Proc. Natl. Acad. Sci. USA Vol. 88, pp. 11236-11240, December 1991 Medical Sciences
Evolution of human immunodeficiency virus type 1 nucleotide sequence diversity among close contacts (human immunodeficiency virus type 1 diversity/human immunodeficiency virus type 1 transmission/direct DNA sequencing)
HAROLD BURGER*tt, BARBARA WEISER*t, KELLI FLAHERTY*, JANET GULLA*, PHI-NGA NGUYEN§, RICHARD A. GIBBS§
AND
*Departments of Medicine and Microbiology, State University of New York, Stony Brook, NY 11794; and
Medicine, Houston, TX 77030
§lnstitute for Molecular Genetics, Baylor College of
Communicated by Dorothy M. Horstmann, September 10, 1991 (received for review June 24, 1991)
ABSTRACT The degree of change in the nucleotide sequence of human immunodeficiency virus type 1 (HIV-1) that occurs when it is transmitted sexually from one individual to another or vertically from mother to child is unknown. Previous studies have shown that most cultured HIV-1 isolates from the same individuals differed in the entire envelope gene nucleotide sequence by up to 2%, although most isolates from unrelated individuals differed by 6-22%. To examine diversity among HIV-1 isolates from close contacts, we determined the nucleotide sequences of viruses from a family with a known epidemiologic profile, in which a woman transmitted HIV-1 heterosexually to her partner and vertically to her daughter. Direct DNA sequence analysis of primary HIV-1 isolates amplified by PCR was used to distinguish the major and minor viral sequences, termed quasispecies, to rapidly determine the predominant sequences and their phylogenetic relationships. The nucleotide sequence diversity of a major portion of the HIV-1 envelope gene was 3.7% between isolates from the woman and her heterosexual partner and 8.5% between isolates from this woman and her daughter, who had been infected for a longer period than the partner. The configuration of the phylogenetic tree demonstrated that the daughter's predominant isolate evolved from a progenitor of her mother's current strain. This study provides evidence of a continuous spectrum of sequence diversity between any two isolates ranging from those derived from the same person to those from close contacts and, ultimately, those from unrelated individuals. These data and methods can be applied to epidemiologic investigations of possible HIV-1 transmission between health care workers and their patients.
we determined the DNA sequences of viruses from a family in which a woman transmitted HIV-1 heterosexually to her partner and vertically to her daughter.¶ Viral nucleotide sequence heterogeneity exists not only between separate isolates of HIV-1 but within each single specimen as well. These heterogeneous populations of HIV-1 genomes within an individual sample, called quasispecies, have complicated the task of determining the predominant HIV-1 sequence in a specimen when traditional methods based on sequencing cloned DNA molecules are employed (13-15). To facilitate rapid determination of the predominant HIV-1 sequences in viruses from this family, direct DNA sequencing was used. Determination of the predominant sequences made it possible to measure their diversity and phylogenetic relationship to each other and to other known HIV-1 sequences. The degree of diversity in the env gene sequences from these close contacts spanned the previous gap between isolates derived from the same individual and those from unrelated individuals, thereby providing evidence for a continuous spectrum of nucleotide sequence diversity among isolates. These data demonstrated the degree of diversity that evolved between donor and recipient isolates when HIV-1 was transmitted between identified intimate contacts. This study illustrates that direct DNA sequencing with measurement of diversity and phylogenetic distances can be used for epidemiologic investigations of possible HIV-1 transmission between close contacts.
MATERIALS AND METHODS Clinical Specimens. The viruses were obtained from an HIV-1-infected woman, her daughter, who was vertically
Nucleotide sequence variation between isolates of human immunodeficiency virus type 1 (HIV-1), particularly in the envelope gene (env), has been studied for its relevance to vaccine design, viral evolution, and pathogenesis (1-10). The considerable sequence heterogeneity among field isolates of HIV-1 can also be measured to assist epidemiologic investigations. Recently, the degree of sequence diversity between HIV-1 isolates from a dentist and his patients was analyzed to help determine whether transmission from this HIV-1infected health care worker to his patients was likely (11). Previous studies of sequence diversity have shown that most cultured HIV-1 isolates from the same individuals, termed "siblings," differed in the entire env gene nucleotide sequence by up to 2%, although most isolates from unrelated individuals, termed "cousins," differed by 6-22% (12). Thus,
infected at birth in 1977, and her heterosexual partner, who began living with her in 1978. The daughter's only risk factor for HIV-1 infection was being the child of an infected mother, and the partner's only risk was heterosexual contact with the mother. The clinical, epidemiologic, and immunologic features of the subjects were described elsewhere (16). Informed consent was given by the woman for herself and her daughter and by her partner. This study was approved by the State University of New York at Stony Brook Institutional Review Board. Direct DNA Sequencing. HIV-1 isolates were obtained from the three infected subjects in December 1988 to determine and compare their predominant env sequences. HIV-1 was
comparisons of diversity in the env gene left a gap in the 2-6% range (12). To examine the diversity in HIV-1 isolates from close contacts, including one with a known time of infection,
Abbreviation: HIV-1, human immunodeficiency virus type 1. tPresent address: Wadsworth Center for Research, New York State Department of Health, Albany, NY 12201, and Department of Medicine, Albany Medical College, Albany, NY 12201. tTo whom reprint requests should be addressed at present address. $The sequences reported in this paper have been deposited in the GenBank data base [accession nos. M77278 (mother), M77229 (daughter), and M77230 (partner)].
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Proc. Natl. Acad. Sci. USA 88 (1991)
cultivated briefly in peripheral blood mononuclear cells to amplify replication-competent viruses, and virus was harvested at the first peak of reverse transcriptase activity (17, 18). DNA was extracted, and an =1400-base-pair DNA fragment, representing variable and conserved regions of the HIV-1 env gene, including the third variable domain (V3 loop), was amplified by PCR. DNA extraction and PCR amplification were done in parallel on uninfected peripheral blood mononuclear cells as a control. Viral DNA was used as templates for PCRs with env-specific oligonucleotide primers. The upstream primer 5'-CAG CAC AGT ACA ATG TAC ACA TGG-3' corresponded to bases 6949-6972 of clone HXB2 (21), and the downstream primer 5'-ATG GTG AGT ATC CCT GCC TAA CTC-3' was complementary to bases 8342-8366. To directly sequence the DNA of the amplified fragments, the double-stranded PCR products were converted to a single-stranded mixture in a second PCR-like reaction that used only a single oligonucleotide primer (19, 20). The dideoxynucleotide sequencing reactions utilized the same two primers used for PCR, and then additional primers were synthesized 'as new sequence was determined. The oligonucleotide primers were end-labeled with 32P and extended with T7 DNA polymerase (Sequenase). More than half the env gene from each isolate, 1353 bases corresponding to nucleotides 6981-8334 of clone HXB2 (12, 21), was analyzed by direct DNA sequencing. Nudeotide Sequence Alinment. The predominant HIV-1 env nucleotide sequences obtained from the mother, her daughter, and the mother's sexual partner, were aligned with the sequence of clone HXB2 (21) as a reference by using
b
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Baylor College of Medicine Molecular Biology Information Resource (EUGENE) software. Phylgnetic Tree Constrtion. A phylogenetic tree was constructed by progressive alignment of the sequences of the familial HIV-1 isolates from'the mother (SBM, Stony Brook mother), her daughter (SBID, Stony Brook daughter), and the mother's sexual partner (SBP,' Stony Brook partner). Minimum-length evolutionary trees were based on partial envelope sequences from the third variable domain to =w580 nucleotides into the gp4l region. Sequences SBM, SBD, and SBP were aligned with 12 other sequences from the Human Retroviruses and AIDS data base (12). The alignment was done by using a multiple alignment sequence editor (MASE, D. Faulkner and J. Jurka, Molecular Biology Computer Research Resource at the Dana-Farber Cancer Institute). The trees were constructed by using the multiple parsimony (MULPARS) algorithm of PAUP (phylogenetic analysis using parsimony, D. L. Swofford, Illinois Natural History Survey). Distances were calculated by adding the horizontal branch lengths between any two points on the tree and dividing that number by the towl number of sites. The total sites represent the number of variable and conserved sites in the alignment minus any gapped regions. RESULTS Amplification by PCR of HIV-1 env sequences from infected peripheral blood mononuclear cells produced D)NA fragments of "'1400 bases in samples from the'three infected family members, but no detectable HIV-1 DNA was amplified from uninfected control cells (Fig. la). The nucleotide
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FIG. 1. PCR amplification and direct DNA sequencing of HIV-1 env sequences. (a) PCR amplification of an "1400-base-pair fragment from the HIV-1 env gene. Lanes: 1, molecular weight markers are a mixture of Hae III digested #X174 and HindIII-cut A DNA; 2, U05% of the amplification products of one reaction resolved on a 1% agarose gel; 3, control aliquot from a reaction where the template was DNA from uninfected human peripheral blood mononuclear cells. (b) Example of direct DNA sequence analysis of PCR-amplified HIV-1 DNA from mother (M), partner (P), and daughter (D). The region shown reveals how the direct DNA sequencing can simultaneously identify both regions of difference between separate samples and heterogeneity within a sample. Three of the differences are unambiguous bWse replacements shown by arrows 1-3: position 1, thymine residue in M is a cytosine in P and D; position 2, cytosine residue in M is thymine in P and D; and position 3, thymine residue in M and P is guanine in D. The other two differences (arrows 4 and 5) are positions where a mixture of bases are present within single samples. At position 4 a cytosine residue is present as the single predominant species in M and P but is mixed with a thymine residue in D; at-position S an adenine residue is present in M, but in P and D mixtures of adenine and guanine occur. The gel also shows that the relative amounts of the adenine and guanine bases at position 5 in P and D differ, showing an excess of guanine in P and an excess of adenine in D.
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Medical Sciences: Burger
et
of HIV-1 env DNA amplified from each family member's sample was determined by direct DNA sequencing. The direct DNA sequencing gels clearly demonstrated the presence of predominant and minor HIV-1 species within each isolate as well as sequence differences between samples from different family members, mother, partner, and daughter (Fig. lb). At the overwhelming majority of base positions (>99%), the identity of the predominant sequence from each isolate was unambiguous, reflecting a 10-fold or greater intensity of signal from one of the four DNA bases. At a few positions an approximately equal representation of two bases was observed (Fig. lb), but overall a predominant sequence was clearly discernible. The predominant sequences of the HIV-1 isolates obtained from the family members were aligned with the sequence of clone HXB2 (21) as a reference (Fig. 2). None of the predominant sequences showed any interruption in the open reading frame due to the presence of stop codons or frameshifting mutations. The percentage of nucleotide diversity in the sequenced region of env was calculated: the nucleotide sequence from the daughter differed by 8.5% from her mother's sequence and differed by 8.3% from the partner's sequence. The mother's and partner's sequences differed by 3.7%. The primary amino acid sequences were deduced (Fig. 3). Based on the predominant nucleotide sequences from the three subjects, a phylogenetic tree was constructed by progressive alignment (Fig. 4). The configuration of the phylogenetic tree illustrates that the daughter's predominant isolate evolved from a progenitor of her mother's current strain rather than one from the partner. The tree also indicates that sequence
the partner's strain is more closely related to the mother's
strain than it is to any other known sequence (12). The daughter had been infected >11 years, whereas the mother's sexual partner had been infected for a shorter period when the HIV-1 isolates were obtained. The configuration of the phylogenetic tree is thus consistent with the clinical and epidemiologic documentation that the daughter was vertically infected and that the mother transmitted HIV-1 to her child before she transmitted it to her partner (16).
DISCUSSION We studied the evolution of HIV-1 sequence diversity among related isolates by directly sequencing the HIV-1 env gene from three family members with a known epidemiologic relationship. Previous studies of HIV-1 sequence diversity among related isolates have focused on blood transfusionassociated infection. One study reported very limited sequence heterogeneity between env genes from a donor and several neonatal transfusion recipients (22), another study found 4-8% diversity between strains from hemophiliacs who received one batch of pooled plasma (23), and a third study found that variation in the third hypervariable domain (V3 loop) of env increased with time (24). We had the opportunity to sequence donor and recipient isolates to examine the divergence that occurred over a period of years after HIV-1 was transmitted to close contacts heterosexually and vertically, two of the most common routes of transmission in the worldwide epidemic (25, 26). The levels of diversity in these close contacts are noteworthy for several reasons. They span the previously described gap of =2-6% diversity between the env genes of strains cultured from the same individuals (siblings) and those isolated from unrelated individuals (cousins) (12). These data suggest that there is a continuous spectrum of nucleotide
diversity between any two isolates ranging from those derived from the same person to those derived from close contacts and, ultimately, to those derived from unrelated individuals. Generation of diversity in the V3 loop of gpl20 is of particular interest because this region contains the sequence
Proc. Natl. Acad. Sci. USA 88 (1991)
al.
CTCM CTGrrAAATGcAGTCTAGCAGAAGAAOAOGTAGTAA
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OATrCTCCAATTrCACGOACAATCTAMACCATAATAGTACAOCTGAACACATCTGTAG -GA- C P.------A-------- AA--- ---------------D.----- AA --------A----------------------- -- ---- GA-A ----AA--- --------------------- T-A--AGA-G ----4.----- A---------
AAATAATYVTACAAACCCAACAACAATACAAAAAAGAATCCGTATcCACAGAGCAC
P.--a------- T-------- --------------- --AA-----..----- --D.----------------------------------C-----G--ATC----..--------
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ACATT ATAGAGCAAAATGGAATAACACT¶'TAAACAGATAG ATAGCAAATTAAGAOAAC
-GAAC-T-----T--- - -C----------- A--P.--C - C--D.------- C-T-A ---------------------G--T-----T---------- CAG --M1. --C-------GA---------------------- G----G--G-GAA-------------
AACGGAAATAATAAAACAATAAITCTrAAOCXAA7CcTC&GAOOGACCCAGAAATrO .
----------- G-------T
T-----------T----------
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TAACGCACAGr'TTPAAT1TGCGAGGG0AATrmr'CACTTAATIAACACAAC'rrTTrA
P .---T --- --A- ---- -- ------ -- -------- -- ------ -- ------ - ----- ---- D . --- -A-- --C--- --- --- - ----- ---- ---- --- --- -----CA ----- -^----A--- . ---T----- ---- ---- - ----- ---- ---- -- -------------A-- ---A ---- ---- -
ATACTACTT'GTAATAGTA . .. CTTICAGTACGAG0cAAAACACTGAGA
P .-- -- -- - - - - --A--G- -A- -at -- ------A-GG-............... .....C.GGA........-..A. -CG - A A- --g--- ---A-GT -----A--- --- G- --AC-- ----AD .----- -- -- --- AA^-'G----M.- -- -- -- -- --- AC - TW -A - -gta - -- - -- A -GC .. .. .. .. ... C - G-AGG^AA-TAAC GTGACACAA7CACCC7CCCATC, AARACATTAUACTG OAaATa GP...T-T ......-AD .A-- T-T-C----- T--- -A-- -- -- -- -- -- ---------G-- - --- - --- - -- G- -- -- - A- -T - ---- -- - --- -- -- -------G- ---- -- -- - - ---G----- M .C-A-T-T . .... -- -- -
---- -- -- - --- - ----- -- - --- ---- - --- - -----
G--A-A-LAACMG.TATGCCCCTICCCATCAGTC:ACAAATTAG ANNTCATCASAT~ATCAG P.- ------ -- ------ -- -- -- -- -----A-- ------ -- ---- ---- ---- ---- ---- D.- -- - ----- -- ------ -- --- --------A-- --T--- ---- -- - ----- - ----- - ----M------- -- ------ -- ---- -- -------A-- ---- -- ------ -- --- --- - ----- -- ....... GAGA GCC............................... GGCTGCTATTAACAAGAGA7GG=T^AACAATAG A -- -- -- -- -- -- -- -- ---- ---A--CA--C-T-A- t .............. P-.----- -AD.- -- -- -- -- -- -- -- -- -- -- -- -- -------A-- -T--CA-TA- -aat&&t&&act --AA -- -- -- -- ---- ---G- ----A-A--CA--C- --A-t .............. M-.----- -ATCTTCAGAC GGAGGTN_ ~ A~ T T A T T C CP.-
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D.- ---- -- --C- -C- -------- -- ---- -- ------ -- ------ ---- ---- ---- ---- M.- ------ ----C--- -- -- -- - ----- ---- ---- ------ -- ------ ---- ---- -- -- AAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAA GGCACAAGAG =TGG;C P-------------------------------------------------------------
D .--- --A-A-C-- ---- ---- ---- ---- -- ------ -- ------ ---- -- ---- - ----- M .----- -- ---G--- ---- -- -- -----A------ ------ -------- ------ -- ---- Aa&GAGA ~CAGlrGGAATACGGAGC7TITCCTT~roGTNGGAGGAGCAGC ___-- _-----G------__--_ P------------D .-A----- -- ------ -- -- -- ---- -- -----G--C- -T-- -- ---- -- ---- ---- -- M .------- -- ------ ---- -- ------ ------A-- ---- ---- -------- -- ---- -- -
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G7TAaG'c;AGCAGCAGAACAATTGCTGAGOGCTAT5GAGGCGCAACAGCA~tTrC
P .--- -- ------A---- -- ---- ---- ---_-__-___-____-____-____-____-___-_-_-_-_D .----- -------A-- ---- ------C---- -- ---A--- -- -- ---------- -- ---- -- -- M .--- ---------A---- -- ---- -- C----- ------ ---- -------- ---- -- ---- -- -
AACTCACAGTCTGGGGATCAAGCAGCT'CCAAGCAGAT'CCTIAGCTC=hAJAATACC
P .--- ---- - ----- -- ------ --- --- -----G------ -G-- --G --- -- ---- ---- -- - --G-- --- --- --G----C- -- -- -- -- --D.- ---- -- -- -- -- -- -- -- -- -- -- -----M .--- -- ------ -- ---- ---- ---- -- - ----G-------G-- --G--- ---- ---- --- --7WAAAAOGTCTOGCT1CATTTGCACCACTG TAAAGGAT'CAACAGCTC P..-G-G-- ----- -- -- ----- -G-- ---A--- -- -- -- -- -- -- -- - ------C- -- -- -- --D .------- -- -- ------ ---G --- --- ----- -- -- ------ -- ---- ---- ---- -- -- 1. -G-G--- ---- ---- -- -- G-----A--- -------- -- ---- -- ------ -- ---- -- -
CTG CCT;GAASGCTAGTGAGTAATAAATCTSC7GACAGACTGG)lATCACACG.A
P.------------ ---T-------------------------T-----T------ A---T-D.---------------------------------------A-TC-T--T---G--A--- T-M-----------------------------------------T-----T---G--A---T-aaMAco^GTvTACACTCCvTUATM C P.---------C -------A---------G-A ------------------ T--AA-------D.---------C ------- --------------- A------ UT-C ---- T--CAA------M.---------C -------A---------G-A------------------ T--AA -------AAGAATCGCAAAACCAGCAAGUI~A=GTA A P.-----------------A--------------------------A--G------------D.-----------------A-------------------- C------A-G ------ G-G ---M.-----------------A-------------------- C--------G------------CAAGTTTrG=AA7=GTTTAACATAAC^AAXTGCGOG7ATAATAT TAA P-----------------O-- ---_________________- _______ D.----------------------G----T----A ------------------ A--------M.-------------G-------- G---------- -----------------G-A --------~ TT^oSAITTC~^T TCA TGA$ATSA5TO P.-A--------------- A----G----------------------------D--. -----------------_-______________-___ M.--:-------------- A------------ ---------------------FIG. 2. Nucleotide sequence alignment. The predominant HIV-1 env nucleotide sequences obtained, from the mother (M)' her daughter (D), and the mother's sexual partner (P) were aligned with clone HXB2 (21) as a reference by using Baylor College of Medicine Molecular Biology Information Resource (EUGENE) software. The sequence of the reference is displayed above the sequences from the subjects. Dashed fines indicate no change compared with the reference. Altered bases are indicated by capital letters, and gaps in the alignment'are indicated by dots.
principal neutralizing determinant of HIV-1 and is, therefore, a candidate for an HIV-1 vaccine immunogen (9, 27). In
Proc. Natl. Acad. Sci. USA 88 (1991)
Medical Sciences: Burger et al. P. VVSTQLLLNGSLAEEEVVIRSDNFTNNAKTI IVQLNESVQINCIRPNNNTRKSISIGPGR D .- --------------------E--------------- T-E---T ------S-R-----M .----------------------------------- K-A-E ---T------- R--N---P. AFYTTGEIIGDIRQAHCNLSRANWANTLKKIVEKLREQF . KNKTIVFNQSSGGDPEIVM4H D. --VAAR---- K--- --- I-ITK-N---RL--S--Q ---G-------- H--------- TM.- -----D-------------- TK-N ----QRG -------------------------P. NFNCGGEFFYCNSTQLFNSTWYSNNTWNGTGG ...... ENITLPCRIKQI INMWQEVGKA D. T-----------T-K------NDSS---VERSSKAEENDIL--Q------V--------M. S----------- T-K ----- TW-S -----E-NN .... T----------- V---------
P. MYAPPIRGQIRCSSNITGLILTRDGGNNTNVT .... EIFRPGGGDMRDNWRSELYKYKVV D .- -------L---------- L-------- N-N-NNNT ----A----------------- I M .- ----------------------- K- --E-....-----------------------P. KIEPLGVAPTKAKRRVVQREKRAVGIGAVFLGFLGAAGSTMGAASITLTVQARLLLSGIV D. Q----------------------------LF--------------L-------Q-----M. R-----I ------------H---------M------------------------------P. QQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTAVP D .--------------------------------- L----K--------------------M.--------------------------------------P. WNSSWSNKSLDQIWNNMTWMQWEREIENYTSLIYNLIEESQNQQEKNEQELLELDKWASL D. --A -H----ND--D------ D---NK--DS--Q ------------- D--K--E --M. --A ---------- D---------------------------------- D---------P. WNWFSITNWLWYIKIFIIIVGGLIGLRIVFAVLS D .------ SK --------- M -----V ---------M. -S ----------- R---M---------------
FIG. 3. Amino acid sequence alignment. Amino acid sequences from the mother (M), her daughter (D), and the mother's sexual partner (P), deduced from their predominant env nucleotide sequences. Dashed lines indicate no change, and gaps in the alignment are indicated by dots. The sequence of the V3 loop is in boldface type.
addition, studies of vertical transmission have suggested that antibodies directed toward the principal neutralizing determinant may protect against infection of children born to HIV-1-infected women (28, 29). Recent analysis of a large number of HIV-1 isolates revealed that the principal neutralizing determinant is less variable than originally thought (9, 30). The primary amino acid sequences (Fig. 3) ofthe V3 loop from the three related isolates studied here were compared with the consensus sequence obtained from 527 North American and European sequences (30). The partner's sequence had two amino acid substitutions, the mother's sequence had three substitutions, and the daughter's sequence had eight A
.006-BR BRU
changes as compared with the 35 amino acids comprising the consensus V3 loop. The partner's and mother's V3 loop sequences varied little from the consensus sequence or from each other's sequences, whereas the daughter's sequence diverged significantly. Because the mother was infected longer than either the partner or daughter (16), it seems unlikely that evolution over time was the only factor influencing the daughter's sequence heterogeneity in the principal neutralizing determinant. These V3 loop sequence data are compatible with the hypothesis that the vertically infected daughter was infected with virus that escaped neutralization by maternal antibody (28, 29), either because of low neutralizing titer or mutation. In this study direct DNA sequencing could distinguish visually the major and minor viral DNA quasispecies. Direct DNA sequencing has been used previously to characterize heterogeneous mutations in the human genome and is particularly well suited to determine predominant sequences for studies of phylogenetic and epidemiologic relationships among HIV-1 strains. The advantage of this procedure for these studies is the relative speed with which it can determine the predominant sequence of a large fragment of the HIV-1 genome, in this case >1300 bases. By using direct DNA sequencing the need for DNA cloning is obviated, and it is therefore not necessary to do many independent sequencing reactions to distinguish variants generated by Taq DNA polymerase during PCR from the preexisting major and minor viral DNA quasispecies (13-15). Several factors may be involved in generating sequence heterogeneity after HIV-1 is transmitted between close contacts, including duration of infection, route of transmission, and host immune response, but the mechanisms involved are not well understood at this time. These data support the hypothesis that the degree of diversity may be a function of the duration of infection and the epidemiologic relatedness of the hosts (10). It should therefore be possible to use these data and methods in epidemiologic investigations of possible HIV-1 transmissions between close contacts. It will be necessary to examine serial samples from other sets of close contacts with different periods of infection and clinical situations to determine the pattern and rate of viral evolution with time. We thank the subjects for participating in this study. We thank Gerald Myers for helpful discussion and construction of the phylogenetic tree; K. MacInnes and J. Lawrence for tree analysis; J. Belmont, L. Donehower, and G. MacGregor for helpful information; C. Spiropoulos for technical help; and B. Baldassano and K. Ronca
MN AL
JRCSF
JH3 (Japan) .020
SBD .026
11239
SF2
OYI (Gabon)
FIG. 4. Phylogenetic tree of HIV-1 env gene sequences. A phylogenetic tree was constructed by progressive alignment of the sequences of the familial HIV-1 isolates from the mother (SBM, Stony Brook mother), her daughter (SBD, Stony Brook daughter), and the mother's sexual partner (SBP, Stony Brook partner). Sequences SBM, SBD, and SBP were aligned with 12 other sequences from the Human Retroviruses and AIDS data base (12).
for manuscript preparation. The DNA sequences have been contributed to the HIV sequence data base at the Los Alamos National Laboratory. This work was supported by National Institutes of Health Grants U01 A125893 (to H.B. and B.W.) and U01 A130243 (to R.A.G.) and the W. M. Keck Center for Computational Biology at Baylor College of Medicine. 1. Wong-Staal, F., Shaw, G. M., Hahn, B. H., Salahuddin, S. Z., Popovic, M., Markham, P., Redfield, R. & Gallo, R. C. (1985) Science 229, 759-762. 2. Benn, S., Rutledge, R., Folks, T., Gold, J., Baker, L., McCormick, J., Feorino, P., Piot, P., Quinn, T. & Martin, M. (1985) Science 230, 949-951. 3. Hahn, B. H., Shaw, G. M., Taylor, M. E., Redfield, R. R., Markham, P. D., Salahuddin, S. Z., Wong-Staal, F., Gallo, R. C., Parks, E. S. & Parks, W. P. (1986) Science 232, 15481553. 4. Starcich, B. R., Hahn, B. H., Shaw, G. M., McNeely, P. D., Modrow, S., Wolf, H., Parks, W. P., Josephs, S. F., Gallo, R. C. & Wong-Staal, F. (1986) Cell 45, 637-648. 5. Fisher, A. G., Ratner, L., Mitsuya, H., Marselle, L. M., Harper, M. E., Broder, S., Gallo, R. C. & Wong-Staal, F. (1986) Science 233, 655-659.
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6. Alizon, M., Wain-Hobson, S., Montagnier, L. & Sonigo, P. (1986) Cell 46, 63-74. 7. Saag, M. S., Hahn, B. H., Gibbons, J., Li, Y., Parks, E. S., Parks, W. P. & Shaw, G. M. (1988) Nature (London) 334, 4n40-44. 8. Cordonnier, A., Montagnier, L. & Emerman, M. (1989) Nature (London) 340, 571-574. 9. LaRosa, G. J., Davide, J. P., Weinhold, K., Waterbury, J. A., Profy, A. T., Lewis, J. A., Langlois, A. J., Dreesman, G. R., Boswell, R. N., Shadduck, P., Holley, L. H., Karplus, M., Bolognesi, D. P., Matthews, T. J., Emini, E. A. & Putney, S. D. (1990) Science 249, 932-935. 10. Smith, T. F., Srinivasan, A., Schochetman, G., Marcus, M. & Myers, G. (1988) Nature (London) 333, 573-575. 11. CDC (1991) Morbid. Mortal. Wkly. Rep. 40, 21-33. 12. Myers, G., Rabson, A., Josephs, S., Smith, T., Berzofsky, J. & Wong-Staal, F. (1990) Human Retroviruses and AIDS (Los Alamos National Lab., Los Alamos, NM). 13. Meyerhans, A., Cheynier, R., Albert, J., Seth, M., Kwok, S., Sninsky, J., Morfeldt-Manson, L., Asjo, B. & Wain-Hobson, S. (1989) Cell 58, 901-910. 14. Hirsch, V. M., Johnson, P. R. & Zack, P. M. (1990) in Vaccines 90: Modern Approaches to New Vaccines Including Prevention of AIDS, eds. Lemer, R., Ginsberg, H., Chanck, R. M. & Brown, F. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 379-382. 15. Goodenow, M., Huet, T., Saurin, W., Kwok, S., Sninsky, J. & Wain-Hobson, S. (1989) J. AIDS 2, 344-352. 16. Burger, H., Belman, A. L., Grimson, R., Kaell, A., Flaherty, K., Gulla, J., Gibbs, R., Nguyen, P.-N. & Weiser, B. (1990) Lancet 336, 134-136. 17. Barre-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T., Charmaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., BrunVezinet, F., Rouzioux, C., Rozenbaum, W. & Montagnier, L. (1983) Science 220, 868-870. 18. Gallo, R. C., Salahuddin, S. Z., Popovic, M., Shearer, G. M., Kaplan, M., Haynes, B. F., Palker, T. J., Redfield, R., Oleske,
19. 20. 21.
22. 23. 24.
25. 26. 27.
28.
29. 30.
J., Safai, B., White, G., Foster, P. & Markham, P. D. (1984) Science 224, 500-502. Gibbs, R. A., Nguyen, P.-N. & Caskey, C. T. (1991) in Methods in Molecular Biology, ed. Mathew, C. (Humana Press, Clifton, NJ), in press. Gibbs, R. A., Nguyen, P.-N., McBride, L. J., Koepf, S. M. & Caskey, C. T. (1989) Proc. Natd. Acad. Sci. USA 86, 19191923. Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B., Josephs, S. J., Doran, E. R., Ragalski, J. A., Whitehorn, E. A., Baumeister, K., Ivanoff, L., Petteway, S. R., Jr., Pearson, M. L., Lautenberger, J. A., Papas, T. S., Ghryeb, J., Chang, N. T., Gallo, R. C. & Wong-Staal, F. (1985) Nature (London) 313, 277-284. McNeatney, T., Westervelt, P., Thielan, B. J., Trowbridge, D. B., Garcia, J., Whittier, R. & Ratner, L. (1990) Proc. Natd. Acad. Sci. USA 87, 1917-1921. Balfe, P., Simmonds, P., Ludlam, C., Bishop, J. & Brown, A. J. L. (1990) J. Virol. 64, 6221-6233. Wolfs, T., deJong, J., van den Berg, H., Tunagel, J., Krone, W. & Goudsmit, J. (1990) Proc. Natl. Acad. Sci. USA 87, 99389942. Pizzo, P. A. (1990) J. Infect. Dis. 161, 316-325. Chin, J. (1990) Lancet 336, 221-224. Javaherian, K., Langlois, A., McDanal, C., Ross, K., Eckler, L., Jellis, C., Profy, A., Rusche, J., Boogmesi, D., Putney, S. & Matthews, M. (1989) Proc. Natd. Acad. Sci. USA 86, 6768-6772. Rossi, P., Moschese, V., Broliden, P., Fundaro, C., Quinti, I., Plebani, A., Giaquinto, C., Tovo, P., Ljunn, K., Rosen, J., Wigzeli, H., Jondal, M. & Wahren, B. (19B Proc. Natl. Acad. Sci. USA 86, 8055-8058. Devash, Y., Calvelli, T., Wood, D., Reagn, K. & Rubinstein, A. (1990) Proc. Natl. Acad. Sci. USA 87, 3445-3. Myers, G., Korber, B., Berzofsky, J. & Smith, R. (1991) Human Retroviruses and AIDS (Los Alamos National Lab., Los Alamos, NM).
Evolution of human immunodeficiency virus type 1 nucleotide sequence diversity among close contacts (human immunodeficiency virus type 1 diversity/human immunodeficiency virus type 1 transmission/direct DNA sequencing)
HAROLD BURGER*tt, BARBARA WEISER*t, KELLI FLAHERTY*, JANET GULLA*, PHI-NGA NGUYEN§, RICHARD A. GIBBS§
AND
*Departments of Medicine and Microbiology, State University of New York, Stony Brook, NY 11794; and
Medicine, Houston, TX 77030
§lnstitute for Molecular Genetics, Baylor College of
Communicated by Dorothy M. Horstmann, September 10, 1991 (received for review June 24, 1991)
ABSTRACT The degree of change in the nucleotide sequence of human immunodeficiency virus type 1 (HIV-1) that occurs when it is transmitted sexually from one individual to another or vertically from mother to child is unknown. Previous studies have shown that most cultured HIV-1 isolates from the same individuals differed in the entire envelope gene nucleotide sequence by up to 2%, although most isolates from unrelated individuals differed by 6-22%. To examine diversity among HIV-1 isolates from close contacts, we determined the nucleotide sequences of viruses from a family with a known epidemiologic profile, in which a woman transmitted HIV-1 heterosexually to her partner and vertically to her daughter. Direct DNA sequence analysis of primary HIV-1 isolates amplified by PCR was used to distinguish the major and minor viral sequences, termed quasispecies, to rapidly determine the predominant sequences and their phylogenetic relationships. The nucleotide sequence diversity of a major portion of the HIV-1 envelope gene was 3.7% between isolates from the woman and her heterosexual partner and 8.5% between isolates from this woman and her daughter, who had been infected for a longer period than the partner. The configuration of the phylogenetic tree demonstrated that the daughter's predominant isolate evolved from a progenitor of her mother's current strain. This study provides evidence of a continuous spectrum of sequence diversity between any two isolates ranging from those derived from the same person to those from close contacts and, ultimately, those from unrelated individuals. These data and methods can be applied to epidemiologic investigations of possible HIV-1 transmission between health care workers and their patients.
we determined the DNA sequences of viruses from a family in which a woman transmitted HIV-1 heterosexually to her partner and vertically to her daughter.¶ Viral nucleotide sequence heterogeneity exists not only between separate isolates of HIV-1 but within each single specimen as well. These heterogeneous populations of HIV-1 genomes within an individual sample, called quasispecies, have complicated the task of determining the predominant HIV-1 sequence in a specimen when traditional methods based on sequencing cloned DNA molecules are employed (13-15). To facilitate rapid determination of the predominant HIV-1 sequences in viruses from this family, direct DNA sequencing was used. Determination of the predominant sequences made it possible to measure their diversity and phylogenetic relationship to each other and to other known HIV-1 sequences. The degree of diversity in the env gene sequences from these close contacts spanned the previous gap between isolates derived from the same individual and those from unrelated individuals, thereby providing evidence for a continuous spectrum of nucleotide sequence diversity among isolates. These data demonstrated the degree of diversity that evolved between donor and recipient isolates when HIV-1 was transmitted between identified intimate contacts. This study illustrates that direct DNA sequencing with measurement of diversity and phylogenetic distances can be used for epidemiologic investigations of possible HIV-1 transmission between close contacts.
MATERIALS AND METHODS Clinical Specimens. The viruses were obtained from an HIV-1-infected woman, her daughter, who was vertically
Nucleotide sequence variation between isolates of human immunodeficiency virus type 1 (HIV-1), particularly in the envelope gene (env), has been studied for its relevance to vaccine design, viral evolution, and pathogenesis (1-10). The considerable sequence heterogeneity among field isolates of HIV-1 can also be measured to assist epidemiologic investigations. Recently, the degree of sequence diversity between HIV-1 isolates from a dentist and his patients was analyzed to help determine whether transmission from this HIV-1infected health care worker to his patients was likely (11). Previous studies of sequence diversity have shown that most cultured HIV-1 isolates from the same individuals, termed "siblings," differed in the entire env gene nucleotide sequence by up to 2%, although most isolates from unrelated individuals, termed "cousins," differed by 6-22% (12). Thus,
infected at birth in 1977, and her heterosexual partner, who began living with her in 1978. The daughter's only risk factor for HIV-1 infection was being the child of an infected mother, and the partner's only risk was heterosexual contact with the mother. The clinical, epidemiologic, and immunologic features of the subjects were described elsewhere (16). Informed consent was given by the woman for herself and her daughter and by her partner. This study was approved by the State University of New York at Stony Brook Institutional Review Board. Direct DNA Sequencing. HIV-1 isolates were obtained from the three infected subjects in December 1988 to determine and compare their predominant env sequences. HIV-1 was
comparisons of diversity in the env gene left a gap in the 2-6% range (12). To examine the diversity in HIV-1 isolates from close contacts, including one with a known time of infection,
Abbreviation: HIV-1, human immunodeficiency virus type 1. tPresent address: Wadsworth Center for Research, New York State Department of Health, Albany, NY 12201, and Department of Medicine, Albany Medical College, Albany, NY 12201. tTo whom reprint requests should be addressed at present address. $The sequences reported in this paper have been deposited in the GenBank data base [accession nos. M77278 (mother), M77229 (daughter), and M77230 (partner)].
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Proc. Natl. Acad. Sci. USA 88 (1991)
cultivated briefly in peripheral blood mononuclear cells to amplify replication-competent viruses, and virus was harvested at the first peak of reverse transcriptase activity (17, 18). DNA was extracted, and an =1400-base-pair DNA fragment, representing variable and conserved regions of the HIV-1 env gene, including the third variable domain (V3 loop), was amplified by PCR. DNA extraction and PCR amplification were done in parallel on uninfected peripheral blood mononuclear cells as a control. Viral DNA was used as templates for PCRs with env-specific oligonucleotide primers. The upstream primer 5'-CAG CAC AGT ACA ATG TAC ACA TGG-3' corresponded to bases 6949-6972 of clone HXB2 (21), and the downstream primer 5'-ATG GTG AGT ATC CCT GCC TAA CTC-3' was complementary to bases 8342-8366. To directly sequence the DNA of the amplified fragments, the double-stranded PCR products were converted to a single-stranded mixture in a second PCR-like reaction that used only a single oligonucleotide primer (19, 20). The dideoxynucleotide sequencing reactions utilized the same two primers used for PCR, and then additional primers were synthesized 'as new sequence was determined. The oligonucleotide primers were end-labeled with 32P and extended with T7 DNA polymerase (Sequenase). More than half the env gene from each isolate, 1353 bases corresponding to nucleotides 6981-8334 of clone HXB2 (12, 21), was analyzed by direct DNA sequencing. Nudeotide Sequence Alinment. The predominant HIV-1 env nucleotide sequences obtained from the mother, her daughter, and the mother's sexual partner, were aligned with the sequence of clone HXB2 (21) as a reference by using
b
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Baylor College of Medicine Molecular Biology Information Resource (EUGENE) software. Phylgnetic Tree Constrtion. A phylogenetic tree was constructed by progressive alignment of the sequences of the familial HIV-1 isolates from'the mother (SBM, Stony Brook mother), her daughter (SBID, Stony Brook daughter), and the mother's sexual partner (SBP,' Stony Brook partner). Minimum-length evolutionary trees were based on partial envelope sequences from the third variable domain to =w580 nucleotides into the gp4l region. Sequences SBM, SBD, and SBP were aligned with 12 other sequences from the Human Retroviruses and AIDS data base (12). The alignment was done by using a multiple alignment sequence editor (MASE, D. Faulkner and J. Jurka, Molecular Biology Computer Research Resource at the Dana-Farber Cancer Institute). The trees were constructed by using the multiple parsimony (MULPARS) algorithm of PAUP (phylogenetic analysis using parsimony, D. L. Swofford, Illinois Natural History Survey). Distances were calculated by adding the horizontal branch lengths between any two points on the tree and dividing that number by the towl number of sites. The total sites represent the number of variable and conserved sites in the alignment minus any gapped regions. RESULTS Amplification by PCR of HIV-1 env sequences from infected peripheral blood mononuclear cells produced D)NA fragments of "'1400 bases in samples from the'three infected family members, but no detectable HIV-1 DNA was amplified from uninfected control cells (Fig. la). The nucleotide
M
D
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T CA G-T C A G'T C A G .i1,itb
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FIG. 1. PCR amplification and direct DNA sequencing of HIV-1 env sequences. (a) PCR amplification of an "1400-base-pair fragment from the HIV-1 env gene. Lanes: 1, molecular weight markers are a mixture of Hae III digested #X174 and HindIII-cut A DNA; 2, U05% of the amplification products of one reaction resolved on a 1% agarose gel; 3, control aliquot from a reaction where the template was DNA from uninfected human peripheral blood mononuclear cells. (b) Example of direct DNA sequence analysis of PCR-amplified HIV-1 DNA from mother (M), partner (P), and daughter (D). The region shown reveals how the direct DNA sequencing can simultaneously identify both regions of difference between separate samples and heterogeneity within a sample. Three of the differences are unambiguous bWse replacements shown by arrows 1-3: position 1, thymine residue in M is a cytosine in P and D; position 2, cytosine residue in M is thymine in P and D; and position 3, thymine residue in M and P is guanine in D. The other two differences (arrows 4 and 5) are positions where a mixture of bases are present within single samples. At position 4 a cytosine residue is present as the single predominant species in M and P but is mixed with a thymine residue in D; at-position S an adenine residue is present in M, but in P and D mixtures of adenine and guanine occur. The gel also shows that the relative amounts of the adenine and guanine bases at position 5 in P and D differ, showing an excess of guanine in P and an excess of adenine in D.
11238
Medical Sciences: Burger
et
of HIV-1 env DNA amplified from each family member's sample was determined by direct DNA sequencing. The direct DNA sequencing gels clearly demonstrated the presence of predominant and minor HIV-1 species within each isolate as well as sequence differences between samples from different family members, mother, partner, and daughter (Fig. lb). At the overwhelming majority of base positions (>99%), the identity of the predominant sequence from each isolate was unambiguous, reflecting a 10-fold or greater intensity of signal from one of the four DNA bases. At a few positions an approximately equal representation of two bases was observed (Fig. lb), but overall a predominant sequence was clearly discernible. The predominant sequences of the HIV-1 isolates obtained from the family members were aligned with the sequence of clone HXB2 (21) as a reference (Fig. 2). None of the predominant sequences showed any interruption in the open reading frame due to the presence of stop codons or frameshifting mutations. The percentage of nucleotide diversity in the sequenced region of env was calculated: the nucleotide sequence from the daughter differed by 8.5% from her mother's sequence and differed by 8.3% from the partner's sequence. The mother's and partner's sequences differed by 3.7%. The primary amino acid sequences were deduced (Fig. 3). Based on the predominant nucleotide sequences from the three subjects, a phylogenetic tree was constructed by progressive alignment (Fig. 4). The configuration of the phylogenetic tree illustrates that the daughter's predominant isolate evolved from a progenitor of her mother's current strain rather than one from the partner. The tree also indicates that sequence
the partner's strain is more closely related to the mother's
strain than it is to any other known sequence (12). The daughter had been infected >11 years, whereas the mother's sexual partner had been infected for a shorter period when the HIV-1 isolates were obtained. The configuration of the phylogenetic tree is thus consistent with the clinical and epidemiologic documentation that the daughter was vertically infected and that the mother transmitted HIV-1 to her child before she transmitted it to her partner (16).
DISCUSSION We studied the evolution of HIV-1 sequence diversity among related isolates by directly sequencing the HIV-1 env gene from three family members with a known epidemiologic relationship. Previous studies of HIV-1 sequence diversity among related isolates have focused on blood transfusionassociated infection. One study reported very limited sequence heterogeneity between env genes from a donor and several neonatal transfusion recipients (22), another study found 4-8% diversity between strains from hemophiliacs who received one batch of pooled plasma (23), and a third study found that variation in the third hypervariable domain (V3 loop) of env increased with time (24). We had the opportunity to sequence donor and recipient isolates to examine the divergence that occurred over a period of years after HIV-1 was transmitted to close contacts heterosexually and vertically, two of the most common routes of transmission in the worldwide epidemic (25, 26). The levels of diversity in these close contacts are noteworthy for several reasons. They span the previously described gap of =2-6% diversity between the env genes of strains cultured from the same individuals (siblings) and those isolated from unrelated individuals (cousins) (12). These data suggest that there is a continuous spectrum of nucleotide
diversity between any two isolates ranging from those derived from the same person to those derived from close contacts and, ultimately, to those derived from unrelated individuals. Generation of diversity in the V3 loop of gpl20 is of particular interest because this region contains the sequence
Proc. Natl. Acad. Sci. USA 88 (1991)
al.
CTCM CTGrrAAATGcAGTCTAGCAGAAGAAOAOGTAGTAA
CAGTAGTATCAA
rA
P.--C-C------------- -
D. --- ---- -- ---- -- ------- ----- ------ ---- --GG-- ---- - ----G-- D .----M.- ---- ---- -- -- -- -----T- --- ---- - --- ---- - --- --- --- ---- -- - --- -- -
OATrCTCCAATTrCACGOACAATCTAMACCATAATAGTACAOCTGAACACATCTGTAG -GA- C P.------A-------- AA--- ---------------D.----- AA --------A----------------------- -- ---- GA-A ----AA--- --------------------- T-A--AGA-G ----4.----- A---------
AAATAATYVTACAAACCCAACAACAATACAAAAAAGAATCCGTATcCACAGAGCAC
P.--a------- T-------- --------------- --AA-----..----- --D.----------------------------------C-----G--ATC----..--------
----T--AAA----..--M.-----------------------------0-------
---
rA
TAGATA .. .AATATGACAAGACA1
CACOOAGAGCAT';IT!ACA
----
--------------
--------------TA-----C ---n,GC-A- -G---- -ata---G ---- A
14.-----
-
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-0
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C ---- G-C ---eat---G----A----------------
TA
ACATT ATAGAGCAAAATGGAATAACACT¶'TAAACAGATAG ATAGCAAATTAAGAOAAC
-GAAC-T-----T--- - -C----------- A--P.--C - C--D.------- C-T-A ---------------------G--T-----T---------- CAG --M1. --C-------GA---------------------- G----G--G-GAA-------------
AACGGAAATAATAAAACAATAAITCTrAAOCXAA7CcTC&GAOOGACCCAGAAATrO .
----------- G-------T
T-----------T----------
-
T--C -------------- T---------M. ----T.. . --G---------- G------ --T-T-------------------- G-------
TAACGCACAGr'TTPAAT1TGCGAGGG0AATrmr'CACTTAATIAACACAAC'rrTTrA
P .---T --- --A- ---- -- ------ -- -------- -- ------ -- ------ - ----- ---- D . --- -A-- --C--- --- --- - ----- ---- ---- --- --- -----CA ----- -^----A--- . ---T----- ---- ---- - ----- ---- ---- -- -------------A-- ---A ---- ---- -
ATACTACTT'GTAATAGTA . .. CTTICAGTACGAG0cAAAACACTGAGA
P .-- -- -- - - - - --A--G- -A- -at -- ------A-GG-............... .....C.GGA........-..A. -CG - A A- --g--- ---A-GT -----A--- --- G- --AC-- ----AD .----- -- -- --- AA^-'G----M.- -- -- -- -- --- AC - TW -A - -gta - -- - -- A -GC .. .. .. .. ... C - G-AGG^AA-TAAC GTGACACAA7CACCC7CCCATC, AARACATTAUACTG OAaATa GP...T-T ......-AD .A-- T-T-C----- T--- -A-- -- -- -- -- -- ---------G-- - --- - --- - -- G- -- -- - A- -T - ---- -- - --- -- -- -------G- ---- -- -- - - ---G----- M .C-A-T-T . .... -- -- -
---- -- -- - --- - ----- -- - --- ---- - --- - -----
G--A-A-LAACMG.TATGCCCCTICCCATCAGTC:ACAAATTAG ANNTCATCASAT~ATCAG P.- ------ -- ------ -- -- -- -- -----A-- ------ -- ---- ---- ---- ---- ---- D.- -- - ----- -- ------ -- --- --------A-- --T--- ---- -- - ----- - ----- - ----M------- -- ------ -- ---- -- -------A-- ---- -- ------ -- --- --- - ----- -- ....... GAGA GCC............................... GGCTGCTATTAACAAGAGA7GG=T^AACAATAG A -- -- -- -- -- -- -- -- ---- ---A--CA--C-T-A- t .............. P-.----- -AD.- -- -- -- -- -- -- -- -- -- -- -- -- -------A-- -T--CA-TA- -aat&&t&&act --AA -- -- -- -- ---- ---G- ----A-A--CA--C- --A-t .............. M-.----- -ATCTTCAGAC GGAGGTN_ ~ A~ T T A T T C CP.-
--
-
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D.- ---- -- --C- -C- -------- -- ---- -- ------ -- ------ ---- ---- ---- ---- M.- ------ ----C--- -- -- -- - ----- ---- ---- ------ -- ------ ---- ---- -- -- AAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAA GGCACAAGAG =TGG;C P-------------------------------------------------------------
D .--- --A-A-C-- ---- ---- ---- ---- -- ------ -- ------ ---- -- ---- - ----- M .----- -- ---G--- ---- -- -- -----A------ ------ -------- ------ -- ---- Aa&GAGA ~CAGlrGGAATACGGAGC7TITCCTT~roGTNGGAGGAGCAGC ___-- _-----G------__--_ P------------D .-A----- -- ------ -- -- -- ---- -- -----G--C- -T-- -- ---- -- ---- ---- -- M .------- -- ------ ---- -- ------ ------A-- ---- ---- -------- -- ---- -- -
--
--
---
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--
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GAAGCACTA5WGCGCAGCCTCAATGCACGCTGACGGTACAGGCCAGACAATTATTGCTIG
P.- -- -- -- -- -- -- -- -- - -- -----A--- -- -- -- -- -- -- -- -- ----T-- -- -- -- -- C--- -D.- -- ------ -- ---- -- ---G---T--- ---- -- ------ -- -- ------ -- --CM .-- -- -- -- -- - - - - ---- -G-- ---A- - ----- -- -- -- -- -- -- ----T-- -- -- -- ---
G7TAaG'c;AGCAGCAGAACAATTGCTGAGOGCTAT5GAGGCGCAACAGCA~tTrC
P .--- -- ------A---- -- ---- ---- ---_-__-___-____-____-____-____-___-_-_-_-_D .----- -------A-- ---- ------C---- -- ---A--- -- -- ---------- -- ---- -- -- M .--- ---------A---- -- ---- -- C----- ------ ---- -------- ---- -- ---- -- -
AACTCACAGTCTGGGGATCAAGCAGCT'CCAAGCAGAT'CCTIAGCTC=hAJAATACC
P .--- ---- - ----- -- ------ --- --- -----G------ -G-- --G --- -- ---- ---- -- - --G-- --- --- --G----C- -- -- -- -- --D.- ---- -- -- -- -- -- -- -- -- -- -- -----M .--- -- ------ -- ---- ---- ---- -- - ----G-------G-- --G--- ---- ---- --- --7WAAAAOGTCTOGCT1CATTTGCACCACTG TAAAGGAT'CAACAGCTC P..-G-G-- ----- -- -- ----- -G-- ---A--- -- -- -- -- -- -- -- - ------C- -- -- -- --D .------- -- -- ------ ---G --- --- ----- -- -- ------ -- ---- ---- ---- -- -- 1. -G-G--- ---- ---- -- -- G-----A--- -------- -- ---- -- ------ -- ---- -- -
CTG CCT;GAASGCTAGTGAGTAATAAATCTSC7GACAGACTGG)lATCACACG.A
P.------------ ---T-------------------------T-----T------ A---T-D.---------------------------------------A-TC-T--T---G--A--- T-M-----------------------------------------T-----T---G--A---T-aaMAco^GTvTACACTCCvTUATM C P.---------C -------A---------G-A ------------------ T--AA-------D.---------C ------- --------------- A------ UT-C ---- T--CAA------M.---------C -------A---------G-A------------------ T--AA -------AAGAATCGCAAAACCAGCAAGUI~A=GTA A P.-----------------A--------------------------A--G------------D.-----------------A-------------------- C------A-G ------ G-G ---M.-----------------A-------------------- C--------G------------CAAGTTTrG=AA7=GTTTAACATAAC^AAXTGCGOG7ATAATAT TAA P-----------------O-- ---_________________- _______ D.----------------------G----T----A ------------------ A--------M.-------------G-------- G---------- -----------------G-A --------~ TT^oSAITTC~^T TCA TGA$ATSA5TO P.-A--------------- A----G----------------------------D--. -----------------_-______________-___ M.--:-------------- A------------ ---------------------FIG. 2. Nucleotide sequence alignment. The predominant HIV-1 env nucleotide sequences obtained, from the mother (M)' her daughter (D), and the mother's sexual partner (P) were aligned with clone HXB2 (21) as a reference by using Baylor College of Medicine Molecular Biology Information Resource (EUGENE) software. The sequence of the reference is displayed above the sequences from the subjects. Dashed fines indicate no change compared with the reference. Altered bases are indicated by capital letters, and gaps in the alignment'are indicated by dots.
principal neutralizing determinant of HIV-1 and is, therefore, a candidate for an HIV-1 vaccine immunogen (9, 27). In
Proc. Natl. Acad. Sci. USA 88 (1991)
Medical Sciences: Burger et al. P. VVSTQLLLNGSLAEEEVVIRSDNFTNNAKTI IVQLNESVQINCIRPNNNTRKSISIGPGR D .- --------------------E--------------- T-E---T ------S-R-----M .----------------------------------- K-A-E ---T------- R--N---P. AFYTTGEIIGDIRQAHCNLSRANWANTLKKIVEKLREQF . KNKTIVFNQSSGGDPEIVM4H D. --VAAR---- K--- --- I-ITK-N---RL--S--Q ---G-------- H--------- TM.- -----D-------------- TK-N ----QRG -------------------------P. NFNCGGEFFYCNSTQLFNSTWYSNNTWNGTGG ...... ENITLPCRIKQI INMWQEVGKA D. T-----------T-K------NDSS---VERSSKAEENDIL--Q------V--------M. S----------- T-K ----- TW-S -----E-NN .... T----------- V---------
P. MYAPPIRGQIRCSSNITGLILTRDGGNNTNVT .... EIFRPGGGDMRDNWRSELYKYKVV D .- -------L---------- L-------- N-N-NNNT ----A----------------- I M .- ----------------------- K- --E-....-----------------------P. KIEPLGVAPTKAKRRVVQREKRAVGIGAVFLGFLGAAGSTMGAASITLTVQARLLLSGIV D. Q----------------------------LF--------------L-------Q-----M. R-----I ------------H---------M------------------------------P. QQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTAVP D .--------------------------------- L----K--------------------M.--------------------------------------P. WNSSWSNKSLDQIWNNMTWMQWEREIENYTSLIYNLIEESQNQQEKNEQELLELDKWASL D. --A -H----ND--D------ D---NK--DS--Q ------------- D--K--E --M. --A ---------- D---------------------------------- D---------P. WNWFSITNWLWYIKIFIIIVGGLIGLRIVFAVLS D .------ SK --------- M -----V ---------M. -S ----------- R---M---------------
FIG. 3. Amino acid sequence alignment. Amino acid sequences from the mother (M), her daughter (D), and the mother's sexual partner (P), deduced from their predominant env nucleotide sequences. Dashed lines indicate no change, and gaps in the alignment are indicated by dots. The sequence of the V3 loop is in boldface type.
addition, studies of vertical transmission have suggested that antibodies directed toward the principal neutralizing determinant may protect against infection of children born to HIV-1-infected women (28, 29). Recent analysis of a large number of HIV-1 isolates revealed that the principal neutralizing determinant is less variable than originally thought (9, 30). The primary amino acid sequences (Fig. 3) ofthe V3 loop from the three related isolates studied here were compared with the consensus sequence obtained from 527 North American and European sequences (30). The partner's sequence had two amino acid substitutions, the mother's sequence had three substitutions, and the daughter's sequence had eight A
.006-BR BRU
changes as compared with the 35 amino acids comprising the consensus V3 loop. The partner's and mother's V3 loop sequences varied little from the consensus sequence or from each other's sequences, whereas the daughter's sequence diverged significantly. Because the mother was infected longer than either the partner or daughter (16), it seems unlikely that evolution over time was the only factor influencing the daughter's sequence heterogeneity in the principal neutralizing determinant. These V3 loop sequence data are compatible with the hypothesis that the vertically infected daughter was infected with virus that escaped neutralization by maternal antibody (28, 29), either because of low neutralizing titer or mutation. In this study direct DNA sequencing could distinguish visually the major and minor viral DNA quasispecies. Direct DNA sequencing has been used previously to characterize heterogeneous mutations in the human genome and is particularly well suited to determine predominant sequences for studies of phylogenetic and epidemiologic relationships among HIV-1 strains. The advantage of this procedure for these studies is the relative speed with which it can determine the predominant sequence of a large fragment of the HIV-1 genome, in this case >1300 bases. By using direct DNA sequencing the need for DNA cloning is obviated, and it is therefore not necessary to do many independent sequencing reactions to distinguish variants generated by Taq DNA polymerase during PCR from the preexisting major and minor viral DNA quasispecies (13-15). Several factors may be involved in generating sequence heterogeneity after HIV-1 is transmitted between close contacts, including duration of infection, route of transmission, and host immune response, but the mechanisms involved are not well understood at this time. These data support the hypothesis that the degree of diversity may be a function of the duration of infection and the epidemiologic relatedness of the hosts (10). It should therefore be possible to use these data and methods in epidemiologic investigations of possible HIV-1 transmissions between close contacts. It will be necessary to examine serial samples from other sets of close contacts with different periods of infection and clinical situations to determine the pattern and rate of viral evolution with time. We thank the subjects for participating in this study. We thank Gerald Myers for helpful discussion and construction of the phylogenetic tree; K. MacInnes and J. Lawrence for tree analysis; J. Belmont, L. Donehower, and G. MacGregor for helpful information; C. Spiropoulos for technical help; and B. Baldassano and K. Ronca
MN AL
JRCSF
JH3 (Japan) .020
SBD .026
11239
SF2
OYI (Gabon)
FIG. 4. Phylogenetic tree of HIV-1 env gene sequences. A phylogenetic tree was constructed by progressive alignment of the sequences of the familial HIV-1 isolates from the mother (SBM, Stony Brook mother), her daughter (SBD, Stony Brook daughter), and the mother's sexual partner (SBP, Stony Brook partner). Sequences SBM, SBD, and SBP were aligned with 12 other sequences from the Human Retroviruses and AIDS data base (12).
for manuscript preparation. The DNA sequences have been contributed to the HIV sequence data base at the Los Alamos National Laboratory. This work was supported by National Institutes of Health Grants U01 A125893 (to H.B. and B.W.) and U01 A130243 (to R.A.G.) and the W. M. Keck Center for Computational Biology at Baylor College of Medicine. 1. Wong-Staal, F., Shaw, G. M., Hahn, B. H., Salahuddin, S. Z., Popovic, M., Markham, P., Redfield, R. & Gallo, R. C. (1985) Science 229, 759-762. 2. Benn, S., Rutledge, R., Folks, T., Gold, J., Baker, L., McCormick, J., Feorino, P., Piot, P., Quinn, T. & Martin, M. (1985) Science 230, 949-951. 3. Hahn, B. H., Shaw, G. M., Taylor, M. E., Redfield, R. R., Markham, P. D., Salahuddin, S. Z., Wong-Staal, F., Gallo, R. C., Parks, E. S. & Parks, W. P. (1986) Science 232, 15481553. 4. Starcich, B. R., Hahn, B. H., Shaw, G. M., McNeely, P. D., Modrow, S., Wolf, H., Parks, W. P., Josephs, S. F., Gallo, R. C. & Wong-Staal, F. (1986) Cell 45, 637-648. 5. Fisher, A. G., Ratner, L., Mitsuya, H., Marselle, L. M., Harper, M. E., Broder, S., Gallo, R. C. & Wong-Staal, F. (1986) Science 233, 655-659.
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6. Alizon, M., Wain-Hobson, S., Montagnier, L. & Sonigo, P. (1986) Cell 46, 63-74. 7. Saag, M. S., Hahn, B. H., Gibbons, J., Li, Y., Parks, E. S., Parks, W. P. & Shaw, G. M. (1988) Nature (London) 334, 4n40-44. 8. Cordonnier, A., Montagnier, L. & Emerman, M. (1989) Nature (London) 340, 571-574. 9. LaRosa, G. J., Davide, J. P., Weinhold, K., Waterbury, J. A., Profy, A. T., Lewis, J. A., Langlois, A. J., Dreesman, G. R., Boswell, R. N., Shadduck, P., Holley, L. H., Karplus, M., Bolognesi, D. P., Matthews, T. J., Emini, E. A. & Putney, S. D. (1990) Science 249, 932-935. 10. Smith, T. F., Srinivasan, A., Schochetman, G., Marcus, M. & Myers, G. (1988) Nature (London) 333, 573-575. 11. CDC (1991) Morbid. Mortal. Wkly. Rep. 40, 21-33. 12. Myers, G., Rabson, A., Josephs, S., Smith, T., Berzofsky, J. & Wong-Staal, F. (1990) Human Retroviruses and AIDS (Los Alamos National Lab., Los Alamos, NM). 13. Meyerhans, A., Cheynier, R., Albert, J., Seth, M., Kwok, S., Sninsky, J., Morfeldt-Manson, L., Asjo, B. & Wain-Hobson, S. (1989) Cell 58, 901-910. 14. Hirsch, V. M., Johnson, P. R. & Zack, P. M. (1990) in Vaccines 90: Modern Approaches to New Vaccines Including Prevention of AIDS, eds. Lemer, R., Ginsberg, H., Chanck, R. M. & Brown, F. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 379-382. 15. Goodenow, M., Huet, T., Saurin, W., Kwok, S., Sninsky, J. & Wain-Hobson, S. (1989) J. AIDS 2, 344-352. 16. Burger, H., Belman, A. L., Grimson, R., Kaell, A., Flaherty, K., Gulla, J., Gibbs, R., Nguyen, P.-N. & Weiser, B. (1990) Lancet 336, 134-136. 17. Barre-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T., Charmaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., BrunVezinet, F., Rouzioux, C., Rozenbaum, W. & Montagnier, L. (1983) Science 220, 868-870. 18. Gallo, R. C., Salahuddin, S. Z., Popovic, M., Shearer, G. M., Kaplan, M., Haynes, B. F., Palker, T. J., Redfield, R., Oleske,
19. 20. 21.
22. 23. 24.
25. 26. 27.
28.
29. 30.
J., Safai, B., White, G., Foster, P. & Markham, P. D. (1984) Science 224, 500-502. Gibbs, R. A., Nguyen, P.-N. & Caskey, C. T. (1991) in Methods in Molecular Biology, ed. Mathew, C. (Humana Press, Clifton, NJ), in press. Gibbs, R. A., Nguyen, P.-N., McBride, L. J., Koepf, S. M. & Caskey, C. T. (1989) Proc. Natd. Acad. Sci. USA 86, 19191923. Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B., Josephs, S. J., Doran, E. R., Ragalski, J. A., Whitehorn, E. A., Baumeister, K., Ivanoff, L., Petteway, S. R., Jr., Pearson, M. L., Lautenberger, J. A., Papas, T. S., Ghryeb, J., Chang, N. T., Gallo, R. C. & Wong-Staal, F. (1985) Nature (London) 313, 277-284. McNeatney, T., Westervelt, P., Thielan, B. J., Trowbridge, D. B., Garcia, J., Whittier, R. & Ratner, L. (1990) Proc. Natd. Acad. Sci. USA 87, 1917-1921. Balfe, P., Simmonds, P., Ludlam, C., Bishop, J. & Brown, A. J. L. (1990) J. Virol. 64, 6221-6233. Wolfs, T., deJong, J., van den Berg, H., Tunagel, J., Krone, W. & Goudsmit, J. (1990) Proc. Natl. Acad. Sci. USA 87, 99389942. Pizzo, P. A. (1990) J. Infect. Dis. 161, 316-325. Chin, J. (1990) Lancet 336, 221-224. Javaherian, K., Langlois, A., McDanal, C., Ross, K., Eckler, L., Jellis, C., Profy, A., Rusche, J., Boogmesi, D., Putney, S. & Matthews, M. (1989) Proc. Natd. Acad. Sci. USA 86, 6768-6772. Rossi, P., Moschese, V., Broliden, P., Fundaro, C., Quinti, I., Plebani, A., Giaquinto, C., Tovo, P., Ljunn, K., Rosen, J., Wigzeli, H., Jondal, M. & Wahren, B. (19B Proc. Natl. Acad. Sci. USA 86, 8055-8058. Devash, Y., Calvelli, T., Wood, D., Reagn, K. & Rubinstein, A. (1990) Proc. Natl. Acad. Sci. USA 87, 3445-3. Myers, G., Korber, B., Berzofsky, J. & Smith, R. (1991) Human Retroviruses and AIDS (Los Alamos National Lab., Los Alamos, NM).
