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Journal of Virology, May 1999, p. 3551-3559, Vol. 73, No. 5
Laboratory of Molecular Virology and
Epidemiology,
Received 27 April 1998/Accepted 19 January 1999
It has been suggested that immune-pressure-mediated positive
selection operates to maintain the antigenic polymorphism on the third
variable (V3) loop of the gp120 of human immunodeficiency virus type 1 (HIV-1). Here we present evidence, on the basis of sequencing 147 independently cloned env C2/V3 segments from a single
family (father, mother, and their child), that the intensity of
positive selection is related to the V3 lineage. Phylogenetic analysis
and amino acid comparison of env C2/V3 and gag
p17/24 regions indicated that a single HIV-1 subtype E source had
infected the family. The analyses of unique env C2/V3
clones revealed that two V3 lineage groups had evolved in the parents.
Group 1 was maintained with low variation in all three family members
regardless of the clinical state or the length of infection, whereas
group 2 was only present in symptomatic individuals and was more
positively charged and diverse than group 1. Only virus isolates
carrying the group 2 V3 sequences infected and induced syncytia in MT2 cells, a transformed CD4+-T-cell line. A statistically
significant excess of nonsynonymous substitutions versus synonymous
substitutions was demonstrated only for the group 2 V3 region. The data
suggest that HIV-1 variants, possessing the more homogeneous group 1 V3
element and exhibiting the non-syncytium-inducing phenotype, persist in
infected individuals independent of clinical status and appear to be
more resistant to positive selection pressure.
Nonsynonymous mutations in
protein-coding loci are often eliminated from the population because
they can cause deleterious effects on protein function or reduce the
fitness of the organisms. This functional constraint suppresses
nonsynonymous substitution (Ka), and synonymous
substitution (Ks) generally exceeds
Ka on protein-coding genes (32),
including the gag, pol, and env genes of human immunodeficiency virus type 1 (HIV-1) (21, 50). In a host-parasite relationship, however, new nonsynonymous mutations in a
particular region can often be maintained in the population, where they
confer a selective advantage on the organism (positive selection). The
existence of the positive selection force that increases amino acid
polymorphism was first noticed on the hypervariable regions of the
major histocompatibility complex and immunoglobulin heavy chain
(25, 26, 57) and soon afterwards for the surface antigen
sites of many pathogens, including HIV-1 (4, 18, 22, 30, 38, 50,
52, 53, 64, 67).
The third variable (V3) loop of the HIV-1 envelope gp120 is highly
immunogenic, and neutralizing antibodies from infected individuals can
recognize this region (45, 61). Therefore, it is conceivable
that new nonsynonymous mutations on the V3 loop-coding locus confer a
selective advantage on HIV-1 via avoidance of antibody recognition.
Consistently, Ka exceeds
Ks in the V3 region (4, 22, 49, 50, 52, 53,
64, 67), and Ka in the V3 region is
reported to correlate with the duration of the immunocompetent period
(33). These data suggest that immune-pressure-mediated positive selection is operating on the V3 region to maintain antigenic polymorphism.
The V3 loop, on the other hand, is a critical determinant to specify
coreceptor usage for HIV-1 entry (8, 9, 48a, 55). This
indicates that V3 sequence variation is influenced by a functional constraint as well as by positive selection. In this regard, V3 loop
amino acid sequences of non-syncytium-inducing (NSI)/macrophage-tropic variants consist of a less diverse and less positively charged population than that of syncytium-inducing (SI)/T-cell line-tropic variants (7, 17, 36, 37, 51, 65). The NSI variants generally
predominate in the asymptomatic period of HIV-1 infection in vivo and
use CCR5 as an entry coreceptor of infection (2, 3, 9-11,
14). In contrast, the SI variants often appear in association
with disease progression and acquire the ability to infect cells
expressing CXCR4 or other chemokine receptors (3, 10, 13, 16,
54).
In this study, we examined whether the intensity of positive selection
is related to NSI and SI V3 genotype. To address this issue, we
determined the nucleotide sequences of 147 independent V3 clones from
uncultured peripheral blood mononuclear cells (PBMCs) and from virus
isolates derived from an HIV-1 subtype E-infected Japanese family
(48), divided them into two subgroups based on the presence
of basic amino acid substitutions and on the extent of variation, and
ascertained the NSI and SI phenotypes of virus isolates bearing the two
V3 subpopulations in MT2 cell infection assays. Analyses of
Ka and Ks values within
each group at different sampling points or for different individuals
suggest that the V3 subpopulation for the NSI phenotype is maintained
with lower variation than that for the SI phenotype in infected
individuals and that positive selection operates less extensively on
the NSI V3 subpopulation. This is the first systematic comparison of
gp120 V3 sequence evolution with the biological changes in HIV-1
isolates, obtained following horizontal and vertical transmission in a
single family.
Epidemiologic and clinical information on the study
subjects.
The family consisted of a male index patient (NH1), the
female spouse of NH1 (NH2), and their child (NH3). NH1 had no history of blood transfusion, surgical operation, or homosexual activity. He
had a history of sexual contacts with female prostitutes in Thailand in
1989 and 1990. He was positive for HIV-1 antibodies in September 1992. He had chronic fatigue and lost 10 kg of weight between 1991 and 1992. He had developed AIDS-related pulmonary complications (Centers for
Disease Control and Prevention [CDC] category C3) at the time of
blood collection in June 1993 for the present study. The
CD4+ lymphocyte count was 40 × 106/liter
at the sampling time. NH1 died of AIDS-related pulmonary complications
in March 1994.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Evolution and Biological Characterization of Human
Immunodeficiency Virus Type 1 Subtype E gp120 V3 Sequences
following Horizontal and Vertical Virus Transmission in a
Single Family
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Nucleotide sequencing of HIV env C2/V3 and p17/p24 gag regions from uncultured PBMCs. Whole blood of the three family members was collected in June 1993. For NH2, blood specimens were also collected in March 1996 (NH2-II) and January 1997 (NH2-III). PBMCs were isolated by Ficoll-Hypaque (Pharmacia LKB, Uppsala, Sweden) density centrifugation, and an env V3 region and flanking regions (324 bp) were amplified from the PBMCs by nested PCR. The first amplification step was performed for 30 cycles with outer primers MK369 and MK616 (43). Five microliters of the first PCR product were used for the second amplification step with inner primers V3-A and V3-C (23). The PCR products were cloned directly into pCRII cloning vectors (Invitrogen, NV Leek, The Netherlands). Fifteen to 22 of the cloned DNAs for each infected individual at a sampling point were sequenced on both strands with either an ALFII automated DNA sequencer (Pharmacia LKB) or an ABI PRISM 310 automated sequencer (Perkin-Elmer, Norwalk, Conn.).
A region covering a part of the long terminal repeat U5, the whole p17 gag gene, and a part of the p24 gag gene (520 bp) was amplified by nested PCR from uncultured PBMCs in June 1993. The outer primers were JA152 and JA155, and the inner primers were JA153 and JA154 (31). After the PCR, the primers were removed from the PCR products with a Centricon-100 (Amicon). The purified PCR products were sequenced directly on both strands with an ABI PRISM 310 automated sequencer.HIV isolation, env C2/V3 nucleotide sequencing, and infection of MT2 cells. Virus isolation was achieved by coculture of freshly isolated PBMCs (5 × 106) from the family members with an equal number of phytohemagglutinin (PHA)-stimulated PBMCs from HIV-1-seronegative individuals. At 2- to 3-day intervals, the cells were fed with RPMI 1640 with 10% fetal bovine serum and 20 U of recombinant interleukin-2/ml. At 1-week intervals, fresh PHA-stimulated PBMCs were added to the culture. Culture supernatants were taken for reverse transcriptase (RT) assay (63) every 2 to 3 days during a 40-day period after coculture.
Total RNA was extracted from 50 µl of the RT-positive culture supernatant (5), and one-fifth of the RNA was subjected to synthesis of complementary DNA with the primer C3E-130B, 5'-AGA AAA ATT CCC CTC TAC AAT TAA-3', and avian myeloblastosis virus RT (Takara Shuzo Co., Kyoto, Japan) followed by PCR with primers C2E-110A, 5'-TTC AAT GGG ACA GGG CCA TGT-3', and C3E-130B. The amplified DNAs corresponding to the HIV-1 env C2/V3 region (324 bp) were either sequenced directly or after cloning into pCRII, as described above. A transformed CD4+-T-cell line, MT2 (2 × 105 cells), was incubated in 0.2 to 0.3 ml of medium containing 2 × 105 to 5 × 105 cpm of 32P-RT activity (63) for 12 h at 37°C, and the cells were grown in 1 to 2 ml of the corresponding medium in 24-well plates. Half of the volume of the culture medium was replaced by fresh medium every 2 to 3 days, and a portion was used for RT assay. The cultures were terminated 25 days after infection, during which time syncytium formation was monitored under a light microscope.Analyses of molecular evolution of HIV sequences. For the reference group and the outgroup of the phylogenetic tree, corresponding sequences of HIV-1 subtypes A through H and simian immunodeficiency virus (SIV)CPZGAB (CPZGAB) were collected from the Los Alamos National Laboratory database (39). The nucleotide sequences were aligned with sequences from the NH family with CLUSTAL W version 1.7 (60). The alignment was corrected manually to ensure that gaps did not alter the reading frame. A distance matrix of nucleotide substitutions was estimated from the alignment according to the method of Tajima and Nei (56), and phylogenetic trees were constructed from the matrix by the neighbor-joining method (47) with the NEIGHBOR program of the PHYLIP package version 3.5c (15a). Bootstrap analyses (15) were done for the tree with the DNABOOT and CONSENCE programs of the PHYLIP package, with 100 resamplings. The trees were rooted by CPZGAB or 18 available V3 sequences of HIV-1 subtype E from Thailand, the Central African Republic, and England (39) and were drawn with NJPLOT. The maximum-likelihood and the parsimonious trees were constructed from aligned sequences with the DNAML and DNAPARS programs (J. Felsenstein), respectively. For DNAML, the value for the transition/transversion ratio was set at 1.5 (24). Only the neighbor-joining trees are presented in this article, because trees generated by the other two algorithms gave the same topology.
Nucleotide substitution per site was estimated with PHYLIP for each pairwise sequence comparison on the basis of the Kimura two-parameter method (28). Nonsynonymous and synonymous substitutions (Ka and Ks) were estimated according to the method of Nei and Gojobori (40) with MEGA version 1.0 (29a). Means and variances of Ka and Ks for all pairwise comparisons of a sample population were calculated according to the method of Nei and Jin (41), in which patristic distances were evaluated by unweighted pair group method with arithmetic mean (UPGMA) trees from each value calculated by the NEIGHBOR program. Welch's t test was used to evaluate the statistical significance of differences between Ka and Ks values (30).Nucleotide sequence accession numbers. The DDBJ database accession numbers for the nucleotide sequences reported here are D78024 to D78070 and AB014775 to AB014874 for the env C2/V3 region and AB015938 to AB015942 for the gag p17/p24 region.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Genetic links of HIV-1 within the NH family. Epidemiological information suggested that HIV-1 infections in the NH family were caused by intrafamilial transmission of viruses of Thai origin (see Materials and Methods). To ascertain whether the HIV-1 quasispecies in the family were evolutionarily closely related, we determined the phylogenetic relationships of the env C2/V3 and p17 gag sequences (1, 24, 42) from the family.
Fifteen to 17 clonal nucleotide sequences were determined for the env C2/V3 region (324 bp) from uncultured PBMCs of each infected individual in June 1993. Figure 1A shows the neighbor-joining tree with the sequences from the family and the reference sequences of HIV-1 group M (subtypes A through G) (39). The tree shows that the env C2/V3 sequences from the family form a monophyletic group (bootstrap value, 81/100). This family cluster was most closely related to HIV-1 subtype E sequences from Thailand (TN2432 and 92TH022.4; bootstrap value, 100/100), whereas it was distant from subtype E from the Central African Republic (CAR4017 and CAR4071) and from the other HIV-1 subtypes. The monophyletic relationship of the family sequences was reproducible when 18 of the other reported HIV-1 subtype E sequences from Thailand, the Central African Republic, and England (39) were included in the tree or when the trees were constructed with other algorithms, including parsimony and maximum likelihood (data not shown).
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env V3 genotype of provirus in uncultured PBMCs of the family. Epidemiological information suggested that NH1 was infected in Thailand during the early epidemic of subtype E infections in the country (62). Subtype E env C2/V3 sequences in this period were highly homogeneous among asymptomatic carriers (12, 27, 34, 43). This enabled us to generate the NSI consensus of the subtype E V3 loop from 21 NSI virus isolates from the early 1990s in Thailand (12, 27). To delineate NSI and SI structural characteristics of the V3 loops from the family members, deduced amino acid sequences were compared to the consensus.
The V3 loop sequences from NH1 with AIDS could be divided into two groups (Fig. 2A, NH1). One population (group 1) was characterized by the presence of a GPGQ motif at the tip of the V3 loop and by the lack of basic amino acid substitutions with respect to the consensus. Group 1 consisted of a minor population (4 of 17) in NH1, whereas it represented the only population detected in the asymptomatic carriers, NH2 (15 of 15) and NH3 (15 of 15) (Fig. 2A, NH2 and NH3). The group 1 sequences were similar to each other or to the NSI consensus. The three family members shared variants carrying an identical V3 loop sequence of group 1 (Fig. 2A).
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env V3 genotype and MT2 tropism of HIV-1 isolates from the family. HIV-1 isolates were obtained from the family, and their V3 nucleotide sequences and abilities to induce syncytia on MT2 cells were determined. A virus isolate from NH1 (HIV-1NH1) in June 1993 had a group 2 V3 genotype with a GPGR motif and basic substitutions at positions 8, 11, and 18 (Fig. 2B, NH1, PBMC). A major population in HIV-1NH1 (Fig. 2B, NH1) was similar to a sequence in uncultured PBMCs of NH1 (Fig. 2A, NH1). No sequences with a GPGQ motif were identified in HIV-1NH1. HIV-1NH1 replicated in PHA-stimulated PBMCs and MT2 cells. RT activity appeared in the MT2 culture supernatant on day 9 after infection and peaked on day 12, and concomitantly syncytia were observed in the culture. V3 sequences in the supernatant of RT-positive MT2 culture also contained a GPGR motif and basic substitutions at positions 8, 11, and 18 (Fig. 2B, NH1, MT2). All of the 58 C2/V3 nucleotide sequences from the virus isolates clustered with the sequences from uncultured PBMCs under the same node in the neighbor-joining tree (data not shown).
Virus isolates from NH2 and NH3 (HIV-1NH2 and HIV-1NH3) in June 1993 had a group 1 V3 genotype with a GPGQ motif and a low net positive charge (Fig. 2B, NH2 and NH3). Major V3 sequence populations in uncultured PBMCs of NH2 and NH3 were still dominant in HIV-1NH2 and HIV-1NH3. The virus isolates grew in PHA-stimulated PBMCs, whereas MT2 was nonpermissive for these isolates: no RT activity in culture supernatant and no syncytium formation were detected during a 25-day cultivation after infection.Nucleotide diversity in env C2/V3 region and PCR
fidelity.
Table 1 summarizes the
number of env C2/V3 nucleotide sequences obtained from
uncultured PBMCs of the NH family in 1993, 1996, and 1997, along with
the nucleotide diversity of the group 1 and group 2 quasispecies. Among
a total of 89 C2/V3 clones sequenced, 87 clones had distinct nucleotide
sequences over the entire 324-bp region and 86 clones had open reading
frames. Among the 86 clones, 51 and 35 sequences encoded group 1 and
group 2 V3 loops, respectively. The mean nucleotide substitution values
per site (28) in the C2/V3 segments were slightly
lower for group 1 than for group 2, i.e., they ranged from
0.006 ± 0.003 to 0.020 ± 0.010 for group 1 and from
0.014 ± 0.011 to 0.041 ± 0.017 for group 2. The total nucleotide diversity in each patient was higher in individuals with
AIDS (NH1, NH2-II, and NH2-III) than in asymptomatic carriers (NH2 and
NH3).
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C, AT, and three AG) in five
different clones were identified, all of which were nonsynonymous
substitutions located at different positions. Within 19 clones from a
103-copy template (6,156 bases sequenced), four
substitutions (TC and three AG) were noted, three of which were
nonsynonymous substitutions and one of which was a synonymous
substitution, mapped to different positions in the four different
clones. The estimated Taq polymerase error rates under our
experimental conditions were 6.5 × 10
4 to 7.3 × 104 mutation frequency/bp/60 cycles. The values were
comparable to previous results obtained for the HIV-1 tat
region (3.2 × 104 mutation frequency/bp/35 cycles)
(35). Thus, the contribution of Taq polymerase to
the frequency of base substitutions observed in the present study
(Table 1) was less than 12%.
V3 sequence variation and Ka/Ks values. A neighbor-joining tree was constructed with the V3 nucleotide sequences (105 bp) of the independent C2/V3 clones (Fig. 3). The tree was rooted by 18 of the HIV-1 subtype E V3 sequences from Thailand and the Central African Republic (39). The tree was divided into two major clusters showing distinct branch lengths. The clustering pattern was reproducible regardless of the algorithms used for constructing trees (data not shown). Sequences of one cluster had shorter branches than did those of the other, suggesting the presence of a sibling sequence population that was less diversified from a putative ancestral V3 sequence of the intrafamilial infection. Notably, the cluster with shorter branches consisted of sequences from all clinical stages (NH1, NH2, NH2-II, NH2-III, and NH3), and they all encoded the group 1 V3 loop with a GPGQ motif and a low positive net charge. In contrast, the cluster with longer branches consisted of only those sequences from patients with AIDS (NH1, NH2-II, and NH2-III), and they all encoded the group 2 V3 loop with a GPGR motif and a high positive net charge. When the neighbor-joining tree was constructed on the flanking sequences of the V3 loop, no relation was detected between the branch length and the group 1 or 2 lineages (data not shown).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
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Discussion
References
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Compared to the mechanisms to yield new mutants via error-prone viral RT, genetic recombination, and rapid turnover of virus in vivo, the selection process that maintains genetic variation is less well understood. The present work has provided for the first time evidence that the intensity of positive selection may be related to the V3 lineage.
To understand V3 evolution in the Japanese family NH, we started to examine the probable infection pathway of virus. Two independent pieces of evidence support the possibility that HIV-1 isolates in the Japanese family had evolved from a common source. First, all of the env and gag nucleotide sequences from the three family members were of HIV-1 subtype E and had a monophyletic relationship (Fig. 1). Second, all of the HIV Env and Gag sequences from this family possessed amino acid residues unique to the family. Taken together with the epidemiological and clinical information, as well as the nucleotide diversity of the env C2/V3 region (Table 1), this evidence caused us to conclude that a single source of HIV-1 subtype E of Thai origin had infected NH1, who had transmitted the viruses to NH2, who had transmitted the viruses to NH3 at some time between 1989 and 1992.
Among the V3 loop amino acid sequences from the family, two major subgroups, group 1 with a GPGQ motif and group 2 with a GPGR motif, were identified (Fig. 2). Structural similarities to the subtype E NSI consensus, predominance in asymptomatic mother and child, and the identity to the sequences of NSI virus isolates from the family all indicate that the group 1 V3 loops are those of NSI variants. Of note is the fact that the group 1 sequences were identified in NH1 with AIDS and were consistently found after NH2 had developed AIDS, suggesting that this quasispecies can persist in infected individuals independent of clinical status. Group 2, on the other hand, was only present in symptomatic individuals and shared many structural features with the reported sequences of the subtype E SI variants. Consistently, virus isolates carrying the group 2 V3 sequences infected and induced syncytia in MT2 cells. Thus, at least two V3 lineage groups had evolved in the two parents during disease progression, one of which was related to the NSI phenotype and the other to the SI phenotype.
Interestingly, all of the group 1 sequences showed relatively low variation irrespective of the clinical status of the infected individuals or the length of infection, whereas all of the group 2 sequences formed a cluster with long branches and were identified only in patients with AIDS (Fig. 3). The low variation for the group 1 sequences were unlikely to be due to template resampling during PCR and cloning processes, because (i) most group 1 sequences were unique over the entire 324-bp C2/V3 region sequenced (Table 1), (ii) Taq polymerase error contributed less than 12% to the observed substitution frequencies in the C2/V3 region, and (iii) the relatively high degree of sequence homogeneity was seen mainly in the V3 loop and not in the flanking regions (48). An alternative and more likely explanation is that the HIV-1 variants possessing group 1 V3 loops existed as a quasispecies with a relatively homogeneous V3 loop element. The conclusion is compatible with the general observation that V3 loop sequences of NSI isolates consist of a less diverse population than those of SI isolates (7, 17, 29, 36, 37, 51, 65).
The difference in variations in the group 1 and group 2 sequence populations could have been caused by two factors. First, mutation rates may be higher in the group 2 V3 region than in the group 1 V3 region. Second, there may be a stronger positive selection in the group 2 V3 sequence population than in that of group 1. Although the former possibility needs to be assessed further, we obtained evidence that the latter mechanism is involved in generating the genotype-dependent difference in V3 variation. Calculation and plotting of Ka and Ks values for each pairwise comparison within the same V3 lineage group revealed that group 1 and group 2 exhibited distinct distribution patterns of the Ks/Ka plots. In group 2, Ka exceeded Ks for most pairs (Fig. 4). Statistical tests of the difference between means of Ka and Ks ruled out the possibility that this observation was due to chance (Table 2). These results strongly argue against the plain model where the group 2 nucleotide diversity would be caused simply by the high error rate of RT or by the rapid turnover of the viruses in patients with little immune response. Although these possibilities alone may increase the nucleotide diversity (Ka + Ks), they should not cause the excess of Ka over Ks because of the functional constraint on the protein-coding locus (32). Thus, the present data indicate that there had been a driving force which increased the probability of fixing nonsynonymous substitutions during the evolution of the group 2 lineage.
In contrast to the group 2 population, Ks exceeded Ka for most pairs in group 1. The Ka/Ks ratios were consistently less than 1.0 in group 1, except at one sampling point, where the difference between Ka and Ks values was not statistically significant. In addition, the mean Ka value for group 1 was consistently lower than that for group 2 at the same sampling point of the same individual. These data indicate that positive selection did not play a major role in the nucleotide diversity of the group 1 V3 lineage. The conclusion is compatible with the observation that the V3 region of NSI gp120 appears to be hidden from the blocking antibodies, being a less effective target for sera of HIV-1-infected individuals (6). In addition, the less rapid and efficient progeny production of NSI than SI variants (58, 59) might have resulted in the smaller extent of antigen presentation or recognition by host immune surveillance. Thus, the differences in intensity of positive selection among the group 1 and 2 V3 lineages may be attained by differences in susceptibility to the host immune responses.
The above-mentioned differences may all be attributed primarily to the differential abilities of viruses to use entry coreceptors. The V3 region has been implicated in the interaction of HIV-1 envelope with chemokine receptors expressed on the target cells (8, 9, 48a, 55). The ability to interact with a particular molecule would become a strong functional constraint which decreases the amino acid variation of the responsible region. However, once the viruses obtain the competence, V3 diversity would be maintained by a balance between the functional constraint and the immune pressure driving positive selection. Experiments assessing the ability of each group 1 or group 2 V3 loop to specify CCR5 and/or CXCR4 usage are currently in progress by constructing a series of infectious V3 recombinants.
Group 2 V3 loop amino acid sequences from NH1, NH2-II, and NH2-III frequently shared unique amino acids, which were not seen in group 1 (Fig. 2A). Either convergent evolution or the direct transmission of SI variants from NH1 to NH2 can explain the observation. In this regard, it is likely that viruses transmitted from NH1 to NH2 were the NSI variants, because neither SI V3 genotypes nor SI viruses were identified when NH2 was asymptomatic (Fig. 2). Therefore, the similarities between groups 2 in the two individuals appear to imply that there is a convergent evolutionary pattern in the V3 loop in the context of subtype E gp120 during disease progression. The possibility is supported by the observation that many of these substitutions also recurred in AIDS patients in Thailand (66), regardless of the predominant role of NSI variants for person-to-person transmission (46, 68).
In conclusion, HIV-1 V3 sequence evolution appears to be bimodal in an infected individual; a V3 sequence lineage for NSI phenotype persists independent of clinical status and is maintained with a relatively low variation due to the dominant role of the functional constraint. In contrast, a V3 sequence lineage for SI phenotype emerges with disease progression and is maintained with a high variation, because of the dominant role of positive selection.
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ACKNOWLEDGMENTS |
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We thank Simeon Aidoo for help with sequencing and Keith Peden for providing pLAI.
This work was supported by grants from the Ministry of Health and Welfare of Japan, the Ministry of Education, Science and Culture of Japan, and the Science and Technology Agency of the Japanese Government.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Molecular Virology and Epidemiology, AIDS Research Center, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku, Tokyo 162-8640, Japan. Phone: (81)-3-52851111. Fax: (81)-3-52851177. E-mail: hirosato{at}nih.go.jp
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REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Albert, J.,
J. Wahlberg,
T. Leitner,
D. Escanilla, and M. Uhlen.
1994.
Analysis of a rape case by direct sequencing of the human immunodeficiency virus type 1 pol and gag genes.
J. Virol.
68:5918-5924 |
| 2. |
Alkhatib, G.,
C. Combadiere,
C. Broder,
Y. Feng,
P. Kennedy,
P. Murphy, and E. Berger.
1996.
CC CKR5: a RANTES, MIP-1  , MIP-1  receptor as a fusion cofactor for macrophage-tropic HIV-1.
Science
272:1955-1958[Abstract].
|
| 3. | Bjorndal, A., H. Deng, M. Jansson, J. Fiore, C. Colognesi, A. Karlsson, J. Albert, G. Scarlatti, D. Littman, and E. Fenyo. 1997. Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J. Virol. 71:7478-7487[Abstract]. |
| 4. | Bonhoeffer, S., E. C. Holmes, and M. A. Nowak. 1995. Causes of HIV diversity. Nature 376:125[Medline]. |
| 5. |
Boom, R.,
C. J. A. Sol,
M. M. M. Salimans,
C. L. Jansen,
P. M. E. Wertheim-van Dillen, and J. van der Noordaa.
1990.
Rapid and simple method for purification of nucleic acids.
J. Clin. Microbiol.
28:495-503 |
| 6. |
Bou-Habib, D.,
G. Roderiquez,
T. Oravecz,
P. Berman,
P. Lusso, and M. Norcross.
1994.
Cryptic nature of envelope V3 region epitopes protects primary monocytotropic human immunodeficiency virus type 1 from antibody neutralization.
J. Virol.
68:6006-6013 |
| 7. |
Chesebro, B.,
K. Wehrly,
J. Nishio, and S. Perryman.
1992.
Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism.
J. Virol.
66:6547-6554 |
| 8. |
Cho, M. W.,
M. K. Lee,
M. C. Carney,
J. F. Berson,
R. W. Doms, and M. A. Martin.
1998.
Identification of determinants on a dualtropic human immunodeficiency virus type 1 envelope glycoprotein that confer usage of CXCR4.
J. Virol.
72:2509-2515 |
| 9. | Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu, C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C. Gerard, and J. Sodroski. 1996. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135-1148[Medline]. |
| 10. |
Connor, R.,
K. Sheridan,
D. Ceradini,
S. Choe, and N. Landau.
1997.
Change in coreceptor use correlates with disease progression in HIV-1-infected individuals.
J. Exp. Med.
185:621-628 |
| 11. | Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di Marzio, S. Marmon, R. Sutton, C. Hill, C. Davis, S. Peiper, T. Schall, D. Littman, and N. Landau. 1996. Identification of a major co-receptor for primary isolates of HIV-1. Nature 381:661-666[Medline]. |
| 12. | De Wolf, F., E. Hogervorst, J. Goudsmit, E. M. Fenyo, H. Rubsamen-Waigmann, H. Holmes, B. Galvao-Castro, E. Karita, C. Wasi, S. D. Sempala, E. Baan, F. Zorgdrager, V. Lukashov, S. Osmanov, C. Kuiken, M. Cornelissen, and the World Health Organization Network for HIV Isolation and Characterization. 1994. Syncytium-inducing and non-syncytium-inducing capacity of human immunodeficiency virus type 1 subtypes other than B: phenotypic and genotypic characteristics. AIDS Res. Hum. Retroviruses 10:1387-1400[Medline]. |
| 13. |
Doranz, B.,
J. Rucker,
Y. Yi,
R. Smyth,
M. Samson,
S. Peiper,
M. Parmentier,
R. Collman, and R. Doms.
1996.
A dual-tropic primary HIV-1 isolate that uses fusin and the -chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors.
Cell
85:1149-1158[Medline].
|
| 14. | Dragic, T., V. Litwin, G. Allaway, S. Martin, Y. Huang, K. Nagashima, C. Cayanan, P. Maddon, R. Koup, J. Moore, and W. Paxton. 1996. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667-673[Medline]. |
| 15. | Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783-791. |
| 15a. | Felsenstein, J. 1993. PHYLIP (phylogeny inference package), version 3.5c. Department of Genetics, University of Washington, Seattle, Wash. |
| 16. | Feng, Y., C. Broder, P. Kennedy, and E. Berger. 1996. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872-877[Abstract]. |
| 17. |
Fouchier, R. A. M.,
M. Groenink,
N. A. Kootstra,
M. Tersmette,
H. G. Huisman,
F. Miedema, and H. Schuitemaker.
1992.
Phenotype-associated sequence variation in the third variable domain of the human immunodeficiency virus type 1 gp120 molecule.
J. Virol.
66:3183-3187 |
| 18. | Ganeshan, S., R. E. Dickover, B. T. Korber, Y. J. Bryson, and S. M. Wolinsky. 1997. Human immunodeficiency virus type 1 genetic evolution in children with different rates of development of disease. J. Virol. 71:663-677[Abstract]. |
| 19. |
Gao, F.,
D. L. Robertson,
S. G. Morrison,
H. Hui,
S. Craig,
J. Decker,
P. N. Fultz,
M. Girard,
G. M. Shaw,
B. H. Hahn, and P. M. Sharp.
1996.
The heterosexual human immunodeficiency virus type 1 epidemic in Thailand is caused by an intersubtype (A/E) recombinant of African origin.
J. Virol.
70:7013-7029 |
| 20. | Gao, F., L. Yue, S. Craig, C. L. Thornton, D. L. Robertson, F. E. McCutchan, J. A. Bradac, P. M. Sharp, B. H. Hahn, and the WHO Network for HIV Isolation and Characterization. 1994. Genetic variation of HIV type 1 in four World Health Organization-sponsored vaccine evaluation sites: generation of functional envelope (glycoprotein 160) clones representative of sequence subtypes A, B, C, and E. AIDS Res. Hum. Retroviruses 10:1359-1368[Medline]. |
| 21. |
Gojobori, T.,
E. Moriyama, and M. Kimura.
1990.
Molecular clock of viral evolution, and the neutral theory.
Proc. Natl. Acad. Sci. USA
87:10015-10018 |
| 22. | Gojobori, T., Y. Yamaguchi, K. Ikeo, and M. Mizokami. 1994. Evolution of pathogenic viruses with special reference to the rates of synonymous and nonsynonymous substitutions. Jpn. J. Genet. 69:481-488[Medline]. |
| 23. | Hattori, T., K. Shiozaki, Y. Eda, S. Tokiyoshi, S. Matsushita, H. Inaba, M. Fujimaki, T. Meguro, K. Yamada, M. Honda, K. Nishikawa, and K. Takatsuki. 1991. Characteristics of the principal neutralizing determinant of HIV-1 prevalent in Japan. AIDS Res. Hum. Retroviruses 7:825-830[Medline]. |
| 24. | Holmes, E. C., L. Q. Zhang, P. Simmonds, A. S. Rogers, and A. J. Brown. 1993. Molecular investigation of human immunodeficiency virus (HIV) infection in a patient of an HIV-infected surgeon. J. Infect. Dis. 167:1411-1414[Medline]. |
| 25. | Hughes, A., and M. Nei. 1988. Pattern of nucleotide substitution at major histocompatibility complex class I loci reveals overdominant selection. Nature 335:167-170[Medline]. |
| 26. |
Hughes, A., and M. Nei.
1989.
Nucleotide substitution at major histocompatibility complex class II loci: evidence for overdominant selection.
Proc. Natl. Acad. Sci. USA
86:958-962 |
| 27. | Ichimura, H., S. Kliks, S. Visrutaratna, C.-Y. Ou, M. Kalish, and J. Levy. 1994. Biological, serological, and genetic characterization of HIV-1 subtype E isolates from northern Thailand. AIDS Res. Hum. Retroviruses 10:263-269[Medline]. |
| 28. | Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120[Medline]. |
| 29. |
Kuiken, C. L.,
J.-J. de Jong,
E. Baan,
W. Keulen,
M. Tersmette, and J. Goudsmit.
1992.
Evolution of the V3 envelope domain in proviral sequences and isolates of human immunodeficiency virus type 1 during transition of the viral biological phenotype.
J. Virol.
66:4622-4627 |
| 29a. | Kumar, S., K. Tamura, and M. Nei. 1993. MEGA: molecular evolutionary genetics analysis. The Pennsylvania State University, University Park, Pa. |
| 30. |
Lamers, S.,
J. Sleasman,
J. She,
K. Barrie,
S. Pomeroy,
D. Barrett, and M. Goodenow.
1993.
Independent variation and positive selection in env V1 and V2 domains within maternal-infant strains of human immunodeficiency virus type 1 in vivo.
J. Virol.
67:3951-3960 |
| 31. | Leitner, T., D. Escanilla, S. Marquina, J. Wahlberg, C. Brostrom, H. B. Hansson, M. Uhlen, and J. Albert. 1995. Biological and molecular characterization of subtype D, G, and A/D recombinant HIV-1 transmissions in Sweden. Virology 209:136-146[Medline]. |
| 32. | Li, W.-H., and D. Graur. 1991. Fundamentals of molecular evolution. Sinauer Associates, Inc., Sunderland, Mass. |
| 33. | Lukashov, V., C. Kuiken, and J. Goudsmit. 1995. Intrahost human immunodeficiency virus type 1 evolution is related to length of the immunocompetent period. J. Virol. 69:6911-6916[Abstract]. |
| 34. | McCutchan, F. E., P. A. Hegerich, T. P. Brennan, P. Phanuphak, P. Singharaj, A. Jugsudee, P. W. Berman, A. M. Gray, A. K. Fowler, and D. S. Burke. 1992. Genetic variants of HIV-1 in Thailand. AIDS Res. Hum. Retroviruses 8:1887-1895[Medline]. |
| 35. | Meyerhans, A., R. Cheynier, J. Albert, M. Seth, S. Kwok, J. Sninsky, L. Morfeldt-Manson, B. Asjo, and S. Wain-Hobson. 1989. Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations. Cell 58:901-910[Medline]. |
| 36. |
Milich, L.,
B. Margolin, and R. Swanstrom.
1993.
V3 loop of the human immunodeficiency virus type 1 Env protein: interpreting sequence variability.
J. Virol.
67:5623-5634 |
| 37. | Milich, L., B. Margolin, and R. Swanstrom. 1997. Patterns of amino acid variability in NSI-like and SI-like V3 sequences and a linked change in the CD4-binding domain of the HIV-1 Env protein. Virology 239:108-118[Medline]. |
| 38. |
Mindell, D.
1996.
Positive selection and rates of evolution in immunodeficiency viruses from humans and chimpanzees.
Proc. Natl. Acad. Sci. USA
93:3284-3288 |
| 39. | Myers, G., B. Korber, B. H. Hahn, K.-T. Jeang, J. W. Mellors, F. E. McCutchan, L. E. Henderson, and G. N. Pavlakis. 1995. Human retroviruses and AIDS 1995: a compilation and analysis of nucleic acid and amino acid sequences. Los Alamos National Laboratory, Los Alamos, N.Mex. |
| 40. | Nei, M., and T. Gojobori. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3:418-426[Abstract]. |
| 41. | Nei, M., and L. Jin. 1989. Variances of the average numbers of nucleotide substitutions within and between populations. Mol. Biol. Evol. 6:290-300[Abstract]. |
| 42. |
Ou, C.-Y.,
C. A. Ciesielski,
G. Myers,
C. I. Bandea,
C.-C. Luo,
B. T. M. Korber,
J. I. Mullins,
G. Schochetman,
R. L. Berkelman,
A. N. Economou,
J. J. Witte,
L. J. Furman,
G. A. Satten,
K. A. MacInnes,
J. W. Curran,
H. W. Jaffe,
L. I. Group, and E. I. Group.
1992.
Molecular epidemiology of HIV transmission in a dental practice.
Science
256:1165-1171 |
| 43. | Ou, C.-Y., Y. Takebe, B. G. Weniger, C.-C. Luo, M. L. Kalish, W. Auwanit, S. Yamazaki, H. D. Gayle, N. L. Young, and G. Schochetman. 1993. Independent introduction of two major HIV-1 genotypes into distinct high-risk populations in Thailand. Lancet 341:1171-1174[Medline] (Erratum, 342:250.) |
| 44. | Peden, K., M. Emerman, and L. Montagnier. 1991. Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virology 185:661-672[Medline]. |
| 45. | Profy, A., P. Salinas, L. Eckler, N. Dunlop, P. Nara, and S. Putney. 1990. Epitopes recognized by the neutralizing antibodies of an HIV-1-infected individual. J. Immunol. 144:4641-4647[Abstract]. |
| 46. | Roos, M. T. L., J. M. A. Lange, R. E. Y. Goede, R. A. Coutinho, P. T. A. Schellekens, F. Miedema, and M. Tersmette. 1992. Viral phenotype and immune response in primary human immunodeficiency virus type 1 infection. J. Infect. Dis. 165:427-432[Medline]. |
| 47. | Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425[Abstract]. |
| 48. | Sato, H., K. Taniguchi, Y. Tomita, T. Shiino, T. Miyakuni, and Y. Takebe. 1997. Evidence for the selective pressure to reduce heterogeneity of HIV-1 subtype E envelope V3-loop sequences in an intrafamilial infection case. AIDS 11:396-397[Medline]. |
| 48a. | Sato, H., K. Kato, and Y. Takebe. Functional complementation of the envelope hypervariable V3 loop of human immunodeficiency virus type 1 subtype B by the subtype E V3 loop. Virology, in press. |
| 49. | Scarlatti, G., E. Tresoldi, A. Bjorndal, R. Fredriksson, C. Colognesi, H. Deng, M. Malnati, A. Plebani, A. Siccardi, D. Littman, E. Fenyo, and P. Lusso. 1997. In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression. Nat. Med. 3:1259-1265[Medline]. |
| 50. | Seibert, S., C. Howell, M. Hughes, and A. Hughes. 1995. Natural selection on the gag, pol, and env genes of human immunodeficiency virus 1 (HIV-1). Mol. Biol. Evol. 12:803-813[Abstract]. |
| 51. | Shpaer, E., E. Delwart, C. Kuiken, J. Goudsmit, M. Bachmann, and J. Mullins. 1994. Conserved V3 loop sequences and transmission of human immunodeficiency virus type 1. AIDS Res. Hum. Retroviruses 10:1679-1684[Medline]. |
| 52. | Shpaer, E. G., and J. I. Mullins. 1993. Rates of amino acid change in the envelope protein correlate with pathogenicity of primate lentiviruses. J. Mol. Evol. 37:57-65[Medline]. |
| 53. |
Simmonds, P.,
P. Balfe,
C. Ludlam,
J. Bishop, and A. Brown.
1990.
Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1.
J. Virol.
64:5840-5850 |
| 54. | Simmons, G., D. Wilkinson, J. Reeves, M. Dittmar, S. Beddows, J. Weber, G. Carnegie, U. Desselberger, P. Gray, R. Weiss, and P. Clapham. 1996. Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry. J. Virol. 70:8355-8360[Abstract]. |
| 55. | Speck, R., K. Wehrly, E. Platt, R. Atchison, I. Charo, D. Kabat, B. Chesebro, and M. Goldsmith. 1997. Selective employment of chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids within the envelope V3 loop. J. Virol. 71:7136-7139[Abstract]. |
| 56. | Tajima, F., and M. Nei. 1984. Estimation of evolutionary distance between nucleotide sequences. Mol. Biol. Evol. 1:269-285[Abstract]. |
| 57. | Tanaka, T., and M. Nei. 1989. Positive Darwinian selection observed at the variable-region genes of immunoglobulins. Mol. Biol. Evol. 6:447-459[Abstract]. |
| 58. |
Tersmette, M.,
R. E. Y. de Goede,
B. J. M. Al,
I. N. Winkel,
R. A. Gruters,
H. T. Cuypers,
H. G. Huisman, and F. Miedema.
1988.
Differential syncytium-inducing capacity of human immunodeficiency virus isolates: frequent detection of syncytium-inducing isolates in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex.
J. Virol.
62:2026-2032 |
| 59. |
Tersmette, M.,
R. A. Gruters,
F. De Wolf,
R. E. Y. De Goede,
J. M. A. Lange,
P. T. A. Schellekens,
J. Goudsmit,
H. G. Huisman, and F. Miedema.
1989.
Evidence for a role of virulent human immunodeficiency virus (HIV) variants in the pathogenesis of acquired immunodeficiency syndrome: studies on sequential HIV isolates.
J. Virol.
63:2118-2125 |
| 60. |
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680 |
| 61. | Vogel, T., R. Kurth, and S. Norley. 1994. The majority of neutralizing Abs in HIV-1-infected patients recognize linear V3 loop sequences. Studies using HIV-1MN multiple antigenic peptides. J. Immunol. 153:1895-1904[Abstract]. |
| 62. | Weniger, B. G., Y. Takebe, C.-Y. Ou, and S. Yamazaki. 1994. The molecular epidemiology of HIV in Asia. AIDS 8(Suppl. 2):S13-S28. |
| 63. |
Willey, R. L.,
D. H. Smith,
L. A. Lasky,
T. S. Theodore,
P. L. Earl,
B. Moss,
D. J. Capon, and M. A. Martin.
1988.
In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity.
J. Virol.
62:139-147 |
| 64. | Wolinsky, S. M., B. T. Korber, A. U. Neumann, M. Daniels, K. J. Kunstman, A. J. Whetsell, M. R. Furtado, Y. Cao, D. D. Ho, J. T. Safrit, and R. A. Koup. 1996. Adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection. Science 272:537-542[Abstract]. |
| 65. | Yamashita, A., N. Yamamoto, J. Matsuda, and Y. Koyanagi. 1994. Cell type-specific heterogeneity of the HIV-1 V3 loop in infected individuals: selection of virus in macrophages and plasma. Virology 204:170-179[Medline]. |
| 66. | Yu, X.-F., Z. Wang, C. Beyrer, D. D. Celentano, C. Khamboonruang, E. Allen, and K. Nelson. 1995. Phenotypic and genotypic characteristics of human immunodeficiency virus type 1 from patients with AIDS in northern Thailand. J. Virol. 69:4649-4655[Abstract]. |
| 67. | Zhang, L., R. Diaz, D. Ho, J. Mosley, M. Busch, and A. Mayer. 1997. Host-specific driving force in human immunodeficiency virus type 1 evolution in vivo. J. Virol. 71:2555-2561[Abstract]. |
| 68. | Zhu, T., H. Mo, N. Wang, D. S. Nam, Y. Cao, R. A. Koup, and D. D. Ho. 1993. Genotypic and phenotypic characterization of HIV-1 in patients with primary infection. Science 261:1179-1181. |
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