Between-Host Evolution of Cytotoxic T-Lymphocyte Epitopes in Human Immunodeficiency Virus Type 1: an Approach Based on Phylogenetically Independent Comparisons
This article has been corrected.
VIEW CORRECTION
ABSTRACT
In human immunodeficiency virus type 1 (HIV-1), mutations that escape from cytotoxic T-lymphocyte (CTL) recognition have been documented, and sequence analyses have provided indirect support for the hypothesis that natural selection has favored CTL escape mutants within an infected host. In spite of such evidence for within-host selection by CTL, it has been more difficult to determine how natural selection by host CTL has influenced long-term evolution of HIV-1. We used statistical analysis of published HIV-1 genomic sequences to examine the role of natural selection in between-host evolution of CTL epitopes. Based on a phylogenetic analysis, we identified 21 pairs of closely related genomes isolated from different hosts and examined the pattern of nucleotide substitution in genomic regions encoding well-characterized CTL epitopes. The results revealed that certain CTL epitopes have been subject to repeated positive selection across the population, while others are generally conserved. Furthermore, evidence of positive selection was associated with divergence from the canonical epitope sequence and with an enhanced frequency of convergent amino acid sequence changes in CTL epitopes. The results support the hypothesis that CTL-driven selection has been a major factor in the long-term evolution of HIV-1.
The binding of virus-derived peptides by class I major histocompatibility complex (MHC) and their recognition by cytotoxic T cells (CTL) play an essential role in the elimination of viral infections in vertebrates (15). The vertebrate CTL system in turn is expected to exert natural selection favoring CTL escape mutations in the virus; that is, mutations to CTL epitopes that eliminate binding of the epitope by the class I MHC molecule (2). In the case of immunodeficiency viruses, it has been proposed that escape from CTL recognition plays an important role in pathogenesis (16, 19). With simian immunodeficiency virus (SIV), experimental infection of rhesus monkeys with a known virus inoculum has provided strong evidence of natural selection favoring CTL escape mutations over the course of infection (1, 6, 14, 26). For human immunodeficiency virus type 1 (HIV-1), mutations that escape from CTL recognition have been documented, and a number of case studies have provided support for the hypothesis that natural selection has favored CTL escape mutants (3, 10, 11, 19, 20).
In spite of evidence for within-host selection by CTL, it has been more difficult to determine how natural selection by host CTL has influenced long-term evolution of HIV-1. A case of mother-to-infant transmission of a CTL escape mutation in HIV-1 demonstrates that escape mutations occurring within an individual host can be transmitted to subsequent hosts (11). However, no data are available regarding the frequency of host-to-host transmission of CTL escape mutations and thus the potential for such mutations to increase in frequency and perhaps eventually to become fixed in the viral population. Because the occurrence of known CTL epitopes was found to be negatively associated with a measure of population-wide sequence variability of viral proteins, Yusim et al. (32) inferred that past CTL-driven natural selection had diversified these regions in the process of eliminating CTL epitopes. However, this statistical association may arise from factors other than natural selection on CTL epitopes; for example, a tendency for MHC-bound peptides to be derived from conserved domains of proteins (31) might produce a similar association.
Positive Darwinian selection favoring amino acid changes is predicted to lead to an enhanced rate of nonsynonymous (amino acid-altering) nucleotide substitution in comparison to synonymous substitution (12, 13). Therefore, an examination of the pattern of nucleotide substitution in gene regions encoding CTL epitopes provides a powerful test of the hypothesis that positive selection has acted on these regions. In addition, positive selection may lead to convergent or parallel evolution at the amino acid sequence level (5) because independent occurrences of the same escape mutations may be selected in different viral lineages. Thus, evidence of parallel or convergent amino acid changes can provide additional evidence of positive selection (14).
We used statistical analysis of published HIV-1 genomic sequences to examine the role of natural selection in between-host evolution of CTL epitopes. Based on a phylogenetic analysis, we identified 21 pairs of closely related genomes isolated from different hosts and examined the pattern of nucleotide substitution in genomic regions encoding well-characterized CTL epitopes. Because we compared pairs of closely related sequences, each difference between the members of a pair has arisen since their most recent common ancestor. Thus, the comparisons between pair members were phylogenetically and statistically independent (7). The use of phylogenetically independent comparisons provides a powerful tool in comparative biology (7), increasing the power of statistical analyses to detect overall trends and making it possible to avoid statistical methods that are highly dependent on a particular model of sequence evolution. By examining the pattern of nucleotide substitution in CTL epitope regions, we tested the hypothesis that natural selection exerted by host CTL has promoted amino acid diversification in these regions.
MATERIALS AND METHODS
Sequences analyzed.
We downloaded 316 complete HIV-1 genome sequences from the National Center for Biotechnology Information database and constructed a phylogenetic tree based on aligned amino acid sequences of the Pol protein (Fig. 1). The sequences were aligned using the CLUSTAL W program (29), and the tree was constructed by the neighbor-joining method (27) on the basis of the Poisson-corrected amino acid distance (22), using the MEGA2 program (17). The support for the internal branches in the tree was assessed by bootstrapping (8); 1,000 bootstrap replicates were used. The phylogenetic tree was used to identify 21 “sister pairs” (Fig. 1) of closely related sequences having the following properties: (i) the branch supporting clustering of the two sequences received at least 90% bootstrap support; and (ii) the two sequences were derived from different hosts, known to be epidemiologically unrelated (see Table S1 in the supplemental material). Because sister pairs were chosen, sequence comparisons between pair members were thus phylogenetically and statistically independent (7).
The purpose of the phylogenetic analysis was to identify phylogenetically independent sister pairs of sequences. As long as clustering of each sequence with its sister sequence was well supported, the accuracy of other details of the phylogeny was not essential to our analysis. Thus, we conducted additional phylogenetic analyses in order to verify the support for sister pairs. Using the concatenated sequence of all nonoverlapping protein-coding regions in the genome, we constructed neighbor-joining trees for our 21 putative sister pairs of genomes based on the following two distances: the Poisson-corrected amino acid distance and the number of synonymous substitutions per synonymous site (dS) (23). The tree based on dS values was used to control for the possibility that clustering patterns in the tree based on amino acid sequences reflected convergent evolution at the amino acid level rather than true relationships. In both of these phylogenetic trees, members of the 21 sister pairs clustered together with strong bootstrap support (≥95% in every case) (data not shown).
CTL epitopes.
We analyzed a set of 69 CTL epitope regions (see Table S2 in the supplemental material) derived from the “best list” of CTL epitopes provided by Frahm et al. (9). Although these epitopes were originally described as being in the B clade (9), they were also found in sequences in our data set assigned to other clades (see Tables S1 and S2 in the supplemental material). This list includes only epitopes supported by strong experimental evidence (9). Adjacent or overlapping epitopes were combined as a single epitope region for purposes of analysis. We included in our analyses only epitopes for which at least one of the 42 sequences in the 21 sister pairs was identical to the epitope sequence provided by Frahm et al. (9). Patterns of nucleotide substitution in epitope regions were compared with those in nonepitope regions of the same genes. Both epitope and nonepitope regions were classified as overlapping or nonoverlapping, depending on whether or not the region in question was encoded by an overlapping reading frame.
Nucleotide substitution.
The number of synonymous nucleotide substitutions per synonymous site (dS) and the number of nonsynonymous nucleotide substitutions per nonsynonymous site (dN) were estimated by the method of Nei and Gojobori (23). In preliminary analyses, we applied a number of more complex models to a subset of the data: Li's method (18), the modified Nei and Gojobori method (33), and the Yang and Nielsen method (30). All models produced essentially identical results. Mean dS and dN values estimated by the Nei and Gojobori method were not significantly different from the values estimated by the other methods (paired t tests). Therefore, we report only results using the Nei and Gojobori method, which, because it makes fewer assumptions than the other models, is expected to have a lower variance (24). In most protein-coding genes, dS exceeds dN; this pattern is seen because most nonsynonymous mutations are deleterious and are eliminated by natural selection (12, 22). On the other hand, a pattern of dN values being greater than dS values is evidence of positive Darwinian selection favoring changes at the amino acid level (12, 13).
Convergent changes.
An amino acid sequence difference between the members of a sister pair was counted as convergent (parallel) if the same amino acid difference occurred at the same site in at least one other sister pair. Convergent differences included both amino acid replacements and indels involving the insertion or deletion of a single amino acid residue.
RESULTS
Comparisons within and between sister pairs.
Overall mean dS and dN values were computed for nonoverlapping nonepitope regions, overlapping nonepitope regions, nonoverlapping epitope regions, and overlapping nonepitope regions both within and between sister pairs (Table 1). The mean dS value was greater than the mean dN value in all cases, and this difference was statistically significant for all comparisons except for within-pair comparisons of overlapping epitope regions (Table 1). This result implies that, on average, both epitope and nonepitope regions are subject to purifying selection acting at nonsynonymous sites; in other words, selection acting to eliminate mutations harmful to protein structure (22).
For the 69 epitope regions, there was a significant positive correlation between mean dN value in within-pair comparisons and mean dN value in between-pair comparisons (r = 0.809; P < 0.001) (Fig. 2A). This result implies that functional constraints on CTL epitopes were generally similar over the shorter evolutionary time spans represented by within-pair comparisons and the longer time spans represented by between-pair comparisons.
There was also a significant positive correlation between mean dS value in within-pair comparisons and mean dS value in between-pair comparisons (r = 0.443; P < 0.001; Fig. 2B). However, the two correlations were significantly different (P = 0.003; two-tailed test). The weaker correlation in the case of dS than in the case of dN may simply reflect the greater stochastic error in the former due to the smaller number of synonymous sites than of nonsynonymous sites. Nonetheless, the positive correlation between the mean dS value in within-pair comparisons and that in between-pair comparisons suggests that mutation rates in the CTL epitopes were generally similar over the shorter evolutionary time spans represented by within-pair comparisons and the longer time spans represented by between-pair comparisons.
Selection on CTL epitopes.
In spite of the fact that the overall mean dS value exceeded the mean dN value in within-pair comparisons (Table 1), there were certain within-pair comparisons in all regions in which dN was greater than dS (Fig. 3). In nonoverlapping regions, there was a significant difference between epitopes and nonepitope regions with respect to the proportion of individual comparisons with dN values greater than dS values, the proportion with dS values equal to dN values, and the proportion with dS values greater than dN values (χ2 = 135.8, 2 df; P < 0.001) (Fig. 3). Similarly, in overlapping regions, there was a significant difference between epitopes and nonepitope regions with respect to the proportion of individual comparisons with dN values greater than dS values, the proportion with dS values equal to dN values, and the proportion with dS values greater than dN values (χ2 = 44.5, 2 df; P < 0.001) (Fig. 3). In each case, the proportions of comparisons with dN values greater than dS values and with dS values greater than dN values were higher in nonepitopes than in epitopes, while a much higher proportion of comparisons of epitopes showed dS values that were equal to dN values (Fig. 3). As noted by Yusim and colleagues (32), the conservation of CTL epitopes in comparison with nonepitope regions may largely be an artifact due to the process by which CTL epitopes have been identified.
Comparison among the 69 CTL epitope regions showed that within-pair comparisons with dN values that were greater than dS values were not equally apportioned among the regions. Rather, certain epitope regions had very high proportions of such comparisons, while in other epitopes, dN did not exceed dS in any comparison (Fig. 4A; also see Table S2 in the supplemental material). The mean value for dN − dS was significantly different among epitope regions by a one-way analysis of variance (F68, 1,293 = 1.49; P = 0.007). A nonparametric Kruskal-Wallis test for differences in the median value for dN − dS among epitopes likewise yielded significant results (P = 0.001). On the basis of these comparisons, we identified 18 epitope regions subject to persistent positive selection, as evidenced by consistently high proportions (>20%) of comparisons with dN values greater than dS values (Table 2). Conversely, we identified 10 epitope regions subject to strong constraint at the amino acid level, as evidenced by the absence of comparisons with dN values that were greater than dS values (Table 2).
In order to test for convergent evolution of CTL epitopes, we examined the proportion of amino acid sequence differences (including both amino acid replacements and indels) between sister pairs that also occurred in other sister pairs. Of 436 amino acid sequence differences in CTL epitope regions between sister pairs, 148 (33.9%) were convergent. The proportions of convergent differences were similar in nonoverlapping epitope regions (127 of 383 or 33.2%) and in overlapping epitope regions (21 of 53 or 39.6%). The proportions of convergent changes in nonepitope regions were similar: 308 of 1,072 (35.4%) in nonoverlapping regions and 111 of 300 (37.0%) in overlapping regions. None of these proportions were significantly different from one another by χ2 tests.
The proportion of amino acid changes that were convergent differed markedly among epitope regions (Fig. 4B). In several cases, epitope regions with high proportions of comparisons with dN values greater than dS values also had high proportions of convergent change. For example, among the epitope regions with the highest proportions of dN values that were greater than dS values were regions 8 and 9 of Gag, regions 1 and 14 of Env, and region 2 of Nef (Fig. 4A and Table 2). Each of these epitope regions also showed a high proportion of convergent changes (Fig. 4B). An apparent exception to this trend was CTL epitope region 16 of Gag, which showed no comparisons with dN values greater than dS values yet 100% of changes were convergent (Fig. 4); however, in this region, a total of only three amino acid sequence changes were observed, all of which were convergent.
In order to test further the hypothesis that CTL-driven selection has favored amino acid changes in epitopes, we examined the pattern of correlation among variables relating to the nucleotide substitution pattern and variables relating to the amino acid sequence changes in epitopes. Because these variables were intercorrelated in complex ways, we used partial correlation to assess independent associations between a set of independent variables relating to the nucleotide substitution pattern and dependent variables reflecting amino acid changes in epitopes (Table 3). (These analyses were applied to 68 epitopes, because one epitope showed no amino acid difference in within-pair comparisons of any of the 21 sister pairs [Fig. 4B]).
The first dependent variable we examined was the proportion of sequences in the 21 sister pairs that conserved the immunologically defined “best epitope” sequence (Table 3). In the case of this variable, there were highly significant negative partial correlations with dS values within pairs and with the proportion of within-pair comparisons showing dN values that were greater than dS values (Table 3). The correlation with dS values within pairs implies that epitopes with high mutation rates were more likely to lose the “best epitope” sequence. However, the significant correlation with the proportion of comparisons with dN values greater than dS values is evidence that positive Darwinian selection plays a role in loss of the “best epitope” sequence that is independent of the mutation rate.
In addition, we examined partial correlations between the same dependent variable set and the proportion of convergent amino acid sequence differences between sister pairs. In this case, the single significant partial correlation was a positive correlation with the proportion of comparisons with dN values greater than dS values (Table 3). This correlation reflects the fact that epitopes with a high proportion of dN values greater than dS values tended to have high proportions of convergent change (Fig. 4). It implies that positive selection is a factor enhancing the likelihood of convergent changes at the amino acid level in CTL epitopes.
DISCUSSION
Experimental studies (1, 6, 14, 26) have established that natural selection driven by host CTL is a major factor in the diversification of SIV within an individual infected host, but it has been much more difficult to study the effect of CTL-driven selection in natural populations of the related virus HIV-1. Because hosts differ with respect to the class I MHC molecules they encode, it expected that different hosts will target different CTL epitopes. Under these conditions, it is expected that the pattern of CTL-driven positive selection on viral proteins will be episodic (28); that is, different genomic regions may be targeted by selection in different portions of the phylogeny. As a consequence, the statistical signal of positive Darwinian selection on viral proteins may be difficult to detect.
We used a novel approach to this question based on the comparison of phylogenetically independent, closely related pairs of HIV-1 genomes. Overall, CTL epitopes, like the rest of the viral genome, showed evidence of purifying selection. Nonetheless, there were differences among CTL epitopes with respect to the nature of the selection acting on them. Certain epitopes appeared to be consistently subject generally to purifying selection, while others showed evidence of repeated positive Darwinian selection. In spite of the potentially episodic nature of selection on CTL epitopes, our approach was able to identify epitopes on which positive selection has acted independently in different portions of the viral phylogeny.
Evidence of positive selection was statistically associated with an enhanced frequency of convergent amino acid sequence changes in CTL epitopes. In experimental studies with rhesus monkeys, the same escape mutations in a CTL epitope in the Tat protein were selected independently in different hosts, thus demonstrating convergent evolution of CTL escape in this experimental system (14). The present results suggest that a similar process is occurring in natural populations of HIV-1 and thus that repeated independent occurrence of the same amino acid replacements is likely to be a characteristic of the CTL epitopes subject to the strongest selective pressure due to host immune recognition. This finding is consistent with the results of work by Moore et al. (20), who showed that there are significant population-wide associations between certain amino acid polymorphisms at known CTL epitopes in the reverse transcriptase protein and occurrence in the host of the restricting HLA allele, a pattern suggestive of repeated independent occurrences of the same escape mutations.
It has been proposed that a CTL vaccine against HIV-1 should target conserved CTL epitopes in order to prevent escape from vaccine-primed immunity (4, 21). Alternatively, it has been argued that the epitopes showing the strongest CTL selection and thus selection of escape mutants might be chosen for a vaccine, since immune responses targeting these epitopes might be most effective in eliminating the virus (25). In either case, understanding the long-term evolution of CTL epitopes in HIV-1 will provide important background information for the choice of epitopes to be included in a CTL vaccine. The present study supports the hypothesis that CTL-driven selection has been an important factor in long-term evolution of HIV-1. Our results also show that phylogenetically independent comparisons between closely related pairs of sequences from natural populations provide a fruitful approach for uncovering the patterns of natural selection shaping the evolution of this and other viral pathogens.
FIG. 1.
FIG. 2.
FIG. 3.
FIG. 4.
TABLE 1.
| Comparison or region (no. of comparisons) | dS value | dN value | P value (paired t test) |
|---|---|---|---|
| Within pairs | |||
| Nonepitope | |||
| Nonoverlap (8) | 0.052 ± 0.007 | 0.019 ± 0.003 | <0.001 |
| Overlap (8) | 0.036 ± 0.006 | 0.018 ± 0.003 | <0.025 |
| Epitope | |||
| Nonoverlap (63) | 0.057 ± 0.005 | 0.018 ± 0.001 | <0.001 |
| Overlap (6) | 0.043 ± 0.019 | 0.019 ± 0.007 | NS |
| Between pairs | |||
| Nonepitope | |||
| Nonoverlap (8) | 0.479 ± 0.044 | 0.119 ± 0.016 | <0.001 |
| Overlap (8) | 0.335 ± 0.053 | 0.151 ± 0.022* | <0.025 |
| Epitope | |||
| Nonoverlap (63) | 0.439 ± 0.023 | 0.752 ± 0.008 | <0.001 |
| Overlap (6) | 0.273 ± 0.058 | 0.177 ± 0.053* | <0.05 |
a
Each dS or dN value is given as mean ± standard error. *, mean dN significantly different from that for nonoverlap epitopes (between pairs) at a simultaneous significance level of 5% (Dunnett's test).
TABLE 2.
| Type of selection,d protein, or epitope regiona | AAb | Sequencec | HLA allele |
|---|---|---|---|
| Positive | |||
| Gag | |||
| 1 | 11-19 | GELDRWEKI | B*4002 (B61) |
| 18-26 | KIRLRPGGK | A*0301 (A3) | |
| 8 | 216-224 | HPVHAGPIA | B*07 (B7) |
| 9 | 240-250 | TSTLQEQIGWF | B*5701 (B57) |
| 14 | 329-337 | DCKTILKAL | B*0801 (B8) |
| 17 | 427-434 | TERQANFL | B*4002 (B61) |
| 433-442 | FLGKIWPSYK | A*0201 (A2) | |
| 18 | 481-489 | KELYPLTSL | B*4001 (B60) |
| Pol | |||
| 5 | 511-521 | RMRGAHTNDVK | A*03 (A3) |
| Env | |||
| 1 | 2-10 | RVKEKYQHL* | B*0801 (B8) |
| 6 | 209-217 | SFEPIPIHY | A*2902 (A29) |
| 7 | 298-307 | RPNNNTRKSI | B*0702 (B7) |
| 8 | 309-317 | HIGPGRAFY | A*3002 (A30) |
| 10 | 419-427 | RIKQIINMW | A*3201 (A32) |
| 11 | 557-565 | RAIEAQQHL | Cw*0304 (Cw10) |
| 12 | 585-594 | RGPGRAFVTI | A*0201 (A2) |
| 585-593 | RYLKDQQLL | A*2402 (A24) | |
| 13 | 606-614 | TAVPWNASW | B*3501 (B35) |
| 14 | 698-707 | VFAVLSIVNR | A*3303 (A33) |
| 15 | 770-780 | RLRDLLLIVTR | A*0301 (A3) |
| 777-785 | IVTRIVELL | A*6802 (A68) | |
| 786-795 | GRRGWEALKY | B*2705 (B27) | |
| Nef | |||
| 2 | 68-77 | FPVTPQVPLR | B*0702 (B7) |
| 73-82 | QVPLRPMTYK | A*0301 (A3) | |
| 83-91 | AAVDLSHFL | Cw*0802 (Cw8) | |
| 84-92 | AVDLSHFLK | A*03 (A3) | |
| 90-97 | FLKEKGGL | B*0801 (B8) | |
| 92-100 | KEKGGLEGL | B*4002 (B61) | |
| B*4001 (B60) | |||
| Negative | |||
| Gag | |||
| 5 | 180-188 | TPQDLNTML | B*0702 (B7) |
| 10 | 260-267 | EIYKRWII | B*0801 (B8) |
| 11 | 269-277 | SFNCGGEFF | B*1516 (B63) |
| 16 | 405-413 | CRAPRKKGC | B*14 (B14) |
| Pol | |||
| 1 | 59-67 | ITLWQRPLV* | A*6802 (A68) |
| A*7401 (A19) | |||
| 2 | 173-181 | LFLDGIDKA | B*81 (B81) |
| 19 | 651-660 | VTDSQYALGI | B*1503 (B72) |
| 21 | 715-723 | GPKVKQWPL | B*0801 (B8) |
| Tat | |||
| 1 | 2-11 | EPVDPRLEPW* | B*5301 (B53) |
| Env | |||
| 9 | 375-383 | SFNCGGEFF | B*1516 (B63) |
a
Epitope regions (each of which may include more than one overlapping epitope) are numbered in sequence from left to right (N- to C-terminal) in Fig. 4.
b
Amino acid (AA) position of epitopes is defined according to reference sequence AF033819.
c
Epitopes in overlapping regions are marked with asterisks (*).
d
Positively selected epitopes were those in which dN values that were greater than dS values were observed in at least 20% of comparisons; negatively selected epitopes were those in which a dN value greater than the dS value was not observed in any comparison (see Fig. 4A).
TABLE 3.
| Independent variable | Correlation with dependent variable | ||
|---|---|---|---|
| Prop. best epitope (P value) | Prop. convergent change (P value) | ||
| dS within pairs | −0.479 (0.00006) | 0.072 (NS) | |
| dN within pairs | 0.082 (NS) | −0.055 (NS) | |
| dS between pairs | −0.046 (NS) | −0.128 (NS) | |
| dN between pairs | −0.113 (NS) | 0.133 (NS) | |
| Prop. dN > dS | −0.544 (0.000003) | 0.291 (0.019) | |
a
Fourth-order partial correlation coefficients between each independent variable and the dependent variable, simultaneously controlling for all four other independent variables.
b
Prop. best epitope, proportion of sequences in the 21 sister pairs that conserved the immunologically defined “best epitope” sequence; prop. convergent change, proportion of convergent change; prop. dN > dS, proportion of within-pair comparisons showing dN values that were greater than dS values; NS, not significant.
Acknowledgments
This research was supported by grant GM43940 from the National Institutes of Health.
Supplemental Material
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Information & Contributors
Information
Published In
Copyright
© 2004 American Society for Microbiology.
History
Received: 19 February 2004
Accepted: 4 June 2004
Published online: 1 November 2004
Contributors
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2004. Between-Host Evolution of Cytotoxic T-Lymphocyte Epitopes in Human Immunodeficiency Virus Type 1: an Approach Based on Phylogenetically Independent Comparisons. J Virol 78:.
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