VIROLOGY
190,
674-681
Ugandan
(1992)
HIV-1 V3 Loop Sequences
Closely Related to the U.S./European
JAN ALBERT,**’ LENA FRANZiN,++ MARIANNE JANSSON,*+ GABRIELLA SCARLAlTI,§=” E. KATABIRA,lI F. MUBIRO, MARIA RYDAKER,1- PAOLO ROSSI,+# ULF PEmERSSON,t
Consensus
PETER K. KATAAHA,1 AND HANS WIGZELL*+
*Department of Virology, Alational Bacteriological Laboratory, Stockholm, Sweden; tDepartment of Medical Genetics, University of Uppsala, Uppsala, Sweden; +Departments of immunology and §Virology, Karolinska Institute, Stockholm, Sweden; ‘IDepartment of Pediatrics I, University of Milan, Milan, Italy; Olakasero Blood Bank, Kampala, Uganda; 7Department of Medicine, Medical School, Makerere University, Kampala, Uganda; and #Department of Pediatrics, University of Rome “Tor Vergata, ” Rome, Italy Received
March
9, 1992;
accepted
June 29, 1992
The third variable (V3) loop of the human immunodeficiency virus type 1 (HIV-l) envelope protein is an important determinant for virus neutralization and cell tropism. V3 loop sequences from uncultured lymphocytes obtained in 1990 from 22 Ugandan HIV-l-infected patients could, with the exception of two patients’ sequences, be divided into two groups (A and B) on the basis the V3 loop size and sequence. The V3 loop consensus sequences from both groups showed a high degree of homology to a U.S./European consensus, a characteristic also reflected by the results of peptide serology. In the case of group B the difference in sequence was only five amino acids. In contrast, the V3-flanking regions for both groups showed greater homology to an earlier (1986/l 987) Ugandan consensus. The discovery of these two new Ugandan V3 loop genotypes, which are closely related to the U.S./European consensus, has implications for the understanding of the evolution of HIV-l and for the future design of a vaccine for use in Africa. o 1992 Academic
Press,
Inc.
sharply in sequence from the U.S./European consensus (Myers et al., 1991; Oram et al., 1991). Most of these isolates were obtained before or during the mid1980’s (Myers eta/., 1991; Oram eta/., 1991) and studies on current African strains are scarce. However, Myers et a/. (199 1) recently presented a consensus of mostly unpublished African V3 loop sequences, which again indicate that African HIV-1 strains are distinct from and more polymorphic than U.S./European strains. As a vaccine against HIV-l is urgently needed in Africa, it is important to determine the V3 loop sequences of additional African HIV-l isolates, especially from more recently infected individuals. The Ugandan HIV-1 sequences reported in this study appear remarkable in two aspects: they have unusually homologous V3 loop sequences compared to most earlier published African sequences and their V3 loop sequences show greater similarity to the U.S./European consensus than to an earlier (1986/1987) Ugandan consensus.
INTRODUCTION A major target for neutralizing antibodies to the human immunodeficiency virus type 1 (HIV-l) is located in a disulphide-bonded loop in the third variable (V3) region of the external envelope glycoprotein gp120 (Rusche et al,, 1988; Palker et a/., 1988). The V3 loop has recently been shown to be also an important determinant of HIV-1 cell tropism (Takeuchi et a/., 1991; Hwang eta/,, 1991). A functional vaccine against HIV-l infection will probably be have to rely, at least in part, on the induction of V3-specific antibodies. Thus, the V3 loop has been intensely studied (LaRosa et al., 1990; Simmonds et a/., 1990; Wolfs et a/., 1990; Epstein et al., 1991; Myers et a/., 1991; Wahlberg et al., 1991; Zwart et a/., 1991). However, most of these investigations have involved U.S./European rather than African HIV-l. It has been possible to establish a U.S./European V3 loop consensus from a compilation of sequences from 527 U.S. and European individuals (Myers eta/,, 1991). In contrast, it has been difficult to arrive at an equally unambiguous African consensus. Virus isolated from African individuals have generally appeared more polymorphic in the V3 region and have often also diverged Sequence data from this article have been deposited EMBUGenBank Data Libraries under Accession Nos. through M98978, LOO61 4 through LOO61 8. ’ To whom reprint requests should be addressed. 0042~6822/92 Copyright All rights
$5.00
0 1992 by Academic Press. Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Patients Twenty-two HIV-l -infected individuals attending the AIDS clinic at the Mulago Hospital in Kampala, Uganda, in 1990 were included in the study. The patients were grouped according to the World Health Or-
with the M98894
674
UGANDAN
HIV-1
ganization (WHO) classification system (World Health Organization, 1990). Their clinical status ranged from asymptomatic infection to AIDS; one patient was classified as WHO stage 1, seven patients as WHO stage 2, seven patients as WHO stage 3, and four patients as WHO stage 4. The status of three patients was unknown. Polymerase
chain reaction
(PCR)
Twenty milliliters of heparinized blood was collected from each patient and peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque (Pharmacia) centrifugation (Albert and Fenyb, 1990) within 48 hr after the samples had been drawn. The PBMC were lysed directly and the V3 domain amplified using the polymerase chain reaction (PCR) with nested primers (JA9, JAI 0, JAI 1, and JA12) as previously described (Albert and Fenyd, 1990). These primers hybridize to sequences flanking the V3 domain which are well conserved between available HIV-1 sequences (Myers et a/., 1991). The PCR was performed on uncultured patient PBMC to avoid selection in vitro of certain variants within the HIV-l quasi-species (Meyerhans et a/., 1989). DNA sequencing The specific PCR fragments (approximately 340 bp) were purified on 5% polyacrylamide gels. This DNA was reamplified by asymmetric PCR using JAI 0 and JAl 1 as primers in 100 ,uI reaction mixtures containing 1 ng (approximately) DNA template, 50 mM KCI, 10 mM Tris-HCI, pH 8.3, 1.5 mM MgCI,, 50 PM of each deoxynucleoside triphosphate, 10 pmol excess primer, 0.2 pmol limiting primer, and 0.5 units Amplitaq (Perkin Elmer Cetus). The temperature cycle profile (30 set at 94”, 30 set at 60”, and 45 set at 72”) was repeated 35 times with addition of another 0.5 units enzyme after 20 cycles. The amplified product was ethanol precipitated, annealed to 50 pmol of the limiting primer, and then sequenced directly using the Sequenase 2.0 Sequence Kit (U.S. Biochemical Corp.). Unambiguous sequence was obtained with direct sequencing for only three patients: 3 12 1, 3124 (both DNA strands), and 307 1 (one DNA strand). Because of this heterogeneity, the PCR products from all patients except 3121 and 3124 were cloned. Before cloning, the V3-encoding sequence was reamplified by symmetric PCR, then treated with the Klenow fragment of DNA polymerase I as well as T4 polynucleotide kinase and purified on 5% polyacrylamide gels. The purified PCR products were cloned into pUC19. Positive clones were sequenced using a T7 Sequencing Kit (Pharmacia LKB Biotechnology). A minimum of four clones were sequenced for each of these patients (85 clones
V3 LOOP
SEQUENCES
675
in total), with the exception of 3071 (as direct PCR sequence of one DNA strand was available for this patient, only three clones were sequenced). The nucleotide sequences were translated and compiled to give a consensus amino acid sequence for each patient. Sequence analysis was carried out using the Genetics Computer Group (GCG) Sequence Analysis Software Package Version 7 (Devereux et a/., 1984). Patient sequences were aligned and consensus sequences generated using the GCG Lineup program. However, the patient and group consensus sequences were modified as described in the text to Fig. 1. Phylogenetic tree analyses were performed using the GCG Pileup program, which uses the UPGMA clustering strategy (Sneath and Sokal, 1973) to make a series of progressive, pairwise alignments between the sequences, The individual sequences which most closely resembled the consensus for each of the patients were used in these analyses. Since consensus sequences were difficult to establish for Oram’s patients, the most common variant was used instead. The tree analysis on our and Oram’s sequences included 13 amino acids from the N-terminal flanking sequence, the entire V3 loop, and 8 amino acids from the C-terminal flank. A second tree analysis covering only the V3 loop included also a representative collection U.S. sequences, i.e., the sequences listed in Table II of the LaRosa et al. (1990) report. The program was run using a gap weight of 3.0 and gap length weight of 0.10. Nucleotide
sequence
accession
number
The nucleotide sequence data reported on in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the Accession Number. Peptide
serology
Peptides corresponding to the central part of the different V3 loop consensus sequences (amino acids 308 to 322 of the HIV-l,, envelope) were synthesized using Fmoc chemistry according to SXllberg et a/. (199 1). One representative autologous peptide from each patient was tested. Samples which contained mixed sequences will be analyzed in detail elsewhere. Peptide ELISA was performed with microtiter plates (Nunc, Denmark) coated with 200 ng peptide per well. Sera were diluted in twofold steps in PBS supplemented with 2% goat serum and 0.5% BSA. The plates were incubated first with serum for 60 min, second with goat anti-human IgG conjugated with alkaline phosphatase (1 :1500 dilution) for 30 min, and third with the substrate p-nitrophenyl phosphate for 30 min. All incubations were performed at 37” and the plates were exten-
676
ALBERT
V3 amino acid consensus sequences
Patient no.
GroupA
277
3071
350 vrsenltnnakiiivhlnkSVTINCTRPNNN.TRQSIHIGPGOALWT..~
3074
NVIGDiRQAHCNISRAEWNKTLC@VTg
tNNAKIIIVOLN~UVTITCTRPTTN.KRaGtHMGPGRA~~TI..i.DXtGDIRQAYCN~SekakW~~TLabVAN~
3079
enltnnaktiivqlnebvtInCtRP'(nN.tROsTI:IGpGOAiyTT..;rniIGdIraAHCN~SKA~WgKTL~VAkKLKDLFK~
3109
~~~~NIIVQLNE~VTITCMrPY~NNNkR~SVHIGPGRA~T...~NIIGDIRCIAHCNfSKVGWDKTLQhVRgklk
3110
naslaeeeiiirseNLTNNAKIiIVQLNE~VpINCTRP~NN.TR5GTHIGPGRA~t
3114
..l.NIIGDIRQAhCNJSRa?WNKTLQ+AKKLgdll..nktt QITGDIRQAHCNISKAEWNKTLQQVAKklrell..nkttiifk
~NLTNNAKIIIVqLNE$'TINCTRPYNN.TRaGIHIGPGRAYYTD...
3120
~TDNAKIIIVQLNE~VAINCTRP~NN.TRKS~h,iGPGQAL~T~..~,~IIGNIRQAhCNisr~kWNKTL~VAkKLgDL~
3121
IRSENLTNNAKTIIVQLN~~VAINCTRPNNN.TRflSTHIGPGOAtYfT....KIIGDIRRAHCEISEAGWNKTLQdVAI;KLGH
3122 Consensus
ET AL.
rsenLTNNAKTIIV$LNEjVkINCTRPANN.TR~sVHLGPGQA1YTT..~~RVIGDIRqAYCSSSEAEWNrTLQaVAVKLGD
A
~nnakiIIVqLNesVtInCtRP~,nN.tR~sthiGPGqA~~~t..,?iiGdIRqAhCn~S?a?WnkTLQ~Vak~
Group B 3072
~NITNNAKNIIVQ~T~PVKINCTRPMNN.TRZSVHIGPGQA~~~T..~DIiGDIRQA~CNVSr?ewnntlqBvvkalrtvfg
3078
~LIVQLTKPVKINCTRP~NNn.TR~SVRIGPGQT~Y~T..BDIIGDIRQAHCn~SRsEWKetlq~~vvkcllrth..wnkt.iiftnss
3082
irsenftnnakNIIVQFTkPVnItCiRPINN.T~~svrIGPGqa~Y~t..~dIIGDIRkAyCeVnkTewn?tlU~v??qlr??f.r?kt.iifans?
3084
naslaedk?mirsenitnnakniIVQFt~~VKINCtRP~NN.TRKsIgIGPGQtCyM..~QNIIGDIRQAhCN~SGTkwnktlq~vakqlrehfqrnkt.iifa
3111 3115
~NIIVQLT~ljVKINCTRPtdNN.TR~SvHIGPGQAi;Y~f..~~IIGDiRaAHCNtfSKvewnktlq.' ~pngslaegKvMIRSENITNNAKNIIVQLtePvIINCTrP~NN.TRKSIHIgPGQA~~R..~DIIGNIR(1AHC~SkkHgi
3116 3118
~RS~NITNNAKNIIVQFk~,~VkINCTRpNNN.TRK~VHIGPG~FY~T..GdIIGnIRrahCe;f~RTeWNrtlqevak
3119
~RSEN1TNNAKNIIVQLKn~VIINCTRPEiNN.TRqSVHIGPGkAiY~T..QGIVGDIRaAYCNVSRaAWNKTLQGVALQLRkhf.enkt.iiftnss
3124
Divergent 3070 3083
:
gslaeqkvmIRSENItNNAKNIlVQfnt~VqINCTRPPINN.TRkSVHIGPGaA~YAT..~eIiGDIRQAhC~SgTAWNKTLQ~ I slaeeeimirsenitnnaknILVQFnE~VQINCTRPNNN.TRKSVRIGPGQTFYAT..~DiIGNIRQAHCN~SGAKKTLqtlvakklqdll..nktti
3117
consensus
IL;
ISDNAKTIIVQFNE-SVTINCTRPNNN.TRjJSVHIGPGQAFY~T..GAIIGSIRQAHC~SRTKWREALQ~VADQLEK B
~AKniiVQft?~V?InCtRP~NN.TRksvhIGPGqaFY~t..gdIiGdIRqAhCn~s???wn?tlq?va
sequences eeeiv?rsenltnnakiiIvqLNESVIINCTRPYNNikiQrTpIG~GqaLFTTr...
IkGikgQAHCNISkaGWNKTLQQVAKklqdnn
~NaRTIIVQL~ESVKItCvRPysN.qRrrTPI~~GQALYTT~~d~~kNIk.QAYCNISvedWNkTLQQVAKkL~
FIG. 1. HIV-1 V3 amino acid consensus sequences derived from 22 Ugandan individuals. Regions where sequence information was available for fewer than all, but at least three, clones are underlined. Large letters represent 100% consensus, small letters at least 50% consensus, question mark less than 50% consensus, and two letters at one position indicate two amino acids with 50% consensus. Gaps introduced to aliqn the sequences are indicated bv a period and In frame stop codons by an asterisk. Amino acids which diverged markedly between the two groups are shaded. Positions are given relative to HIV-l,,.
sively washed between each incubation. The reaction was stopped by 1 M NaOH and the OD measured at 405 nm. Blood donor sera were used as negative controls. RESULTS The HIV-l V3 consensus amino acid sequences from 22 Ugandan patients are shown in Fig. 1. Upon analyzing the sequences, we found that the consensus sequences for 20 of these 22 patients could be divided into two almost equally large groups (A and B) on the basis of the size (34 and 35 amino acids, respectively) and sequence of the V3 loop (Fig. 1). The sequences from two patients (3070 and 3083) differed significantly from groups A and B as well as from each other, but had two characteristics in common. First, they both lacked the conserved GPG sequence at the loop apex (they had GLG and GRG instead). Second, their consensus sequences contained deletions and insertions in the V3 loop, which were not seen in any of the other patients. Although the V3 sequences of these two patients were highly divergent, both most
closely resembled the group A consensus. Furthermore, the members of group A were generally more polymorphic than those in group B. Not only did their sequences diverge more from the group consensus, but insertions, deletions and stop codons were only found among clones derived from members of group A and the divergent 3070 and 3083 (data not shown). No correlation could be found between the type of amino acid sequence and the clinical status, origin, sex, or age of the patients. Next, the V3 consensus sequences of groups A and B were compared with U.S./European and earlier (1986/l 987) Ugandan consensus sequences (Oram et al., 1991) (Figs. 2 and 3). The earlier Ugandan consensus was based on sequences from 13 patients who attended the Mulago hospital in Kampala during 1986/ 87 (Oram et al., 1991). Interestingly, the consensus V3 loop sequences of both group A and B were closer to the U.S./European consensus (77 and 86%, respectively) than to the earlier Ugandan sequences (69 and 54%, respectively). However, the 15 most central amino acids of group A showed a slightly higher homology with the earlier Ugandan consensus than with the
UGANDAN
HIV-1
N-terminal flank KSJEuropean
consensus
V3 LOOP
SEQUENCES
677
v3 loop
C-terminal flank
FTDNAKTIIVQLNESVEIN
CTRPNNNTRKSI~JIGPGRAFYTTG~IIGDIRQAHC
NISRAKWNNTLKQIV~
Group B consensus (Uganda 1990)
I-N---N----FTz$J-k--
-----------VH----Q---A-~D-----------
-“-&--k--,-e-A
Group A consensus (Uganda 1990)
L-N---I---------T--
---.Y----Q-TH----Q-L-.-,~-----------
-m-k-e--K--,-Q-A
Oram consensus (Uganda 1986/87)
L-N-------------T-.
-.--y~-QRTe--x-Q-L--.,kit-d------
s--G-e--K--+-Q-A
FIG. 2. Alignment of HIV-1 V3 amino acid consensus sequences from the U.S./Europe and Uganda. The U.S./European consensus sequence was based on the V3 loop sequences from 527 individuals (Myers et al., 1991) and the flanking sequences from 19 HIV-1 isolates in the Los Alamos database (Myers et a/., 1991). The Ugandan 1986/l 987 consensus sequence was derived from sequences of 13 Ugandan HIV-1 isolates published by Oram et a/. (1991), with minor changes (Cram, J., personal communication). Large letters represent 50-l 000/o consensus, underlined small letters indicate less than 50% consensus (most common amino acid). x indicates that no single most common amino acid could be identified. Amino acid identity between the consensus sequences is represented by a dash. Gaps introduced to align the sequences are indicated by a +.
U.S./European consensus. In contrast, the V3 loops of the group B and U.S./European consensus differed by only five amino acids. Despite this similarity the group B sequences have retained one African trait, a GPGQ sequence at the V3 loop apex instead of GPGR. In contrast, almost half of the group A sequences had a GPGFJ motif at this position, although in other respects they were closer than group B to earlier Ugandan sequences. The comparison between the different consensus sequences also revealed an interesting dissociation between the V3 loop and the flanking regions (Fig. 3). The flanking regions of group A were highly homologous with’the sequences published by Oram et a/. (1991) while the V3 loop was closer to the U.S./European consensus. Furthermore, the flanking sequences of group B were highly divergent from the U.S./European consensus sequence (and the earlier Ugandan sequences), although the V3 loop showed high homology with the U.S./European consensus. A phylogenetic tree analysis (Fig. 4) based on representative individual patient sequences from this and from Oram’s study, gave a division into three major sequence clusters. The first cluster consisted of all group B sequences. The second cluster included all group A sequences and four of Oram’s sequences. However, within the second cluster Oram’s sequences were more divergent than the group A sequences. The third cluster consisted of most of Orams’s sequences and the two divergent sequences (3070 and 3083) from this study. We performed a second tree analysis (data not shown), based only on V3 loop sequences, which also included 20 randomly selected U.S. sequences (LaRosa et a/., 1990). This tree gave a similar pattern with three major clusters. Interestingly, all U.S. sequences, except three, clustered together with the group B sequences. The second cluster now contained only group A sequences. The third cluster contained our two divergent sequences
(3070 and 3083) and all Oram’s sequences, except U455 and U653 which together with three U.S. sequences remained outside the major clusters. The sequences in the third cluster were more divergent than the sequences in the two other clusters. Sera from all patients were tested for reactivity with peptides corresponding to the V3 loop consensus sequences of the autologous virus, group A, group B, HIV-l,,, and a variant of the Oram consensus (Table 1). All sera except six reacted well (titer > 1:600) with the respective autologous peptides. However, these six exceptional sera showed a low level of reactivity to all peptides tested. Interestingly, the sera from the two patients with divergent sequences belonged to this group of weakly reactive sera. The level of cross-reactivity to the respective group consensus peptide (A or B) tended to correlate with the degree of similarity between the autologous peptide and group peptide. Generally the members of the more heterogenous group A gave a more variable response to the autologous and group A peptide, whereas the members of group B gave a stronger and more uniform response to the autologous and group B peptides. A high proportion of the sera also reacted with the HIV-I,, peptide. The reactivity with the Oram consensus sequence peptide was surprisingly infrequent and weak. Similarly, all sera showed weak or no reactivity with peptides derived from other African HIV-1 strains with divergent V3 loop sequences: JYl , 22, and ELI (data not shown).
DISCUSSION This study demonstrates the presence of two previously unrecognized HIV-1 V3 loop genotypes in Uganda. A majority of the sequences (20 of 22) could be divided into two distinct groups whose V3 loop sequences were closer to the U.S./European V3 consensus than to a consensus based on earlier Ugandan
ALBERT
678
Group A Amino acid identity (%) with other sequences
N-teiminal flank
v3 loop
C-terminal flank
Group 6 Amino acid identity (%) with other sequences
ET AL
consensus sequence B and the U.S./European consensussequence. Most previously published V3 loop sequences from African patients show less than 60% identity with the U.S./European V3 loop consensus. However, a few exceptional African HIV-1 strains from Gabon, Zaire, and Tanzania, i.e., HIV-l,,,, HIV-l,,,,) HIV-lSBL,,S (Myers et a/., 1991; Wahlberg et al., 1991) are closer to the U.S./European consensus. Myers et al. (199 1) recently presented a consensus of African V3 loop sequences based on 108 mostly unpublished sequences. Although this consensus contains many highly variable amino acids, it appears to be closer to the U.S./European V3 consensus than most earlier African sequences, including the Ugandan sequences published by Oram et al. (1991). The marked differences between the V3 loop sequences obtained in this study and those previously published by Oram et al. (1991) were unexpected, since the viruses were obtained from patients attending the same hospital although at different time points (1990 and 1986/l 987, respectively). There may be sev-
U1665 U1685 03070 u2999
i?iE
40 -
w2 u31 u5055
20
-
-
--I
N-terminal flank
v3
loop
C-terminal flank
FIG. 3. Percentage identity among the group A, group B, Oram ef a/., and U.S./European amino acid consensus sequences. The comparisons were based on the alignment presented in Fig. 2 and involved 19 amino acids from the N-terminal flank, 35 amino acids from the V3 loop, and 16 amino acids from the C-terminal flank. Identity was scored between amino acids only if both represented a 50-100% consensus. Gaps as well as amino acids representing less than a 50% consensus were scored as positions of nonidentity.
sequences published by Oram et al. (1991). The results of the phylogenetic tree analyses supported the division of our sequences into two groups, both distinct in V3 sequence from those earlier reported by Oram et al. (1990). In addition this analysis showed that the group B sequences and most of the U.S. sequences clustered together. In fact, the V3 loop consensus sequence of group B was more homologous to the U.S./European consensus sequence than the prototypic U.S. strain HIV-l,, (Myers eta/., 1991). Fut-thermore, a Dutch HIV-1 isolate, 168.1 (ZwartetaL, 1991), differed by as little as two amino acids from both the
(
u5059 D3083 83116 B3117 83072 B3111 B3118 83124 B3084 B3078 B3119 83115 B3082 A3121 A3122 A3110 A3114 A3071 A3079 A3120 A3109 41 A3074 u4133 U462 U4132 u455 U653
bRAtI/ lIVERGENT SEQUENCES
SROUPB
3ROUP A/ IRAfl
FIG. 4. Phylogenetic tree analysis with Ugandan V3 sequences from 1986187 (Oram et al., 1991) and 1990 (this study). Symbols: U designates sequences from Oram eta/., while A, B, and D designate, respectively, the group A, group B, and divergent patient sequences from this study.
UGANDAN
REACTIVITYINPEPTIDEELISAOF~~ ALJTOLOGOUS VIRUS, GROUPA,
HIV-1
V3
LOOP
TABLE
1
SEQUENCES
UGANDANSERATOPEPTIDESCORRESPONDINGTOTHEV~ LooPAPEx(HIV-lM,, ENVELOPE POSITIONS 308-322)o~ B, HIV-I,, (RKRIHIGPGRAFYTT), ANDAVARIANTOFTHEORAMCONSENSUS(RQRTPIGLGQALYTT)
GROUP
ELISA titers Autologous
Sera Group
A
679
peptide
3079 3121
RQSTHIGPGQALYTT _________-___-___--___-___--_
3071
---I--.--------
3120
Autologous
Group
A
against
different Group
+
+ ++
++
+ +
-K-I----------
+++ ++t
t++ ttt
+t t+t
--."-L-..---.----"--.--R-F----G..---.R-yW-. --GT-M---R--..--GI----.R-yy.D
++ tt t +t+ ++
t+ +t -
t +tt -
3118
+tt
3124 3116
_..___..___..__
+++ ++
3072 3111 3115 3082 3117 3078 3084
-R----.----.---T.-v---.---.-R --.I---.---...R -T--R-.----.------R-----T----R--R-----T------IG-----T.v-A
3119
-Q------K-
3122 3109 3110 3074 3114 Group 6
Divergent 3070 3083 Note.
V3 loop peptides B
HIV-l,,
+
Oram
et al.
-
tt
-
++t
t
tt tt-
tt ++t tt ++t +tt
-
+++ ttt -
t+t
+tt
+++ ++
+++ t
t -
+++ t+ +++ t +++ ++ +
+tt + ++ + -
t++ t+ +tt + + ++ -
tt +t ++t t + tt t
-
____
t+
+
++
tt
+
IQRTPIGRGQALFTT RRRTPIGLGQALYTT
-
+ +
-
t -
-
RKSVHIGPGQAFYAT
(-) Titer < 150, (+) titer
150-600,
+ (i-+)
titer 600-2400,
(++t)
era1 reasons for these differences. One possibility may be that our patients and Oram’s were unintentionally selected from different subpopulations in Kampala. However, no such selection bias could be identified, except that all of the patients in the previous study had developed AIDS, whereas we also studied individuals with milder disease. A second possibility is that our primers selected for sequences of U.S./European character. We believe this to be unlikely for the following reasons. The primers we used represent sequences flanking the V3 domain which are well conserved between available HIV-l sequences (Myers et al., 1991). Also, the flanking sequences of many patients, especially in group A, were highly homologous with those published by Oram eta/. (199 1). In a larger, partly overlapping, set of Ugandan samples we found that 27 of 31 samples could be amplified with our V3 primers (data not shown). In addition, the strong reactivity of our patients’ sera with the autologous peptides indicates that the amplified sequences were representative of the provirus population harbored by these patients.
+
+
+ -
t
titer > 2400.
Third, the results could indicate that new V3 genotypes were introduced in Uganda between the time Oram’s patients and our patients were infected. It is possible that this time difference in some cases is longer than 3-4 years, since Oram et al. only studied patients with AIDS. However, it is unlikely that U.S./European sequences have been introduced in Uganda, since most patients display a GPGQ V3 loop apex sequence. Fourth, the differences could indicate that Ugandan and U.S./European V3 loop sequences may tend to converge. If so, this may be due to an adaptation of HIV-l to replication in the human host. Subtle changes in the V3 loop have been shown to alter the tropism of HIV-1 for different types of human cells (Takeuchi eta/., 1991; Hwang et a/., 1991). It has been proposed that cleavage of the V3 loop by cellular proteases may play an essential role in the infection process (Hattori et a/., 1989; Clements et al., 1991). Differences in cellular proteases of different species may therefore impose specific selection pressure on HIV-l. Similarly, many earlier African sequences may be biased since they
ALBERT
680
were obtained from virus adapted to growth on cell lines or PBMC (Meyerhans et a/., 1989). In contrast, our sequences were obtained from fresh, uncultured patient PBMC. A high proportion (>90%) of HIV-l strains in the United States and Europe have been shown to belong to the same V3 serotype, i.e., the HIV-l,, and HIV1 ,68., serotype (Devash eta/., 1990; Zwart eta/., 1991). This serological reactivity to the V3 loop is primarily dependent on amino acids at the apex of the loop (Javaherian et al., 1989; Meloen et a/., 1989; Akerblom et al., 1990). Thus, the Ugandan HIV-1 strains presented in this study and U.S./European strains may belong to different V3 serotypes despite sequence similarities, since their loop apex sequences are usually different. However, most of our patients’sera recognized autologous, group and HIV-l,, V3 peptides, but not “typical” African peptides (Oram, JYl, 22, and ELI). This confirms that these viruses are structurally and antigenitally closer to U.S./European HIV-1 strains than to earlier Ugandan and African strains. Interestingly, Carrow et al. (1991) recently reported that Zairian sera also showed stronger reactivity with a V3 loop peptide derived from HIV-l MNthan one from the African HIV-l z2 isolate. The close relationships between V3 loop sequences present in Uganda in 1990, the U.S. and Europe have important implications for understanding the evolution of HIV-l and for attempts to develop a vaccine against HIV-l infection in the African population. ACKNOWLEDGMENTS This work was supported by grants from the World Health Organization (Human Reproductive Programme and Global Programme on AIDS), the Swedish Medical Research Council (Grants 692.16H09935 and B90-16H-07738.06B), the Labour Market Insurance Company (AFA), the Pediatric AIDS Foundation, and lstituto Superiore di Sanitg IV Progetto AIDS. We thank Dr. D. Larhammar. Dr. U. Gyllensten, Professor E. Norrby, Dr. E. M. Fenytl, Dr. G. Myers, and Dr. B. Korber for helpful discussions.
REFERENCES AKERBLOM, L., HINKULA, J., BROLIDEN, P.-A., MAKITALO, B., FRIDBERGER, T., ROSEN, J., VILLACRES-ERIKSSON, M., MOREIN, B., and WAHREN, B. (1990). Neutralizing cross-reactive and non-neutralizing monoclonal antibodies to HIV-1 gpl20. AlDS 4, 953-960. ALBERT, J., and FENYB, E. M. (1990). Simple, sensitive and specific detection of HIV-1 in clinical specimens by polymerase chain reaction with nested primers. J. C/in. Microbial. 28, 1560-l 564. CARROW, E. W.. VUJCIC, L. K., GLASS, W. L., SEAMON, K. B., RASTOGI, S. C.. HENDRY, R. M.. BOULOS, R., NZILA, N., and QUINNAN, G. V., JR. (1991). High prevalence of antibodies to the gpl20 V3 region principal neutralizing determinant of HIV-1 MN in sera from Africa and the Americas. A//X Res. Hum. Retroviruses 7, 831-838. CLEMENTS. G. I., PRICE-JONES, M. J., STEPHENS, P. E., SUTTON, C., SCHULTZ, T. F., CLAPHAM, P. R., MCKEATING, I. A., MCCLURE, M. O.,
ET AL. THOMPSON, S., MARSH, M., WEISS, R. A., and MOORE, J. P. (1991). The V3 loops of the HIV-1 and HIV-2 surface glycoproteins contain proteolytic cleavage sites: A possible function in viral fusion?A/DS Res. Hum. Retroviruses 7, 3-l 6. DEVASH, Y., MATTHEWS, T. J., DRUMMOND, J. E., JAVAHERIAN, K., WATERS, D. J., ARTHUR, L. O., BLA~NER, W. A., and RUSCHE, J. R. (1990). C-terminal fragment of gpl20 and synthetic peptides from five HTLV-III strains: Prevalence of antibodies to the HTLV-III-MN isolate in infected individuals. AIDS Res. Hum. Retroviruses 6, 307-316. DEVEREUX, J., HAEBERLI, P., and SMITHIES, 0. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395. EPSTEIN, L. G., KUIKEN, C., BLUMBERG, B. M., HARTMAN, S., SHARER, L. R., CLEMENT, M., and GOUDSMIT, J. (1991). HIV-l V3 domain variation in brain and spleen of children with AIDS: Tissue-specific evolution within host-determined quasispecies. virology 180, 583-590. HATTORI, T., KOITO, A., TAKASUKI, K., KIDO, H., and KATUNUMA, N. (1989). Involvement of ttyptase-related cellular protease in human immunodeficiency virus type 1 infection. FEBS Letts. 248, 48-52. HWANG, S. S., BOYLE, T. J., LYERLY, K., and CULLEN, B. R. (1991). Identification of the envelope V3 loop as the primary determinant for cell tropism of HIV-l. Science 253, 71-74. JAVAHERIAN, K., LANGLOIS, A. J., MCDANAL, C., Ross, K. L., ECKLER, L. I., JELLIS, C. L., PROFY, A. T., RUSCHE, J. R., BOLOGNESI, D. P., PUTNEY, S. D., et al. (1989). Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein. Proc. Nat/. Acad. Sci. USA 86, 6768-6772. LL\RosA, G. J., DAVIDE. J. P., WEINHOLD, K., WATERBURY. J. A., PROFY, A. T., LEWIS, J. A., LANGLOIS, A. J., DREESMAN, G. R. B. R. N., SHADDUCK, P., HOLLEY, L. H., KARPLUS, M., BOLOGNESI. D. P.. MATTHEWS, T. J., EMINI, E. A., and PUTNEY, S. D. (1990). Conserved sequence and structural elements in the HIV-1 principal neutralizing domain. Science 249, 932-935. MELOEN, R. H., LISKAMP, R. M., and GOUDSMIT, J. (1989). Specificity and function of the individual amino acids of an important determinant of human immunodeficiency virus type 1 that induces neutralizing activity. 1. Gen. Viral. 70, 1505-l 512. MEYERHANS, A., CHEYNIER, R., ALBERT, J., SETH, M., KWOK, S., SNINSKI, . J., MORFELDT-MANSSON, L., ASJO, B., and WAIN-HOBSON, S. (1989). Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations. Cell 58, 901-910. MYERS, G., KORBER, B., BERZOFSKY, J. A., SMITH, R. F., and PAVLAKIS, G. N. (1991). “Human Retroviruses and AIDS, 1991: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences.” Los Alamos National Laboratory. Los Alamos, NM. ORAM, J. D., DOWNING, R. G., ROFF, M.. SERWANKAMBO, N., CLEGG, J. C., FEATHERSTONE, A. S., and BOOTH, J. C. (1991). Sequence analysis of the V3 loop regions of the env gene of human immunodeficiency proviruses. AIDS Res. Hum. Retroviruses 7, 605-614. PALKER, T. J., CLARK, M. E., LANGLOIS, A., MATTHEWS, T. J., WEINHOLD, K. J., RANDALL, R. R., BOLOGNESI, D. P., and HAYNES, B. F. (1988). Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides. Proc. Nat/. Acad. Sci. USA 85, 1932-l 936. RUSCHE, J. R., JAVAHERIAN, K., MCDANAL, C., PETRO, J., LYNN, D. L., GRIMAILA, R., LANGLOIS, A., GALLO, R. C., ARTHUR, L. O., FISCHINGER, P. J., BOLOGNESI, D. P., PUTNEY, S. D., and MATHEWS, T. J. (1988). Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24.amino acid sequence of the viral envelope, gpl20. Proc. Alatl. Acad. Sci. USA 85, 3 198-3202. S&LLBERG, M.. RUD~N. U., MAGNIUS, L. O., NORRBY, E., and WAHREN,
UGANDAN
HIV-1
B. (1991). Rapid “tea-bag” peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids applied for antigenie mapping of viral proteins. Immunol. Left. 30, 59-68. SIMMONDS, P., BALFE, P., LUDLAM, C. A., BISHOP, J. O., and LEIGH BROWN, A. J. (1990). Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1. /. Viral. 64, 5840-5850. SNEATH, P. H. A., and SOKAL, R. R. (1973). In “Numerical Taxonomy,” pp. 230-234. Freeman, San Francisco, CA. TAKEUCHI, Y., AKUTSU. M., MURAYAMA, K., SHIMIZU, N., and HOSHINO, H. (1991). Host range of human immunodeficiency virus type 1: Modification of cell tropism by a single point mutation at the neutralization epitope in the env gene. /. VYrol. 65, 171 O-l 718. WAHLBERG, J., ALBERT, J., LUNDEBERG, J., VON GEGERFELT, A., BROLIDEN, K., UTTER, G., FENYB, E. M.. and UH~EN, M. (1991). Analysis of
V3 LOOP
SEQUENCES
681
the V3 loop in neutralization-resistant human immunodeficiency virus type 1 variants by direct solid phase DNA sequencing. AIDS Res. Hum. Retroviruses 7, 983-989. WOLFS, T. F., DE JONG, J. J., VAN DER BERG, H.. TIJNAGEL, J. M., KRONE, W. J., and GOUDSMIT, J. (1990). Evolution of sequences encoding the principal neutralization epitope of human immunodeficiency virus type 1 is host dependent, rapid, and continuous. Proc. Nat/. Acad. Sci. USA 87, 9938-9942. World Health Organization. (1990). Acquired immunodeficiency syndrome (AIDS): Interim proposal for a WHO staging system for HIV infection and disease. Wkly. fpidem. Rec. 65, 221-224. ZWART, G., LANGEDIJK, H., VAN DER HOEK, L., DE JONG, J. J., WOLFS, T. F., RAMAUTARSING, C., BAKKER, M.. DE RONDE, A., and GOUDSMIT, J. (1991). lmmunodominance and antigenic variation of the principal neutralization domain of HIV-l. \/iro/ogy 181, 481-489.
190,
674-681
Ugandan
(1992)
HIV-1 V3 Loop Sequences
Closely Related to the U.S./European
JAN ALBERT,**’ LENA FRANZiN,++ MARIANNE JANSSON,*+ GABRIELLA SCARLAlTI,§=” E. KATABIRA,lI F. MUBIRO, MARIA RYDAKER,1- PAOLO ROSSI,+# ULF PEmERSSON,t
Consensus
PETER K. KATAAHA,1 AND HANS WIGZELL*+
*Department of Virology, Alational Bacteriological Laboratory, Stockholm, Sweden; tDepartment of Medical Genetics, University of Uppsala, Uppsala, Sweden; +Departments of immunology and §Virology, Karolinska Institute, Stockholm, Sweden; ‘IDepartment of Pediatrics I, University of Milan, Milan, Italy; Olakasero Blood Bank, Kampala, Uganda; 7Department of Medicine, Medical School, Makerere University, Kampala, Uganda; and #Department of Pediatrics, University of Rome “Tor Vergata, ” Rome, Italy Received
March
9, 1992;
accepted
June 29, 1992
The third variable (V3) loop of the human immunodeficiency virus type 1 (HIV-l) envelope protein is an important determinant for virus neutralization and cell tropism. V3 loop sequences from uncultured lymphocytes obtained in 1990 from 22 Ugandan HIV-l-infected patients could, with the exception of two patients’ sequences, be divided into two groups (A and B) on the basis the V3 loop size and sequence. The V3 loop consensus sequences from both groups showed a high degree of homology to a U.S./European consensus, a characteristic also reflected by the results of peptide serology. In the case of group B the difference in sequence was only five amino acids. In contrast, the V3-flanking regions for both groups showed greater homology to an earlier (1986/l 987) Ugandan consensus. The discovery of these two new Ugandan V3 loop genotypes, which are closely related to the U.S./European consensus, has implications for the understanding of the evolution of HIV-l and for the future design of a vaccine for use in Africa. o 1992 Academic
Press,
Inc.
sharply in sequence from the U.S./European consensus (Myers et al., 1991; Oram et al., 1991). Most of these isolates were obtained before or during the mid1980’s (Myers eta/., 1991; Oram eta/., 1991) and studies on current African strains are scarce. However, Myers et a/. (199 1) recently presented a consensus of mostly unpublished African V3 loop sequences, which again indicate that African HIV-1 strains are distinct from and more polymorphic than U.S./European strains. As a vaccine against HIV-l is urgently needed in Africa, it is important to determine the V3 loop sequences of additional African HIV-l isolates, especially from more recently infected individuals. The Ugandan HIV-1 sequences reported in this study appear remarkable in two aspects: they have unusually homologous V3 loop sequences compared to most earlier published African sequences and their V3 loop sequences show greater similarity to the U.S./European consensus than to an earlier (1986/1987) Ugandan consensus.
INTRODUCTION A major target for neutralizing antibodies to the human immunodeficiency virus type 1 (HIV-l) is located in a disulphide-bonded loop in the third variable (V3) region of the external envelope glycoprotein gp120 (Rusche et al,, 1988; Palker et a/., 1988). The V3 loop has recently been shown to be also an important determinant of HIV-1 cell tropism (Takeuchi et a/., 1991; Hwang eta/,, 1991). A functional vaccine against HIV-l infection will probably be have to rely, at least in part, on the induction of V3-specific antibodies. Thus, the V3 loop has been intensely studied (LaRosa et al., 1990; Simmonds et a/., 1990; Wolfs et a/., 1990; Epstein et al., 1991; Myers et a/., 1991; Wahlberg et al., 1991; Zwart et a/., 1991). However, most of these investigations have involved U.S./European rather than African HIV-l. It has been possible to establish a U.S./European V3 loop consensus from a compilation of sequences from 527 U.S. and European individuals (Myers eta/,, 1991). In contrast, it has been difficult to arrive at an equally unambiguous African consensus. Virus isolated from African individuals have generally appeared more polymorphic in the V3 region and have often also diverged Sequence data from this article have been deposited EMBUGenBank Data Libraries under Accession Nos. through M98978, LOO61 4 through LOO61 8. ’ To whom reprint requests should be addressed. 0042~6822/92 Copyright All rights
$5.00
0 1992 by Academic Press. Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Patients Twenty-two HIV-l -infected individuals attending the AIDS clinic at the Mulago Hospital in Kampala, Uganda, in 1990 were included in the study. The patients were grouped according to the World Health Or-
with the M98894
674
UGANDAN
HIV-1
ganization (WHO) classification system (World Health Organization, 1990). Their clinical status ranged from asymptomatic infection to AIDS; one patient was classified as WHO stage 1, seven patients as WHO stage 2, seven patients as WHO stage 3, and four patients as WHO stage 4. The status of three patients was unknown. Polymerase
chain reaction
(PCR)
Twenty milliliters of heparinized blood was collected from each patient and peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque (Pharmacia) centrifugation (Albert and Fenyb, 1990) within 48 hr after the samples had been drawn. The PBMC were lysed directly and the V3 domain amplified using the polymerase chain reaction (PCR) with nested primers (JA9, JAI 0, JAI 1, and JA12) as previously described (Albert and Fenyd, 1990). These primers hybridize to sequences flanking the V3 domain which are well conserved between available HIV-1 sequences (Myers et a/., 1991). The PCR was performed on uncultured patient PBMC to avoid selection in vitro of certain variants within the HIV-l quasi-species (Meyerhans et a/., 1989). DNA sequencing The specific PCR fragments (approximately 340 bp) were purified on 5% polyacrylamide gels. This DNA was reamplified by asymmetric PCR using JAI 0 and JAl 1 as primers in 100 ,uI reaction mixtures containing 1 ng (approximately) DNA template, 50 mM KCI, 10 mM Tris-HCI, pH 8.3, 1.5 mM MgCI,, 50 PM of each deoxynucleoside triphosphate, 10 pmol excess primer, 0.2 pmol limiting primer, and 0.5 units Amplitaq (Perkin Elmer Cetus). The temperature cycle profile (30 set at 94”, 30 set at 60”, and 45 set at 72”) was repeated 35 times with addition of another 0.5 units enzyme after 20 cycles. The amplified product was ethanol precipitated, annealed to 50 pmol of the limiting primer, and then sequenced directly using the Sequenase 2.0 Sequence Kit (U.S. Biochemical Corp.). Unambiguous sequence was obtained with direct sequencing for only three patients: 3 12 1, 3124 (both DNA strands), and 307 1 (one DNA strand). Because of this heterogeneity, the PCR products from all patients except 3121 and 3124 were cloned. Before cloning, the V3-encoding sequence was reamplified by symmetric PCR, then treated with the Klenow fragment of DNA polymerase I as well as T4 polynucleotide kinase and purified on 5% polyacrylamide gels. The purified PCR products were cloned into pUC19. Positive clones were sequenced using a T7 Sequencing Kit (Pharmacia LKB Biotechnology). A minimum of four clones were sequenced for each of these patients (85 clones
V3 LOOP
SEQUENCES
675
in total), with the exception of 3071 (as direct PCR sequence of one DNA strand was available for this patient, only three clones were sequenced). The nucleotide sequences were translated and compiled to give a consensus amino acid sequence for each patient. Sequence analysis was carried out using the Genetics Computer Group (GCG) Sequence Analysis Software Package Version 7 (Devereux et a/., 1984). Patient sequences were aligned and consensus sequences generated using the GCG Lineup program. However, the patient and group consensus sequences were modified as described in the text to Fig. 1. Phylogenetic tree analyses were performed using the GCG Pileup program, which uses the UPGMA clustering strategy (Sneath and Sokal, 1973) to make a series of progressive, pairwise alignments between the sequences, The individual sequences which most closely resembled the consensus for each of the patients were used in these analyses. Since consensus sequences were difficult to establish for Oram’s patients, the most common variant was used instead. The tree analysis on our and Oram’s sequences included 13 amino acids from the N-terminal flanking sequence, the entire V3 loop, and 8 amino acids from the C-terminal flank. A second tree analysis covering only the V3 loop included also a representative collection U.S. sequences, i.e., the sequences listed in Table II of the LaRosa et al. (1990) report. The program was run using a gap weight of 3.0 and gap length weight of 0.10. Nucleotide
sequence
accession
number
The nucleotide sequence data reported on in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the Accession Number. Peptide
serology
Peptides corresponding to the central part of the different V3 loop consensus sequences (amino acids 308 to 322 of the HIV-l,, envelope) were synthesized using Fmoc chemistry according to SXllberg et a/. (199 1). One representative autologous peptide from each patient was tested. Samples which contained mixed sequences will be analyzed in detail elsewhere. Peptide ELISA was performed with microtiter plates (Nunc, Denmark) coated with 200 ng peptide per well. Sera were diluted in twofold steps in PBS supplemented with 2% goat serum and 0.5% BSA. The plates were incubated first with serum for 60 min, second with goat anti-human IgG conjugated with alkaline phosphatase (1 :1500 dilution) for 30 min, and third with the substrate p-nitrophenyl phosphate for 30 min. All incubations were performed at 37” and the plates were exten-
676
ALBERT
V3 amino acid consensus sequences
Patient no.
GroupA
277
3071
350 vrsenltnnakiiivhlnkSVTINCTRPNNN.TRQSIHIGPGOALWT..~
3074
NVIGDiRQAHCNISRAEWNKTLC@VTg
tNNAKIIIVOLN~UVTITCTRPTTN.KRaGtHMGPGRA~~TI..i.DXtGDIRQAYCN~SekakW~~TLabVAN~
3079
enltnnaktiivqlnebvtInCtRP'(nN.tROsTI:IGpGOAiyTT..;rniIGdIraAHCN~SKA~WgKTL~VAkKLKDLFK~
3109
~~~~NIIVQLNE~VTITCMrPY~NNNkR~SVHIGPGRA~T...~NIIGDIRCIAHCNfSKVGWDKTLQhVRgklk
3110
naslaeeeiiirseNLTNNAKIiIVQLNE~VpINCTRP~NN.TR5GTHIGPGRA~t
3114
..l.NIIGDIRQAhCNJSRa?WNKTLQ+AKKLgdll..nktt QITGDIRQAHCNISKAEWNKTLQQVAKklrell..nkttiifk
~NLTNNAKIIIVqLNE$'TINCTRPYNN.TRaGIHIGPGRAYYTD...
3120
~TDNAKIIIVQLNE~VAINCTRP~NN.TRKS~h,iGPGQAL~T~..~,~IIGNIRQAhCNisr~kWNKTL~VAkKLgDL~
3121
IRSENLTNNAKTIIVQLN~~VAINCTRPNNN.TRflSTHIGPGOAtYfT....KIIGDIRRAHCEISEAGWNKTLQdVAI;KLGH
3122 Consensus
ET AL.
rsenLTNNAKTIIV$LNEjVkINCTRPANN.TR~sVHLGPGQA1YTT..~~RVIGDIRqAYCSSSEAEWNrTLQaVAVKLGD
A
~nnakiIIVqLNesVtInCtRP~,nN.tR~sthiGPGqA~~~t..,?iiGdIRqAhCn~S?a?WnkTLQ~Vak~
Group B 3072
~NITNNAKNIIVQ~T~PVKINCTRPMNN.TRZSVHIGPGQA~~~T..~DIiGDIRQA~CNVSr?ewnntlqBvvkalrtvfg
3078
~LIVQLTKPVKINCTRP~NNn.TR~SVRIGPGQT~Y~T..BDIIGDIRQAHCn~SRsEWKetlq~~vvkcllrth..wnkt.iiftnss
3082
irsenftnnakNIIVQFTkPVnItCiRPINN.T~~svrIGPGqa~Y~t..~dIIGDIRkAyCeVnkTewn?tlU~v??qlr??f.r?kt.iifans?
3084
naslaedk?mirsenitnnakniIVQFt~~VKINCtRP~NN.TRKsIgIGPGQtCyM..~QNIIGDIRQAhCN~SGTkwnktlq~vakqlrehfqrnkt.iifa
3111 3115
~NIIVQLT~ljVKINCTRPtdNN.TR~SvHIGPGQAi;Y~f..~~IIGDiRaAHCNtfSKvewnktlq.' ~pngslaegKvMIRSENITNNAKNIIVQLtePvIINCTrP~NN.TRKSIHIgPGQA~~R..~DIIGNIR(1AHC~SkkHgi
3116 3118
~RS~NITNNAKNIIVQFk~,~VkINCTRpNNN.TRK~VHIGPG~FY~T..GdIIGnIRrahCe;f~RTeWNrtlqevak
3119
~RSEN1TNNAKNIIVQLKn~VIINCTRPEiNN.TRqSVHIGPGkAiY~T..QGIVGDIRaAYCNVSRaAWNKTLQGVALQLRkhf.enkt.iiftnss
3124
Divergent 3070 3083
:
gslaeqkvmIRSENItNNAKNIlVQfnt~VqINCTRPPINN.TRkSVHIGPGaA~YAT..~eIiGDIRQAhC~SgTAWNKTLQ~ I slaeeeimirsenitnnaknILVQFnE~VQINCTRPNNN.TRKSVRIGPGQTFYAT..~DiIGNIRQAHCN~SGAKKTLqtlvakklqdll..nktti
3117
consensus
IL;
ISDNAKTIIVQFNE-SVTINCTRPNNN.TRjJSVHIGPGQAFY~T..GAIIGSIRQAHC~SRTKWREALQ~VADQLEK B
~AKniiVQft?~V?InCtRP~NN.TRksvhIGPGqaFY~t..gdIiGdIRqAhCn~s???wn?tlq?va
sequences eeeiv?rsenltnnakiiIvqLNESVIINCTRPYNNikiQrTpIG~GqaLFTTr...
IkGikgQAHCNISkaGWNKTLQQVAKklqdnn
~NaRTIIVQL~ESVKItCvRPysN.qRrrTPI~~GQALYTT~~d~~kNIk.QAYCNISvedWNkTLQQVAKkL~
FIG. 1. HIV-1 V3 amino acid consensus sequences derived from 22 Ugandan individuals. Regions where sequence information was available for fewer than all, but at least three, clones are underlined. Large letters represent 100% consensus, small letters at least 50% consensus, question mark less than 50% consensus, and two letters at one position indicate two amino acids with 50% consensus. Gaps introduced to aliqn the sequences are indicated bv a period and In frame stop codons by an asterisk. Amino acids which diverged markedly between the two groups are shaded. Positions are given relative to HIV-l,,.
sively washed between each incubation. The reaction was stopped by 1 M NaOH and the OD measured at 405 nm. Blood donor sera were used as negative controls. RESULTS The HIV-l V3 consensus amino acid sequences from 22 Ugandan patients are shown in Fig. 1. Upon analyzing the sequences, we found that the consensus sequences for 20 of these 22 patients could be divided into two almost equally large groups (A and B) on the basis of the size (34 and 35 amino acids, respectively) and sequence of the V3 loop (Fig. 1). The sequences from two patients (3070 and 3083) differed significantly from groups A and B as well as from each other, but had two characteristics in common. First, they both lacked the conserved GPG sequence at the loop apex (they had GLG and GRG instead). Second, their consensus sequences contained deletions and insertions in the V3 loop, which were not seen in any of the other patients. Although the V3 sequences of these two patients were highly divergent, both most
closely resembled the group A consensus. Furthermore, the members of group A were generally more polymorphic than those in group B. Not only did their sequences diverge more from the group consensus, but insertions, deletions and stop codons were only found among clones derived from members of group A and the divergent 3070 and 3083 (data not shown). No correlation could be found between the type of amino acid sequence and the clinical status, origin, sex, or age of the patients. Next, the V3 consensus sequences of groups A and B were compared with U.S./European and earlier (1986/l 987) Ugandan consensus sequences (Oram et al., 1991) (Figs. 2 and 3). The earlier Ugandan consensus was based on sequences from 13 patients who attended the Mulago hospital in Kampala during 1986/ 87 (Oram et al., 1991). Interestingly, the consensus V3 loop sequences of both group A and B were closer to the U.S./European consensus (77 and 86%, respectively) than to the earlier Ugandan sequences (69 and 54%, respectively). However, the 15 most central amino acids of group A showed a slightly higher homology with the earlier Ugandan consensus than with the
UGANDAN
HIV-1
N-terminal flank KSJEuropean
consensus
V3 LOOP
SEQUENCES
677
v3 loop
C-terminal flank
FTDNAKTIIVQLNESVEIN
CTRPNNNTRKSI~JIGPGRAFYTTG~IIGDIRQAHC
NISRAKWNNTLKQIV~
Group B consensus (Uganda 1990)
I-N---N----FTz$J-k--
-----------VH----Q---A-~D-----------
-“-&--k--,-e-A
Group A consensus (Uganda 1990)
L-N---I---------T--
---.Y----Q-TH----Q-L-.-,~-----------
-m-k-e--K--,-Q-A
Oram consensus (Uganda 1986/87)
L-N-------------T-.
-.--y~-QRTe--x-Q-L--.,kit-d------
s--G-e--K--+-Q-A
FIG. 2. Alignment of HIV-1 V3 amino acid consensus sequences from the U.S./Europe and Uganda. The U.S./European consensus sequence was based on the V3 loop sequences from 527 individuals (Myers et al., 1991) and the flanking sequences from 19 HIV-1 isolates in the Los Alamos database (Myers et a/., 1991). The Ugandan 1986/l 987 consensus sequence was derived from sequences of 13 Ugandan HIV-1 isolates published by Oram et a/. (1991), with minor changes (Cram, J., personal communication). Large letters represent 50-l 000/o consensus, underlined small letters indicate less than 50% consensus (most common amino acid). x indicates that no single most common amino acid could be identified. Amino acid identity between the consensus sequences is represented by a dash. Gaps introduced to align the sequences are indicated by a +.
U.S./European consensus. In contrast, the V3 loops of the group B and U.S./European consensus differed by only five amino acids. Despite this similarity the group B sequences have retained one African trait, a GPGQ sequence at the V3 loop apex instead of GPGR. In contrast, almost half of the group A sequences had a GPGFJ motif at this position, although in other respects they were closer than group B to earlier Ugandan sequences. The comparison between the different consensus sequences also revealed an interesting dissociation between the V3 loop and the flanking regions (Fig. 3). The flanking regions of group A were highly homologous with’the sequences published by Oram et a/. (1991) while the V3 loop was closer to the U.S./European consensus. Furthermore, the flanking sequences of group B were highly divergent from the U.S./European consensus sequence (and the earlier Ugandan sequences), although the V3 loop showed high homology with the U.S./European consensus. A phylogenetic tree analysis (Fig. 4) based on representative individual patient sequences from this and from Oram’s study, gave a division into three major sequence clusters. The first cluster consisted of all group B sequences. The second cluster included all group A sequences and four of Oram’s sequences. However, within the second cluster Oram’s sequences were more divergent than the group A sequences. The third cluster consisted of most of Orams’s sequences and the two divergent sequences (3070 and 3083) from this study. We performed a second tree analysis (data not shown), based only on V3 loop sequences, which also included 20 randomly selected U.S. sequences (LaRosa et a/., 1990). This tree gave a similar pattern with three major clusters. Interestingly, all U.S. sequences, except three, clustered together with the group B sequences. The second cluster now contained only group A sequences. The third cluster contained our two divergent sequences
(3070 and 3083) and all Oram’s sequences, except U455 and U653 which together with three U.S. sequences remained outside the major clusters. The sequences in the third cluster were more divergent than the sequences in the two other clusters. Sera from all patients were tested for reactivity with peptides corresponding to the V3 loop consensus sequences of the autologous virus, group A, group B, HIV-l,,, and a variant of the Oram consensus (Table 1). All sera except six reacted well (titer > 1:600) with the respective autologous peptides. However, these six exceptional sera showed a low level of reactivity to all peptides tested. Interestingly, the sera from the two patients with divergent sequences belonged to this group of weakly reactive sera. The level of cross-reactivity to the respective group consensus peptide (A or B) tended to correlate with the degree of similarity between the autologous peptide and group peptide. Generally the members of the more heterogenous group A gave a more variable response to the autologous and group A peptide, whereas the members of group B gave a stronger and more uniform response to the autologous and group B peptides. A high proportion of the sera also reacted with the HIV-I,, peptide. The reactivity with the Oram consensus sequence peptide was surprisingly infrequent and weak. Similarly, all sera showed weak or no reactivity with peptides derived from other African HIV-1 strains with divergent V3 loop sequences: JYl , 22, and ELI (data not shown).
DISCUSSION This study demonstrates the presence of two previously unrecognized HIV-1 V3 loop genotypes in Uganda. A majority of the sequences (20 of 22) could be divided into two distinct groups whose V3 loop sequences were closer to the U.S./European V3 consensus than to a consensus based on earlier Ugandan
ALBERT
678
Group A Amino acid identity (%) with other sequences
N-teiminal flank
v3 loop
C-terminal flank
Group 6 Amino acid identity (%) with other sequences
ET AL
consensus sequence B and the U.S./European consensussequence. Most previously published V3 loop sequences from African patients show less than 60% identity with the U.S./European V3 loop consensus. However, a few exceptional African HIV-1 strains from Gabon, Zaire, and Tanzania, i.e., HIV-l,,,, HIV-l,,,,) HIV-lSBL,,S (Myers et a/., 1991; Wahlberg et al., 1991) are closer to the U.S./European consensus. Myers et al. (199 1) recently presented a consensus of African V3 loop sequences based on 108 mostly unpublished sequences. Although this consensus contains many highly variable amino acids, it appears to be closer to the U.S./European V3 consensus than most earlier African sequences, including the Ugandan sequences published by Oram et al. (1991). The marked differences between the V3 loop sequences obtained in this study and those previously published by Oram et al. (1991) were unexpected, since the viruses were obtained from patients attending the same hospital although at different time points (1990 and 1986/l 987, respectively). There may be sev-
U1665 U1685 03070 u2999
i?iE
40 -
w2 u31 u5055
20
-
-
--I
N-terminal flank
v3
loop
C-terminal flank
FIG. 3. Percentage identity among the group A, group B, Oram ef a/., and U.S./European amino acid consensus sequences. The comparisons were based on the alignment presented in Fig. 2 and involved 19 amino acids from the N-terminal flank, 35 amino acids from the V3 loop, and 16 amino acids from the C-terminal flank. Identity was scored between amino acids only if both represented a 50-100% consensus. Gaps as well as amino acids representing less than a 50% consensus were scored as positions of nonidentity.
sequences published by Oram et al. (1991). The results of the phylogenetic tree analyses supported the division of our sequences into two groups, both distinct in V3 sequence from those earlier reported by Oram et al. (1990). In addition this analysis showed that the group B sequences and most of the U.S. sequences clustered together. In fact, the V3 loop consensus sequence of group B was more homologous to the U.S./European consensus sequence than the prototypic U.S. strain HIV-l,, (Myers eta/., 1991). Fut-thermore, a Dutch HIV-1 isolate, 168.1 (ZwartetaL, 1991), differed by as little as two amino acids from both the
(
u5059 D3083 83116 B3117 83072 B3111 B3118 83124 B3084 B3078 B3119 83115 B3082 A3121 A3122 A3110 A3114 A3071 A3079 A3120 A3109 41 A3074 u4133 U462 U4132 u455 U653
bRAtI/ lIVERGENT SEQUENCES
SROUPB
3ROUP A/ IRAfl
FIG. 4. Phylogenetic tree analysis with Ugandan V3 sequences from 1986187 (Oram et al., 1991) and 1990 (this study). Symbols: U designates sequences from Oram eta/., while A, B, and D designate, respectively, the group A, group B, and divergent patient sequences from this study.
UGANDAN
REACTIVITYINPEPTIDEELISAOF~~ ALJTOLOGOUS VIRUS, GROUPA,
HIV-1
V3
LOOP
TABLE
1
SEQUENCES
UGANDANSERATOPEPTIDESCORRESPONDINGTOTHEV~ LooPAPEx(HIV-lM,, ENVELOPE POSITIONS 308-322)o~ B, HIV-I,, (RKRIHIGPGRAFYTT), ANDAVARIANTOFTHEORAMCONSENSUS(RQRTPIGLGQALYTT)
GROUP
ELISA titers Autologous
Sera Group
A
679
peptide
3079 3121
RQSTHIGPGQALYTT _________-___-___--___-___--_
3071
---I--.--------
3120
Autologous
Group
A
against
different Group
+
+ ++
++
+ +
-K-I----------
+++ ++t
t++ ttt
+t t+t
--."-L-..---.----"--.--R-F----G..---.R-yW-. --GT-M---R--..--GI----.R-yy.D
++ tt t +t+ ++
t+ +t -
t +tt -
3118
+tt
3124 3116
_..___..___..__
+++ ++
3072 3111 3115 3082 3117 3078 3084
-R----.----.---T.-v---.---.-R --.I---.---...R -T--R-.----.------R-----T----R--R-----T------IG-----T.v-A
3119
-Q------K-
3122 3109 3110 3074 3114 Group 6
Divergent 3070 3083 Note.
V3 loop peptides B
HIV-l,,
+
Oram
et al.
-
tt
-
++t
t
tt tt-
tt ++t tt ++t +tt
-
+++ ttt -
t+t
+tt
+++ ++
+++ t
t -
+++ t+ +++ t +++ ++ +
+tt + ++ + -
t++ t+ +tt + + ++ -
tt +t ++t t + tt t
-
____
t+
+
++
tt
+
IQRTPIGRGQALFTT RRRTPIGLGQALYTT
-
+ +
-
t -
-
RKSVHIGPGQAFYAT
(-) Titer < 150, (+) titer
150-600,
+ (i-+)
titer 600-2400,
(++t)
era1 reasons for these differences. One possibility may be that our patients and Oram’s were unintentionally selected from different subpopulations in Kampala. However, no such selection bias could be identified, except that all of the patients in the previous study had developed AIDS, whereas we also studied individuals with milder disease. A second possibility is that our primers selected for sequences of U.S./European character. We believe this to be unlikely for the following reasons. The primers we used represent sequences flanking the V3 domain which are well conserved between available HIV-l sequences (Myers et al., 1991). Also, the flanking sequences of many patients, especially in group A, were highly homologous with those published by Oram eta/. (199 1). In a larger, partly overlapping, set of Ugandan samples we found that 27 of 31 samples could be amplified with our V3 primers (data not shown). In addition, the strong reactivity of our patients’ sera with the autologous peptides indicates that the amplified sequences were representative of the provirus population harbored by these patients.
+
+
+ -
t
titer > 2400.
Third, the results could indicate that new V3 genotypes were introduced in Uganda between the time Oram’s patients and our patients were infected. It is possible that this time difference in some cases is longer than 3-4 years, since Oram et al. only studied patients with AIDS. However, it is unlikely that U.S./European sequences have been introduced in Uganda, since most patients display a GPGQ V3 loop apex sequence. Fourth, the differences could indicate that Ugandan and U.S./European V3 loop sequences may tend to converge. If so, this may be due to an adaptation of HIV-l to replication in the human host. Subtle changes in the V3 loop have been shown to alter the tropism of HIV-1 for different types of human cells (Takeuchi eta/., 1991; Hwang et a/., 1991). It has been proposed that cleavage of the V3 loop by cellular proteases may play an essential role in the infection process (Hattori et a/., 1989; Clements et al., 1991). Differences in cellular proteases of different species may therefore impose specific selection pressure on HIV-l. Similarly, many earlier African sequences may be biased since they
ALBERT
680
were obtained from virus adapted to growth on cell lines or PBMC (Meyerhans et a/., 1989). In contrast, our sequences were obtained from fresh, uncultured patient PBMC. A high proportion (>90%) of HIV-l strains in the United States and Europe have been shown to belong to the same V3 serotype, i.e., the HIV-l,, and HIV1 ,68., serotype (Devash eta/., 1990; Zwart eta/., 1991). This serological reactivity to the V3 loop is primarily dependent on amino acids at the apex of the loop (Javaherian et al., 1989; Meloen et a/., 1989; Akerblom et al., 1990). Thus, the Ugandan HIV-1 strains presented in this study and U.S./European strains may belong to different V3 serotypes despite sequence similarities, since their loop apex sequences are usually different. However, most of our patients’sera recognized autologous, group and HIV-l,, V3 peptides, but not “typical” African peptides (Oram, JYl, 22, and ELI). This confirms that these viruses are structurally and antigenitally closer to U.S./European HIV-1 strains than to earlier Ugandan and African strains. Interestingly, Carrow et al. (1991) recently reported that Zairian sera also showed stronger reactivity with a V3 loop peptide derived from HIV-l MNthan one from the African HIV-l z2 isolate. The close relationships between V3 loop sequences present in Uganda in 1990, the U.S. and Europe have important implications for understanding the evolution of HIV-l and for attempts to develop a vaccine against HIV-l infection in the African population. ACKNOWLEDGMENTS This work was supported by grants from the World Health Organization (Human Reproductive Programme and Global Programme on AIDS), the Swedish Medical Research Council (Grants 692.16H09935 and B90-16H-07738.06B), the Labour Market Insurance Company (AFA), the Pediatric AIDS Foundation, and lstituto Superiore di Sanitg IV Progetto AIDS. We thank Dr. D. Larhammar. Dr. U. Gyllensten, Professor E. Norrby, Dr. E. M. Fenytl, Dr. G. Myers, and Dr. B. Korber for helpful discussions.
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UGANDAN
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SEQUENCES
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