Intra- and Interspecific Variation of the CCR5 Gene in Higher Primates

Yun-wu Zhang*,1, Oliver A. Ryder*, and Ya-ping Zhang{dagger},{ddagger}

* Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, California
{dagger} Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China
{ddagger} Laboratory of Molecular Genetics, Yunnan University, Kunming, Yunnan, China

Correspondence: E-mail: oryder{at}ucsd.edu.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We have evaluated the molecular evolution of the chemokine receptor CCR5 in primates. The chemokine receptor CCR5 serves as a major co-receptor for human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection. Knowledge of evolution of the CCR5 molecule and selection on the CCR5 gene may shed light on its functional role. The comparison of differences between intraspecific polymorphisms and interspecific fixed substitutions provides useful information regarding modes of selection during the course of evolution. There is marked polymorphism in the CCR5 gene sequence within different primate species, whereas sequence divergence between different species is small. By using contingency tests, we compared synonymous (SS) and nonsynonymous (NS) CCR5 mutations occurring within and between a broad range of primates. Our results demonstrate that CCR5 evolution did not follow expectations of strict neutrality at the level of the whole gene. The proportion of NS to SS at the intraspecific level was significantly higher than that observed at the interspecific level. These results suggest that most CCR5 NS polymorphisms are slightly deleterious. However, at domains more closely correlated with its known biological functions, there was no obvious evidence to support deviation from neutrality.

Key Words: CCR5 • evolution • primates


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Different modes of selection may result in differing patterns of the nature and extent of genetic variation. Three basic theories dominate attempts to explain the characteristics of mutations: the strictly neutral theory (Kimura 1968; King and Jukes 1969), the slightly deleterious theory (Ohta 1992), and the advantageous theory (Gillespie 1991). Although all three theories agree that most mutations are strongly deleterious and therefore are rapidly eliminated from populations, they differ in consideration of the fitness of the majority of mutations observed in populations. The strictly neutral theory asserts that all readily observable mutations in populations have little or no effect on an organism's fitness, and their evolutionary dynamics are governed solely by genetic drift. The slightly deleterious theory or the nearly neutral theory, which was modified from the strictly neutral theory, proposes that a large fraction of mutations have selection coefficients near the reciprocal of the species effective population size. For such mutations, evolution proceeds as a balance among the forces of mutation pressure, natural selection, and genetic drift. Finally, the advantageous theory considers that a relatively larger proportion of mutations afford fitness advantage to the organism. Under this theory, positive selection plays an important role in DNA evolution (Nachman, Boyer, and Aquadro 1994; Li 1997; Akashi 1999).

According to the strictly neutral theory, the amount of variation within species is expected to be correlated with the rate of divergence between species for different genes (Maynard Smith 1970). Comparison of intra- and interspecific variation serves as a valuable test to examine if mutations follow the neutral model. This approach has been applied broadly, disclosing adaptive (McDonald and Kreitman 1991; Eanes, Kirchner, and Yoon 1993; Karotam, Boyce, and Oakeshott 1995; King 1998; Fay, Wyckoff, and Wu 2001, 2002) or disadvantageous (Ballard and Kreitman 1994; Nachman, Boyer, and Aquadro 1994; Templeton 1996) molecular evolution. However, most of these studies only compared two or three closely related species, and many of them were for Drosophila species or focused on mitochondrial DNA sequences, or both.

In a previous report, we studied sequence evolution of the CCR5 gene among primates. Only one sequence from each species was obtained, and intraspecific variation in primates was not evaluated (Zhang, Ryder, and Zhang 1999). To provide a broader perspective on CCR5 evolution in primates, additional non-human primate CCR5 sequences have been obtained and compared with those available in the expanding collections of GenBank. The CCR5 protein acts not only as a chemokine receptor but also as a major co-receptor for human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection (Alkhatib et al. 1996; Dragic et al. 1996). Separate CCR5 domains are associated with its multiple functions (Atchison et al. 1996; Doranz et al. 1997; Edinger et al. 1997; Wu et al. 1997). Consequently, we have also tested the neutral theory at different CCR5 domains to evaluate evolutionary aspects of virus-host interactions.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We used previously reported methods for polymerase chain reaction (PCR) amplification and sequencing of primate CCR5 genes (Zhang, Ryder, and Zhang 1999). Primate and rodent CCR5 sequences were retrieved from GenBank, using the keyword "CCR5." Patent sequences were excluded, and data from several publications reporting polymorphisms in humans (Ansari-Lari et al. 1997; Carrington et al. 1997, 1999; Cohen et al. 1998) were directly incorporated instead of screening GenBank. We used only the human CCR5 sequence, X91492 (Samson et al. 1996), as a reference. In a preliminary study, one Pan troglodytes sequence (AF011538) and one Saguinus sp. sequence (AF162015) were found to be very different from other conspecific sequences and may have represented misassigned data. To avoid the possibility of sample source error, we excluded them from our analysis. To increase the number of sequences for our comparisons, we included some retrieved sequences that were not complete at the 5'-end (N-terminus) and the 3'-end (C-terminus). The GenBank accession numbers and publication resources of the sequences used in the present study are given in the Supplementary Material online.

When two or more sequences were available from one species, we analyzed the intraspecific variation and hypothesized a possible ancestral CCR5 sequence according to parsimony principles, with the help of MacClade (Maddison and Maddison 1992) and using other closely related species' sequences as outgroups. Indels and nonsense substitutions were found in some sequences, but these sites were excluded from intra- and interspecific variation comparisons. We focused the analyses on two substitution groups: synonymous substitutions (SS) and strict nonsynonymous substitutions (NS) (see below). Genetic diversity at the nucleotide level (Pi value, the average number of nucleotide differences per site between two sequences) within species was estimated by using the Arlequin software package (Schneider et al. 1997). There were five primate lineages studied here: hominoids, colobines, cercopithecines, platyrrhines (New World monkeys), and lemurs. Therefore, we also estimated the CCR5 genetic distance (uncorrected P value) within and between each lineage by using PAUP*4.0 (Sinauer Associates).

The neutral theory predicts that the ratio of synonymous (silent) and nonsynonymous (replacement) nucleotide substitutions will be the same for intraspecific polymorphisms and interspecific fixed substitutions. Based on this rationale, McDonald and Kreitman (1991) formulated a test to examine if mutations follow the neutral model by using a 2 x 2 contingency table to compare the numbers of NS and SS substitutions within and between species. Unlike most tests of the neutral theory, this test does not require that populations have reached equilibrium. By using the McDonald-Kreitman test (G-test with William's correction), we evaluated CCR5 mutations to see if they followed the neutral theory during the course of evolution of higher primates. In this analysis, only species with two or more available sequences were included. Because we do not know exactly if the sequences of Ateles sp. and Saguinus sp. represent one species or different species, they were excluded from this analysis. The lemur data were also excluded from this analysis because of the very early divergence time between lemurs and other primate lineages. Because some sequences are not complete at the N-terminus and the C-terminus, we only used 1,017 bp of the CCR5 gene for comparisons (nucleotide site 22–1038, counting from the start codon ATG), which encodes 339 amino acids (amino acid site 8–346). We used the phylogeny-based analysis for evaluating interspecific variation (Zhang, Rosenberg, and Nei 1998). We first constructed CCR5 gene trees by using different methods (parsimony, distance, maximum likelihood, and Bayesian), using software packages: PAUP (Sinauer Associates), PHYLIP (Felsenstein 1989), MEGA2 (Kumar et al. 2001), or MrBayes (Huelsenbeck and Ronquist 2001). We assumed that one tree constructed by the Neighbor-Joining (NJ) distance method with a gamma-justified Kimura-2-P model (Kimura 1980) represented the actual primate phylogeny. We used both the distance-based Bayesian method (Zhang and Nei 1997; Zhang, Rosenberg, and Nei 1998) and parsimony principles to infer the ancestral CCR5 sequences at all interior nodes of the tree and then counted the numbers of NS and SS on each branch of the tree. For intraspecific variation, we used both the (NJ) distance method and the parsimony method to construct haplotype trees for each species and then counted the numbers of NS and SS on each branch of the trees. The NS and SS substitutions were then pooled from each species.

McDonald and Kreitman (1991) limited the tree category dimension of the contingency test to "fixed" versus "polymorphic." Castelloe and Templeton (1994) evaluated rooting intraspecific gene trees and suggested that a refinement consisting of splitting the intraspecific "polymorphic" class into "tip" class and "interior" class would be meaningful: a tip haplotype is connected to only one other haplotype in the tree, and an interior haplotype is connected to two or more other haplotypes in the tree and thus represents an interior node in a topological sense. Templeton (1996) used this categorization for testing the neutrality of the mitochondrial cytochrome oxidase II gene in hominoid primates. We also followed this categorization with the CCR5 data and tested different mutational categories with the contingency method: intraspecific tip class versus intraspecific interior class and intraspecific interior class versus interspecific fixed mutations. Because of the limited numbers of interior mutations, these comparisons were studied only at the whole-gene level.

The functional importance of different CCR5 domains may affect selection of mutations in these domains: more functionally crucial domains are expected to be less likely to accumulate nonsynonymous mutations. Thus we also explored the distribution patterns of intra- and interspecific variation for different CCR5 functional domains. The CCR5 functional domains were classified as the N-terminus (N-t, corresponding to nucleotide sites 22–93; sites 1–21 were eliminated from our analysis), the seven transmembrane domains combined (TM, including nucleotide sites 94–168, 202–264, 307–372, 433–501, 592–654, 706–771, and 838–906), the three intercellular domains combined (I, nucleotide sites 169–201, 373–432, and 655–705), each of the three extracellular domains: E1 (265–306), E2 (502–591), and E3 (772–837), and the C-terminus (C-t, 907-1038; nucleotide sites 1039–1056 were eliminated from our analysis). As the number of observed nucleotide substitutions on each domain is correlated with the domain length, we evaluated the relative number of substitutions (substitutions/domain length). The McDonald-Kreitman test was also performed to explore if mutations at different domains follow the neutral model.

Adaptive evolution among CCR5 amino acids was tested by using PAML (Yang 1997). Based on contingency test results on different CCR5 domains, we partitioned CCR5 amino acids into two classes: (N-t, E1, E2, E3, C-t) and (TM, I). Six models were used here: M0, M1, M2, M3, M7, and M8; and their specifications are explained in Yang and Swanson (2002).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Our sequences covered five major primate lineages and 68 species (including two rodents) (see Supplementary Material online). Polymorphism screening within the species demonstrated that CCR5 polymorphisms are prevalent in primates and rodents and exist within all species studied (table 1). In many species, the number of NS is almost the same as or even higher than the number of SS. Considering the fact that the number of observed polymorphisms is positively correlated with the number of sequences studied, we have also estimated nucleotide diversity (Pi value), which is relatively independent of the number of sequences sampled. Our results showed that, on average, rodents possess higher genetic diversity (6.16 x 10–3) than primates (1.80 x 10–3 –3.84 x 10–3) (table 1). To minimize the effects of nonrandom sampling on estimates of genetic diversity, we excluded species with fewer than five sequences. As a result, the magnitude of the difference between the genetic diversity of rodents (10.43 x 10–3) and primates (1.80 x 10–3–3.24 x 10–3) was increased.


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Table 1 CCR5 Polymorphism and Genetic Diversity (Pi Value) Within Each Species.

 
With the exception of mutants with indels, all primate CCR5 sequences are 1,059 bp long (including the stop codon TGA) and the deduced protein sequences contain 352 amino acid residues. Substitutions increase with species divergence time. In general, though, primate CCR5 sequence similarities are high, and genetic distances within and between the different primate lineages we analyzed are no more than 0.13. There was no variation observed at all 12 cysteines in the CCR5 protein, which are critical in protein folding (sites 20, 58, 101, 178, 213, 224, 269, 290, 291, 321, 323, 324).

From the deduced ancestral sequences of each species exhibiting polymorphism, CCR5 gene trees were constructed by different methods and with different models. All the trees have similar topologies, and their differences lie within the interpretation of the relationships within genera. A tree constructed by the Neighbor-Joining distance method (see fig. 1) was used here, whose topology conformed well to the generally accepted phylogenetic families and subfamilies of primate phylogeny (e.g., Goodman et al. 1998). We found that different analysis methods generated the same conclusions, so we have listed only results based on node sequences deduced by the distance-based Bayesian method (Zhang and Nei 1997; Zhang et al. 1997). This method provided highly reliable inference of interior node sequences, with probabilities over 96%. The numbers of interspecific NS and SS occurring on each branch are listed in figure 1. In contrast, at the intraspecific level, the parsimony method was more conservative than the NJ method at inferring relationships between haplotypes. Both methods counted the same numbers of NS and SS for most species, and differences of final pooling results between the two methods were small (total NS/SS = 178/121 for the NJ method and 181/124 for the parsimony method). As both methods provided essentially the same results, only results based on the NJ method are presented in figure 1. Detailed haplotype trees with NS and SS information for each species are available from the authors on request. We performed the McDonald-Kreitman test for intra- and interspecific NS and SS comparison in different primate lineages. The proportion of NS to SS at the intraspecific level is always higher than the proportion at the interspecific level, and the differences are statistically significant (fig. 1).



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FIG. 1. McDonald-Kreitman test for intra- and interspecific CCR5 variation comparisons. The CCR5 gene tree was constructed by the NJ method. The numbers of nonsynonymous (NS) and synonymous (SS) substitutions occurring on each branch were listed correspondingly as NS/SS. Total NS/SS numbers at the interspecific level were then summed for different primate lineages. Total NS/SS numbers at the intraspecific level (in parentheses) were pooled from the haplotype tree of each species based on the NJ method (see details in the text). OWMs and NWMs denote the Old World monkeys (including colobines and cercopithecines) and the New World monkeys (platyrrhines)

 
Dividing intraspecific substitutions into tip and interior classes allowed the performance of a contingency test for comparison of different mutational categories. Polymorphisms within interior branches have similar evolutionary properties to those of fixed interspecific mutations; there was no significant difference between them. Conversely, at the intraspecific level, the difference between tip class and interior class was significant, the proportion of NS to SS at tip class exceeding that at interior class (table 2).


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Table 2 Comparison of Different Mutational Categories (Based on NJ Haplotype Tree Data at the Intraspecific Level).

 
The distribution of substitutions within species is strikingly different from that between species (fig, 2). At the intraspecific level, the relative number of NS substitutions is higher than that of SS at all CCR5 domains except at the C-terminus. Both NS and SS occurred relatively evenly along different CCR5 domains, except at the N-terminus (that has the highest relative number of NS) and the E2 (that has the lowest relative number of SS). At the interspecific level, the relative number of NS is higher than that of SS only at the N-terminus and the E2 domain. Intraspecific variation in both NS and SS occurred unevenly along different CCR5 domains; this is especially apparent for the NS.



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FIG. 2. CCR5 mutation distribution patterns at intra- and interspecific levels. The relative number of mutations (mutations/domain length) was used. Mutation patterns were compared at seven classified domains here: the N-terminus (N-t), the seven transmembrane domains altogether (TM), the three intercellular loops altogether (I), each of the three extracellular loops (E1, E2, and E3), and the C-terminus (C-t)

 
The contingency test was performed to compare substitution variation at different CCR5 domains (table 3). Although the proportions of NS to SS at the intraspecific level were significantly higher than those at the interspecific level for the transmembrane domains (TM) and the intercellular domains (I), there was no evidence to show deviation from neutrality at the N-terminal region, two of the three extracellular domains (E1 and E3), and the C-terminal region (C-t). At the second extracellular domain (E2), although results based on parsimony haplotype trees suggested a deviation, the G value (4.54) was near the critical value (G = 3.84, P = 0.05); and results based on NJ haplotype trees did not suggest a deviation (table 3).


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Table 3 Contingency Test at Different CCR5 Domains (Based on NJ Haplotype Tree Data at the Intraspecific Level).

 
We tested different models to detect adaptive evolution among CCR5 amino acids. However, there was only one site, 15Y, showed a nonsynonymous/synonymous substitution rate ratio (w = dN/dS) significantly higher than 1 (at the 99% level). This amino acid residue is located within the N-terminus. Excluding this codon did not change our results (data not shown).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Accuracy of the Analyses and Neutrality Test
The contingency test for neutrality is simple, straightforward, and does not require equilibrium (McDonald and Kreitman 1991; Templeton 1996). However, it requires accurate and unbiased counts of the numbers of mutations in various categories, which in turn requires that the topology and branch lengths of the tree are estimated in an accurate, unbiased fashion (Templeton 1996). In our case, the CCR5 gene tree used here fitted well to the generally accepted clades of the primate phylogeny (e.g., Goodman et al. 1998). We have tested different tree-construction methods and assumed different models. All the trees gave similar topologies, with differences only within genera. Because our analyses focused on higher taxonomic levels (family and above), slight topological incongruities at lower taxonomic levels had little or no effect on the final results. Although different methods inferred slightly different ancestral CCR5 sequences at some interior nodes, they did not change the essence of our results. At the intraspecific level, however, both the NJ and the parsimony methods gave identical or very similar haplotype topologies. Although there were slight differences between the accrued numbers of NS and SS based on different methods, the final conclusions were essentially the same.

Unobserved multiple hits on some classes of mutations could affect the accuracy of branch lengths, and thus could have an impact on the contingency test (Maynard Smith 1994; Templeton 1996). For example, Maynard Smith (1994) pointed out that synonymous substitutions tend to be transitions, and nonsynonymous substitutions are more often transversions. When the genetic distance between species is large, there is a bias toward undercounting the number of transitions, and thus the number of SS. This effect is not important if the between-species differences are not close to saturation (Templeton 1996). However, considering our CCR5 data, even if the number of SS between species were undercounted, a correction would only increase the significance of the outcome. It is unlikely, however, that substitutions within nuclear genes such as CCR5 have reached saturation. We constructed a worse-case scenario by presuming that the NJ method estimated the number of intraspecific NS accurately while the parsimony method estimated the number of intraspecific SS accurately; thus the proportion of NS to SS at the intraspecific level was reduced from 178/121 to 178/124, and the McDonald-Kreitman test still suggested a highly significant deviation from neutrality (G = 19.37, P = 0.00001).

One assumption for the contingency test is that sequences have been sampled at random with respect to different categories of variation. Because most of our sequences were from GenBank, it is hard to estimate their randomness. However, we may tend to "randomize" samples by nesting our analysis on those species with five or more available sequences. The nested analysis results were shown in figure 3 and the result was essentially the same.



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FIG. 3. Nested McDonald-Kreitman test for intra- and interspecific CCR5 variation comparison. Only species with five or more available sequences were included in this analysis. Notations have the same meaning as in figure 1

 
In general, our results demonstrated an explicit rejection of the null hypothesis that within higher primates the CCR5 gene evolved under a strictly neutral model of molecular evolution. There is an excess of NS within species. One plausible explanation for this observed pattern is that most of the NS polymorphisms within the primate CCR5 gene are slightly deleterious. They may exist within populations for brief periods, but they are unlikely to rise in frequency or become fixed. The slightly deleterious mutants may contribute more to polymorphism within species than to differences between species (Ohta 1992). Although recent population expansion could serve as one alternative explanation for the excess of NS within species, it is unlikely in this case. First, there is no evidence that most primates experienced a recent population expansion, except humans (Stoneking 1994; Kaessmann et al. 2001). Second, when human data were excluded from the nested analysis (fig. 3), the results remained the same. Recently, Li et al. (2003) developed a methodology of using multiple data sets that overlap in some extent to test neutral selection. When using human CCR5 data sets from Carrington et al. (1997) as an example, they also found that the hypothesis of strict neutrality cannot explain polymorphism patterns of the CCR5 gene in humans.

By splitting the intraspecific "polymorphic" class into "tip" class and "interior" class, it showed that for an intraspecific haplotype tree, interior haplotypes strongly tend to be older than tip haplotypes, and that interior haplotypes also tend to be more frequent in the gene pool than tip haplotypes (Castelloe and Templeton 1994; Templeton 1996). Because the mutations that are ultimately fixed derive preferentially from the old, common class (interior) and not the young, rare class (tip) under neutrality, polymorphic mutations on interior branches may well have evolutionary properties more akin to fixed interspecific mutations than to polymorphic tip branches (Templeton 1996). Our results did support this hypothesis: the contingency test did not detect differences between intraspecific interior mutation patterns and interspecific fixed mutation patterns. Still, there was a significant difference between the intraspecific tip class and the interior class, suggesting deviation from neutrality (table 2). This finding conforms with Fu and Li's results (1993), who demonstrated that deviations from neutrality can be detected with intraspecific data by discriminating between "external" (tip) versus "internal" (interior) mutations.

Selection at Different CCR5 Domains
The CCR5 protein acts not only as a chemokine receptor but also as a major co-receptor for viruses such as HIV and SIV (Alkhatib et al. 1996; Dragic et al. 1996). Some studies revealed that interactions between CCR5 and HIV/SIV are conformationally complex and involve multiple CCR5 domains; the amino terminal domain and extracellular loop are two critical domains for CCR5 co-receptor function (Atchison et al. 1996; Doranz et al. 1997; Edinger et al. 1997). However, the pattern for chemokine binding is relatively simple, as only the second extracellular loop (E2) seems to be involved (Wu et al. 1997). The carboxyl terminus (C-t) of CCR5 correlates with receptor phosphorylation (Oppermann et al. 1999). Thus different CCR5 domains are associated with different CCR5 functions, and it would be informative to investigate how selection acted on these different domains.

Interestingly, our results showed that CCR5 transmembrane domains and intercellular domains have experienced strong selective constraints. Although we do not know why selective constraints acted on intercellular domains, the anchor function of transmembrane domains for CCR5 expression on the cell surface might be a reasonable explanation for selective constraints on transmembrane domains. Strikingly, for the other CCR5 domains that are more closely correlated with and important for its chemokine receptor and virus co-receptor functions, there was no obvious evidence to show deviation from neutrality. There is conjecture that some amino acids on these functional domains may have experienced positive selection, contradicting the effects of purifying selection if there were any. We tested different models but found that only amino acid residue 15Y on the N-terminus had the sign of positive selection. When this codon was excluded, the results remained the same. These results therefore imply that selective constraints did not act strongly on these domains, or that the CCR5 chemokine receptor function in vivo is not very important, an observation that would be consistent with the finding that deficient CCR5 proteins do not show obvious detrimental effects in humans (O'Brien and Dean 1997). One possible explanation is that the CCR5 chemokine receptor function is at least partially redundant and can be compensated for by other chemokine receptors (Baggiolini, Dewald, and Moser 1997).

CCR5 Polymorphisms and Genetic Diversity
We compared different non-human primate CCR5 sequences to human ones and found that they share high similarity at both the nucleotide and the protein level. Computer simulation of some primate CCR5 sequences has revealed high structural similarity (Yang et al. 2000). These results together imply that different primate CCR5 proteins, at least for closely related species, perform the same or similar functions in vivo. In vitro experiments have revealed that HIV can use a non-human primate CCR5 as a co-receptor, and SIV can also use the human CCR5 as a co-receptor (Chen et al. 1997; Marcon et al. 1997; Pretet et al. 1997), findings consistent with the hypothesis that HIV originated as a result of cross-species transmission of SIV from African apes or monkeys to humans. Evidence has been reported to suggest that HIV-2 originated from sooty mangabey SIVs and that HIV-1 originated from chimpanzee SIVs (Gao et al. 1992, 1999).

Many CCR5 polymorphisms have been found in humans, one of which, {Delta}32-deletion, has been identified to be highly resistant to HIV infection (Dean et al. 1996; Liu et al. 1996). Some other polymorphisms may potentially change the efficiency of CCR5 as an HIV/SIV co-receptor, such as M303, which is a single-nucleotide polymorphism (T -> A at nucleotide site 303, T303A) and causes premature translation termination (Carrington et al. 1997; Quillent et al. 1998). Our sequence comparison results showed that notable CCR5 polymorphisms also exist in non-human primates. One fragment deletion allele, {Delta}24-deletion, has been found in mangabey species (Cercocebus torquatus), resulting in a mutant protein that is not expressed at the cell surface and thus does not function as a viral co-receptor (Palacios et al. 1998). Single-nucleotide polymorphisms causing premature translation termination were also found in one rhesus monkey (Macaca mulatta, AF161958, G569A) and one marmoset (Callithrix jacchus, AF161942, C304T). These polymorphisms might also change the efficiency of CCR5 as a HIV/SIV co-receptor and be useful mutant animal models for a HIV/SIV and CCR5 interaction study.

In an earlier study we found that the CCR5 gene evolved faster in rodents than in primates (Zhang, Ryder, and Zhang 1999). Consistent with that finding, the present study showed that the CCR5 genetic diversity in rodents is higher than that in primates (table 1). Differences of the efficiency of the DNA repair system, generation time, and/or metabolic rate have been used to explain rate variation among different lineages (Wu and Li 1985; Li 1997).

Comparison of intra- and interspecific variation is a powerful test to study mutation models. However, so far most studies involving this test compared only very closely related species; a possible reason for this is that there usually were limited sequences available from divergent species, constraining broader comparisons. Because CCR5 acts as an important co-receptor for HIV/SIV infection (Alkhatib et al. 1996; Dragic et al. 1996) and because HIV originated from SIV (Gao et al. 1992, 1999), researchers have had a great interest in identifying genetic variation between different primate CCR5s that modulate pathogenesis. Although there is no evidence showing that CCR5 variation between nonhuman primate species has an effect on different HIV/SIV pathogenetic mechanisms (e.g., Muller-Trutwin et al. 1999), such an interest has generated so many CCR5 sequences of divergent species that we had the opportunity to compare intra- and interspecific variation across a broad range of primate species.

This study focused on the CCR5 coding sequences themselves. Evidence for selection at this locus could also arise as a result of linked sequences, such as promoters (Bamshad et al. 2002). As comparative genomic studies advance, progress can be anticipated as additional evaluations of co-evolution of pathogens and the host proteins upon which they depend provide new insights into the selective focus that affects genome sequences and their variation.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank Dr. Chung-I Wu and two anonymous reviewers for comments on this manuscript. This work was supported by the Alice B. Tyler Perpetual Trust (Y.W.Z. and O.A.R.), the Chinese Academy of Sciences, and the Natural Science Foundation of China (Y.W.Z. and Y.P.Z.).


    Footnotes
 
1 Present address: Department of Molecular Genetics, City of Hope National Medical Center/Beckman Research Institute, Duarte, California. Back

Keith Crandall, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
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Accepted for publication June 5, 2003.





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