|

I--- IMMUNODOMINANT

LOOP---FIG. 5. Alignment of sequences obtained fromsevenhemophiliacs in theV3andflanking regions. Individual sequencesareindicatedas

ato z. Symbolsare asforFig.3. Differences from thepHXB2 sequenceareshown in lowercase. Allpotential N-linkedglycosylationsites are shown.

from the same source, contained

proviral

sequences in this

area that were distinct from each other, from those of the other cohort members and

p12,

and from published

se-quences(Fig. 6A). However, atthe time ofseroconversion, the V4 sequences of both individuals were similar to those

reported

here for the twoindividuals(Zhangand Simmonds, unpublished observations). This finding rulesout the

possi-bility

thatahigher rate of sequence change ofHIV in these

two individuals was responsible for the current dissimilari-tiesin V4 sequencesfromtherestof the cohort. At present, itisnot clear whether several strains ofHIV-1 werepresent

in the same contaminated batch of factor VIII orwhether p82andp74wereinfectedfromadifferentsourcealtogether. There isnoevidence for other risk factors forHIVinfection in either hemophiliac. The most likely sources are other infectious batches oflocally prepared factor VIII concen-trate. V4 sequencessimilartothoseofp82have beenfound in anotherhemophiliac (datanotshown)whowasnottreated with theimplicatedbatch of factor VIII but who didreceive several otherlocally producedbatches thatwere alsogiven

top82.

Incontrast tothesimilarities observed in the V4

region,

no

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.612.106.509.69.559.2]

ANALYSIS OF SEQUENCE VARIATION IN gpl20 5847

A

p77 |StWLFNSTW|Nd TTesN tTeS? NtEpIt p79 STw Nd ttGSn tTGSN nTEpIT p83 STW ND TTGlN iTGSN N?gnIT p84 STW ND TTGSN TTGSn NTETIT p87 STW Nd TteSN TTgSN NnEtIT p91 STW ND tTgsN tTgSN NTEiIt p82 STW nst| DltqtNSTQ-NkeEN IT p74 STW NN?DTSTWNK?eESgN IT

p12 SNW StSPGEpNNTTGN-- IT HXB2 STUWFNSTW|S S NN ji DT IT

RF STW NSTEGSNNTGGNDT IT

SF-2 NTW RLNHTEGTKGNDT II

B

p77 DGG

rn[N

eieI

TEtI

p79 dGg kn?s??-t ET p83 DGG ?egN?T E. p84 DGG -ENR?eT E. p87 DGG ?ngnk?e? Ei p91 DGG gsNss--N ET

p82

p74

p12

HXB2

RF

SF-2

DGG NSgnksndTT Et DGG tEn?-TENRtT El DGG

?-Ne iI

Ei DGG NSNNES ET

DGG E DTT NTT El

DGG

T NVT NT EV

C

p77 TIIVQLkdpVnITC TRPSNNTRKSIHI--gPGRVFHATGEIIGDIRQAH

CnLSR?dWNNTLkQIVtKLrEQFeN-KTIIFNqSSI

p79 TIIVQLNESWINC ?RPNNNTRKSI??--GPG??FYATG?I?GNIRQAH

CNLSRAEWNNTLKQIVTKL?EQF?N-?TIIFNQSSI

p87 TIIVQLKDpVkINC TRPgNnTRerISI--GPGRAF?A?g?IIGDIRQAH CN?S?AeUnnTLrQIv?kLKEQF?N-kTiIFNQSs p91 TIIVQLKNPVKINC TRPGnNTRKsIpI--GPGRAFvATsqIIGDIRkAH CNLSREEWnnTLKQIVTKLrEQFKN-KTIIFN...

Cons TIIVQLkdpV?InC tRP?nnTRksl?i--gPGraF?Atg?IiGdlRqAH CnLSraeWnnTLkQIvtkLkEQF?N-kTiiFNqSs

Cons TIiVqLNeSVvInC tRPnrntRk?ihi--gpGrafyttg?IiGdirqAh CnisrakWn?TLkqIv?KLrEQF?n-ktliF?qSS

p82 TIIVQLNeSWInC tRPNNNTRKrI?I--GPGrAvYtTeqIIGnIRQAH CNiSRAKWNETLkQIViKLrEQFen-KtIVFkqSS;

p74 TIiVQLNESWINC TRPnNNTRRgIHI--gpGRAFYAtgnIIGDIRQAH CNiSRTKWndTLKqIVTKLREQFgN-sTIVFnqSSI p12 TIIVQLNnSVEINC TRPS?n?RRS?HI--GsGRAFYTiegI?GDVrKAY CTLNGTKWNDTLKLIVAKLREQFGN-KTI?FkPSSI

MN TIIVHLNESVQINC TRPNYNKRKRIHI--GPGRAFYTTKNIIGTIRQAH CNISRAKWNDTLRQIVSKLKEQFKN-KTIIFNQSS RF TIIVQLNASVQINC TRPNNNTRKSITK--GPGRVIYATGQIIGDIRKAH CNLSRAQWNNTLKQIVTKLREQFDN-KTIIFTSSS SF2 TIIVQLNESVAINC TRPNNNTRKSIYI--GPGRAFHTTGRIIGDIRKAH CNISRAQUNNTLEQIVKKLREQFGNNKTIIFNQSS

HX82 TIIVQLNTSVEINC TRPNNNTRKRIRIQRGPGRAFVTIGKI-GNMRQAH CNISRAKWNNTLKQIDSKLREQFGNNKTIIFKQSSJ

FIG. 6. Comparison of hemophiliac and published sequences in the env hypervariableregions V4 (A), V5 (B), and V3 (C). Consensus sequencesfrom individuals believedto havebeeninfected from a common source (p77, p79, p83, p83, p87, andp91)are compared with those ofapparently unrelatedsequences(p82andp74), thoseof a hemophiliac infectedfrom commercial factorVIII, and published sequences. Differences fromtheconsensus within infected individual are indicated in lowercase. ?, No majority consensus at this position in an individual orgroup consensussequence.

relationship between thecohort members is apparent in V5 (Fig. 6B), where sequence variation is much greater. The

rateofsequencechangein the short V5region appears more rapidand does notindicateanyrelatednessbetween the six individuals who have similar V4 sequences. The V3 regions of the members of the hemophiliac cohort differ from the reference HXB2 sequence by a number of amino acids. However, no clear relationship distinguishes the cohort sequences. This is shown bythesimilarity in the consensus of the four confirmed cohort members and the combined

consensus of noncohort members p12, p74, and p82 and published sequences (Fig. 6C). There are only five

differ-encesbetween theconsensus sequences of thetwogroups, and at all but one of these sites, variants within the

con-firmed cohort members exist that match the noncohort sequences (Fig. 6C).

Nature of variation in the hypervariable regions of env.

Both amino acid substitutions and gaps contribute to

varia-tion in the V4 and V5 regions. Many of the gaps in V4 involve repeated sequences suchasTTGSN (p77 and p79). In such cases, some variants have one copy while others have two. Similar variation in the numbers ofcopies of the sequence (F)NSTWmay also befound inp77. Indeed, the existenceof repeatsofthesetwo sequencesinmost individ-uals indicates thatsome sort ofduplicationeventhas taken place. In many sequences, there are minor differences

be-tweenthe twocopies, suggestingthat somesequencechange has taken place after duplication. The exact sequence in-volved in a duplication event may differ. Forexample, the

block NSTW isrepeatedinp82, while inp77thereis oftena

repeat of the longer sequence LFNSTW. In published

se-quences of the viral isolated HIVHTLVIIIB, the BH8 clone has one copy of the sequence FNSTW, while others

(for

example BH10) have two. The widespread occurrence of these repeated sequences (3, 11, 23, 34)and the likelihood that they occur independently in different HIV-infected

VOL.64, 1990

I

on November 10, 2019 by guest

http://jvi.asm.org/

[image:8.612.104.510.78.473.2]

individuals suggest that this sequence is predisposed to duplicationduring eitherreverse transcriptionorRNA syn-thesis. Furthermore, ifduplicationisoccurring repeatedlyin thesamplesthat we haveexamined,it is alsolikelythatonce

formed, theduplicationsare predisposedtodeletion. Alloftheinsertionsordeletions in the V4 andV5regions aremultiples of3 nucleotides,thus

maintaining

thereading frame downstream. Similarly, only one chain termination mutation (p79, sequence 1; Fig. 3) was found in 37 V3 sequences and 71 V4 and V5 sequences. The low rate of inactivating mutations is consistent an absence of pheno-typic mixing. Thisfinding mayreflectthe low copy number (closeto one) ofprovirus in infected PBMCs(30).

Positive selection for sequence change in hypervariable regions of env. There has been nosatisfactoryexplanationof the high rates of mutation in localized regions of the env

gene. It could be argued thatthe cause is

simply

a lack of functional constraints which might limit the amount of variation in regions such as the CD4 binding site (6, 16). However, this view isnotsupportedbyacomparison ofthe rates ofsynonymousandnonsynonymoussubstitutionrates in the different regions of env. Published data and data collected in thislaboratory (P. Balfeetal., unpublished data) give a ratio of synonymous to replacement substitutions

(KsIKa

ratio; 18) for the CD4 binding site of 1.24. For comparison, the Ks/Ka ratio for gag sequences (17) and those ofsamples studiedhereis about6.7, andtheratio for 42 eucaryoticgenes is5.28(18). Thus, theCD4bindingsite does not appear to be understringent constrainttomaintain its amino acid sequence, and the much higher substitution frequency of the hypervariableregionscannotsimply be due to lack of constraint. The KsIKa ratio for the sequences reported here ofthe V3 loop and flanking regions is 0.67, lowerthananypreviouslyreported. Onaverage, the survival ofareplacement mutation is almost twiceas

probable

asthe survival of a synonymous mutation.

Overall,

this

signifies

that selection favors change in this region, although the absence of stopcodonsand the conservationofanumberof aminoacids, includingthecysteineresidues

spanning

the V3 loop, indicate that the extent ofchange possible is limited. Although positive selection for changeisunusual,it has been observed in majorhistocompatibility complex proteins (13) and in mammalian and avianserineprotease inhibitors(12, 15). For the specific requirements ofincreaseddiversity of antigenic recognition by the major

histocompatibility

com-plex molecule, and for defense againsta range of bacterial proteases in the latter example, positive selection appears to confer a selective advantage for the mutated sequences. Given theknown involvement ofV3 in virus neutralization (28), selection forchange in V3 suggests that the selective forceis the immune defense system.

Consequences of sequence change in the hypervariable regions of env. Several areas of the env gene product have beenshown to beantigenicupon natural infection and upon vaccination (8, 9, 19,26-28,36). The V3 loop and regions in gp4l have been positivelyidentified as targets of antibody-mediated neutralization or cytotoxic T-lymphocyte (CTL) killing. Both immune effector functions are sensitive to sequence variation around the crown of the V3 loop, i.e., either side of the relatively well conserved GPGR central sequence. The specificity of the CTL response can be determined in large part by the amino acids immediately downstream of GPGR (36), while residues immediately upstream of GPGR have been shown to be important in antibody-mediated neutralization (10, 19, 22). Considerable sequence variation is found in both of these sites in the

sequences obtained in this

study.

Both

tyrosine

and valine

arefoundat a

position

criticalfor CTL

recognition (36),

not

only in different

samples

but also within the same

sample

(e.g.,p87 and p91;

Fig. 6C).

Ahistidine substitution atthis site (p77) would also be

expected

to

modify

the

epitope.

Other amino acid

changes

are concentrated at the site of B-cellrecognition,withmostsamples

containing

atleasttwo

differentsequences. Therearealsomutations in the central GPGRmotif

(p12,

p74,andp77)thatcould

disrupt

the,Bturn at this site (22) and probably abolish immune

recognition.

Thishighlevelofvariationin V3

predicts

the existence ofa

wide range ofneutralization serotypes in many of the pa-tients. Sera and CTLs from such individuals may be

ex-pected

to be reactive

against

a range of standard viral isolates. Thebroadening specificity of

neutralizing

antibod-ieswithtimeseeninHIV-seropositve individuals

(1)

maybe due to the de novo appearance of V3 serotypes upon

long-term

infection.

Neutralizing

antibodies that bind to V4 and V5

regions

have notbeendescribed, and thecontribution ofthesetwo

regions to the overall antigenicity of

gpl20

is uncertain. However,both

regions

have beenidentifiedas

being

poten-tially antigenic on the basis of surface

probability

and hydrophilicity (23). The absence of linear

epitopes

ineither

region

is shown by the poor

serological reactivity

with

synthetic oligopeptides containing

V4andV5sequences and the low

immunogenicity

of such

peptides

uponvaccination (25). This does notrule outthe

possibility

that V4 and V5 form conformational

epitopes

that are not mimicked

by

synthetic peptides.

Furthermore, the

antigenicity

of both

areasin vivomay bealtered

by

posttranslational

additionsof N-linked

oligosaccharide

groups.

gpl20

is

heavily

glycosy-lated at N-linked but not at 0-linked sites

(14),

and all 24

potential

N-linked sites are

glycosylated

whenrecombinant HIVHTLVIIIB

gpl2O

is

expressed

in mammalian cells

(T.

J.

Gregory,

C. K.

Leonard,

L.

Riddle,

J. R.

Thomas,

R.J.

Harris,

and M. W.

Spellman,

J. Cell. Biochem.

14D:151,

1990). N-linked glycosylation can mask

potential

peptide

epitopes

(2, 32)orthemselves formconformational

epitopes

(2, 7, 33, 35).

Glycosylation

ofV4 and V5 may therefore

serve to mask the

relatively

invariant

intervening

CD4

binding region.

The absence ofmonoclonal antibodiesto

V4,

C4,

andV5 may thus be areflection oftheeffectiveness of

glycosylation

in

masking potential epitopes

rather than a

supposed

low

antigenicity

of the

underlying

peptide

se-quences. The

preponderance

of

glycosylation

sites in the hypervariable regions, and the major alterationin the

posi-tionand numberofsuchsites

by

aminoacidsubstitutionand sequence

reduplication,

could therefore be

interpreted

asan

evolutionary

response

by

HIVtoevade the immunesystem.

ACKNOWLEDGMENTS

We thank F. McOmish andA. Cleland for technicalassistance.

Samples were collected by the staff ofthe Haemophilia Centre, Edinburgh Royal Infirmary.

Theworkwassupported bytheMedicalResearchCouncilAIDS DirectedProgramme.

LITERATURECITED

1. Albert, J.,B.Abrahamson,K.Nagy,E.Aurelius,H.Gaines,G. Nystrom,and E. M.Fenyo. 1990.Rapid developmentof

isolate-specific neutralising antibodies afterprimary HIV-1 infection andconsequent emergenceof virus variants which resist neu-tralisationby autologoussera. AIDS4:107-112.

2. Alexander, S.,andJ.H. Elder.1984.Carbohydrate dramatically

influences immune reactivity of antiserato viral glycoprotein antigens. Science 226:1328-1330.

on November 10, 2019 by guest

http://jvi.asm.org/

ANALYSIS OF SEQUENCE VARIATION IN gp120 5849 3. Alizon, M., S. Wain-Hobson, L. Montagnier, and P. Sonigo.

1986. Genetic variability of the AIDS virus: nucleotide sequence analysis of two isolates from African patients. Cell46:63-74. 4. Carpenter, S., L. H. Evans, M. Sevoian, and B. Chesebro. 1987.

Role of the host immune response in selection of equine infectious anemia virus variants. J. Virol. 61:3783-3789. 5. Clements, J. E., F. S. Pedersen,0. Narayan, and W. A.

Hasel-tine. 1980. Genomic changesassociated withantigenicvariation of visna virusduring persistent infection. Proc. Natl. Acad.Sci. USA 77:4454 -458.

6. Cordonnier, A., L. Montagnier, and M. Emmerman. 1989. Single amino-acidchanges inHIVenvelope affectviraltropism and receptor binding. Nature (London) 340:571-574.

7. Feizi, T., and R. A. Childs. 1987. Carbohydrates as antigenic determinants. Biochem. J. 245:1-12.

8. Gnann, J. W., P. L. Schwimmbeck, J. A. Nelson, A. B. Truax, and M. B. A. Oldstone. 1987. Diagnosis of AIDS by using a 12-amino acid peptiderepresenting animmunodominantepitope of the human immunodeficiency virus. J. Infect. Dis. 156:261-267.

9. Goudsmit, J., C. A. B. Boucher, R. H. Meloen, L. G. Epstein, L. Smit, L. Van Der Hoek, and M. Bakker. 1988. Human antibody response to a strain-specific HIV-1 gpl20 epitope associated with cell fusioninhibition. AIDS 2:157-164. 10. Goudsmit, J., M. C. Debouck, R. H. Meloen, L. Smit, M.

Bakker, D. M. Asher, A.V. Wolff, C.J. Gibbs, and D. C.

Gaidusek.1987. Human immunodeficiency virustype 1 neutral-isation epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees. Proc. Natl.Acad. Sci. USA 85:4478-4482.

11. Hahn, B., G. M. Shaw, M. E. Taylor, R.R. Redfield, P. D. Markham, S. Z. Salahuddin, F. Wong-Staal, R. C.Gallo, E. S. Parks, and W. P.Parks. 1986. Genetic variation in HTLV-III/ LAV over time in patients with AIDS or at risk for AIDS. Science 232:1548-1553.

12. Hill, R. E., and N.D.Hastie. 1987.Accelerated evolution in the reactive centre regions of serine protease inhibitors. Nature (London) 326:96-99.

13. Hughes, A. L., and M. Nei. 1989. Nucleotide substitution at major histocompatibility complex class II loci: evidence for overdominant selection. Proc. Natl. Acad. Sci. USA 86:958-962.

14. Kozarsky, K., M. Penman, L. Basiripour, W. Haseltine, J. Sodroski, andM.Krieger. 1989.Glycosylationandprocessingof thehumanimmunodeficiency virus type 1 envelope protein. J. AIDS 2:163-169.

15. Laskowski, M.,I. Kato, W. Ardelt, J. Cook,A.Denton, M. W. Empie, W. J.Kohr, S.J.Park, K.Parks,B. L.Schatzley,0. L. Schoenberger, M.Tashiro, G.Vichot, H. E.Whatley, A. Wiec-zorek, and M.Wieczorek. 1987. Ovomucoid thirddomains from 100 avianspecies: isolation,sequences, and hypervariabilityof enzyme-inhibitor contact residues. Biochemistry 26:202-221. 16. Lasky, L. A., G. Nakamura, D. H. Smith, C. Fennie, C.

Shi-masaki, E. Patzer, P. Berman, T. Gregory, and D. J. Capon. 1987. Delineation of aregion of the humanimmunodeficiency virus type1gpl20glycoprotein criticalforinteraction with the CD4receptor. Cell50:975-985.

17. Leigh Brown, A., and P. Monaghan. 1988. Evolution of the structuralproteinsofhumanimmunodeficiency virus: selective constraintsonnucleotide substitution. AIDS Res. Human Ret-roviruses 4:399-407.

18. Li, W.-H., C.-I.Wu, and C.-C. Luo. 1985. A new method for estimatingsynonymous andnonsynonymous ratesofnucleotide substitution consideringtherelative likelihoodofnucleotideand codonchanges. Mol. Biol. Evol. 2:150-174.

19. Looney, D. J., A. G. Fisher, S. D.Putney, J. R. Rusche, R. R. Redfield, D.S. Burke, R. C. Gallo, and F. Wong-Staal. 1988. Type-restricted neutralization of molecular clones of human immunodeficiency virus. Science241:357-359.

20. Ludlam, C. A., J. Tucker, C. M. Steel, R. S. Tedder, R. Cheingsong-Popov, R. A. Weiss, D. B.McClelland, I. Philip,and R. J. Prescott. 1985. Human T-lymphotropic virus type III (HTLV-III) infectioninseronegative haemophiliacs after

trans-fusion of factor VIII. Lancetii:233-236.

21. Melbye, M., K. S. Froebel, R. Madhok, R.J. Biggar, P.S. Sarin,S. Sternbjerg, G. D. 0. Lowe, C. D.Forbes, J. J. Goed-ert,R.C.Gallo,and P.Ebbeson.1984.HTLV-IIIseropositivity

inEuropeonhaemophiliacsexposedtoFactor VIIIconcentrate imported from the USA. Lancet ii:1444 1446.

22. Meloen, R. H.,R. M. Liskamp,andJ. Goudsmit. 1989. Speci-ficityandfunctionof the individual amino acids ofanimportant

determinant of HIV-1 that induces neutralising antibody. J. Gen. Virol. 70:1505-1512.

23. Modrow, S., B. H.Hahn, G. M. Shaw,R.C. Gallo,F. Wong-Staal, and H. Wolf. 1986. Computer-assisted analysisof enve-lopeproteinsequencesofsevenhumanimmunodeficiency virus isolates: prediction of antigenic epitopes in conserved and variableregions. J. Virol. 61:570-578.

24. Narayan,O.,D. E.Griffin,and J.Chase. 1977.Antigenic shift of visna virus inpersistentlyinfected sheep. Science 197:376-378. 25. Neurath, A.R., N.Strick,and E.S. Y.Lee. 1990. Bcellepitope

mapping of human immunodeficiency virusenvelope

glycopro-teins with long (19- to 36-residue) synthetic peptides. J. Gen. Virol. 71:85-95.

26. Palker, T. J., T. J. Matthews, M. E. Clark, G. J. Cianciole, R. R.Randall, A.J. Langlois, G. C.White, B. Safai,R. Snyder-man, D. P. Bolognesi, and B. F. Haynes. 1987. A conserved region at theCOOHterminusof human immunodeficiencyvirus gpl20envelope protein contains an immunodominant epitope. Proc. Natl. Acad. Sci. USA 84:2479-2483.

27. Reitz, M. S., C. Wilson, C. Naugle, R. C. Gallo, and M. Robert-Guroff. 1988. Generation of a neutralization-resistant variant ofHIV-1 is duetoselectionforapointmutation in the envelope gene. Cell54:57-63.

28. Rusche,J. R.,K.Javaherian, C. McDanal, J. Petro, D. L.Lynn, R. Grimaila, A. Langlois, R. C. Gallo, L. 0. Arthur, P.J. Fischinger, D. P. Bolognesi, S. D. Putney, and T. J. Matthews. 1988.Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid sequence oftheviral envelope, gpl20. Proc. Natl. Acad. Sci. USA 85:3198-3202. 29. Saag, M. S., B. H. Hahn, J. Gibbons, Y. Li, E. S. Parks, W. P.

Parks, and G. M. Shaw. 1988. Extensive variation of human immunodeficiency virus type-1 in vivo. Nature (London) 334: 440-444.

30. Simmonds, P., P. Balfe, J. F. Peutherer, C. A. Ludlam, J. 0. Bishop, and A. Leigh Brown. 1990. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheralmononuclearcells andatlow copy numbers. J.Virol. 64:864-872.

31. Simmonds, P., F. A. L. Lainson, R. Cuthbert, C. M. Steel, J. F. Peutherer, and C. A. Ludlam. 1988. HIV antigen andantibody detection: variable responses to infection in the Edinburgh haemophiliac cohort. Br. Med. J. 296:593-598.

32. Skehel, J., D. J. Stevens, R. S. Daniels, A. R. Douglas, M. Knossow,I. A. Wilson, and D. C. Wiley. 1984. Acarbohydrate side chainonhaemagglutinins ofHongKonginfluenza viruses inhibits recognition by a monoclonal antibody. Proc. Natl. Acad. Sci. USA 81:1779-1783.

33. Sodora, D. L., G. H. Cohen, and R. J. Eisenberg. 1989. Influ-ence of asparagine-linked oligosaccharides on antigenicity, processing, and cell surface expression ofherpes simplex virus type 1glycoprotein D. J. Virol. 63:5184-5193.

34. Starcich, B. R., B. H. Hahn, G. M. Shaw, P. D. McNeely, S. Modrow, H. Wolf, E. S. Parks, W. P. Parks, S. F. Josephs, R. C. Gallo, and F. Wong-Staal. 1986. Identification and char-acterization of conserved andvariable regions in the envelope gene ofHTLVIII/LAV, the retrovirus of AIDS. Cell 45:637-648.

35. Sugwara, K., F. Kitame, H. Nishimura, and K. Kakamura. 1988. Operationaland topological analysesofantigenic sites on influ-enza C virus glycoprotein and their dependence of glycosyl-ation. J. Gen. Virol. 69:537-547.

36. Takahashi, H., S. Merli, S. D. Putney, R. Houghten, B. Moss, R. N. Germain, and J. A. Berzofsky. 1989. Asingleamino acid interchangeyieldsreciprocalCTL specificities for HIV-1gpl60. Science246:118-121.

VOL.64, 1990

on November 10, 2019 by guest

http://jvi.asm.org/

37. Tsang, T. C., and D. R. Bentley. 1989. An improved method using Sequenase (tm) that is independent of template

concen-tration. Nucleic Acids Res. 16:6238.

38. Willey, R. L., R. A. Rutledge, S. Dias, T. Folks, T. Theodore,

C. E. Buckler, and M. A. Martin. 1986. Identification of

con-served anddivergent domains within the envelopegeneof the

acquired immunodeficiency virus syndrome retrovirus. Proc. Natl. Acad. Sci. USA 83:5038-5042.

39. Williams,P.,P.Simmonds, P. L. Yap,P. Balfe, J.0.Bishop,R.

Brettle,R.Hague, D.Hargreaves, J. Inglis, A. Leigh Brown,J. Peutherer, S. Rebus,andJ. Mok. 1990. Thepolymerase chain reaction in the diagnosis of vertically transmitted HIV infection. AIDS 4:393-398.

40. Winship, P. R.1989. An improvedmethod for directly

sequenc-ing PCR amplified material ussequenc-ing dimethyl sulphoxide. Nucleic Acids Res. 17:1266.

on November 10, 2019 by guest

http://jvi.asm.org/