Structure and function of CC-chemokine receptor 5 homologues derived from representative primate species and subspecies of the taxonomic suborders Prosimii and Anthropoidea

A chemokine receptor from the seven-transmembrane-domain G-protein-coupled receptor superfamily is an essential coreceptor for the cellular entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) strains. To investigate nonhuman primate CC-chemokine receptor 5 (CCR5) homologue structure and function, we amplified CCR5 DNA sequences from peripheral blood cells obtained from 24 representative species and subspecies of the primate suborders Prosimii (family Lemuridae) and Anthropoidea (families Cebidae, Callitrichidae, Cercopithecidae, Hylobatidae, and Pongidae) by PCR with primers flanking the coding region of the gene. Full-length CCR5 was inserted into pCDNA3.1, and multiple clones were sequenced to permit discrimination of both alleles. Compared to the human CCR5 sequence, the CCR5 sequences of the Lemuridae, Cebidae, and Cercopithecidae shared 87, 91 to 92, and 96 to 99% amino acid sequence homology, respectively. Amino acid substitutions tended to cluster in the amino and carboxy termini, the first transmembrane domain, and the second extracellular loop, with a pattern of species-specific changes that characterized CCR5 homologues from primates within a given family. At variance with humans, all primate species examined from the suborder Anthropoidea had amino acid substitutions at positions 13 (N to D) and 129 (V to I); the former change is critical for CD4-independent binding of SIV to CCR5. Within the Cebidae, Cercopithecidae, and Pongidae (including humans), CCR5 nucleotide similarities were 95.2 to 97.4, 98.0 to 99.5, and 98.3 to 99.3%, respectively. Despite this low genetic diversity, the phylogeny of the selected primate CCR5 homologue sequences agrees with present primate systematics, apart from some intermingling of species of the Cebidae and Cercopithecidae. Constructed HOS.CD4 cell lines expressing the entire CCR5 homologue protein from each of the Anthropoidea species and subspecies were tested for their ability to support HIV-1 and SIV entry and membrane fusion. Other than that of Cercopithecus pygerythrus, all CCR5 homologues tested were able to support both SIV and HIV-1 entry. Our results suggest that the shared structure and function of primate CCR5 homologue proteins would not impede the movement of primate immunodeficiency viruses between species.     

Gene conversion between mammalian CCR2 and CCR5 chemokine receptor genes: a potential mechanism for receptor dimerization

The chemokine receptor genes of the CCR cluster on human chromosome 3p21 play important roles in humoral and cellular immune responses. Several of these receptors have been shown to influence human immunodeficiency virus infection and progression to AIDS, and their homologues may play a role in feline immunodeficiency virus infection. We report the isolation and sequencing of a 150-kb domestic cat BAC clone containing the feline CCR genes CCR1, CCR2, CCR3, and CCR5 to further analyze these four receptor genes within the family Felidae. Comparative and phylogenetic analyses reveal evidence for historic gene conversion between the adjacent CCR2 and CCR5 genes in the Felidae and in three independent mammalian orders (Primates, Cetartiodactyla, and Rodentia), resulting in higher than expected levels of sequence similarity between the two paralogous genes within each order. The gene conversion was restricted to the structural (transmembrane) domains of the CCR2 and CCR5 genes. We also discovered a recent gene conversion event between the third extracellular loop of CCR2 and CCR5 genes that was fixed in Asian lions and found at low frequency in African lions (Panthera leo), suggesting that this domain may have an important functional role. Our results suggest that ongoing parallel gene conversion between CCR2 and CCR5 promotes receptor heterodimerization in independent evolutionary lineages and offers an effective adaptive strategy for gene editing and coevolution among interactive immune response genes in mammals.     

Donor- and ligand-dependent differences in C-C chemokine receptor 5 reexpression

N-terminal modifications of the chemokine RANTES bind to C-C chemokine receptor 5 (CCR5) and block human immunodeficiency virus type 1 (HIV-1) infection with greater efficacy than native RANTES. Modified RANTES compounds induce rapid CCR5 internalization and much slower receptor reexpression than native RANTES, suggesting that receptor sequestration is one mode of anti-HIV activity. The rates of CCR5 internalization and reexpression were compared using the potent n-nonanoyl (NNY)-RANTES derivative and CD4(+) T cells derived from donors with different CCR5 gene polymorphisms. NNY-RANTES caused even more rapid receptor internalization and slower reexpression than aminooxypentane (AOP)-RANTES. Polymorphisms in the promoter and coding regions of CCR5 significantly affected the receptor reexpression rate after exposure of cells to NNY-RANTES. These observations may be relevant for understanding the protective effects of different CCR5 genotypes against HIV-1 disease progression.     

Neutral and Nonneutral Mitochondrial Genetic Variation in Deep-Sea Clams from the Family Vesicomyidae

Nucleotide sequences at two mitochondrial genes from 57 individuals representing eight species of deep-sea clams (Vesicomyidae) were examined for variation consistent with the neutral model of molecular evolution. One gene, cytochrome oxidase subunit I (COI), deviated from the expectations of neutrality by containing an excess of intraspecific nonsynonymous polymorphism. Additionally, one species, Calyptogena kilmeri, showed a significant excess of rare polymorphism specifically at the COI locus. In contrast, a second mitochondrial gene, the large-subunit 16S ribosomal RNA gene (16S), showed little deviation from neutrality either between or within species. Together, COI and 16S show no deviation from neutral expectations by the HKA test, produce congruent phylogenetic relationships between species, and show correlated numbers of fixed differences between species and polymorphism within species. These patterns of both neutral and nonneutral evolution within the mitochondrial genome are most consistent with a model where intraspecific nonsynonymous polymorphism at COI is near neutrality. In addition to examining the forces of molecular evolution, we extend hypotheses about interspecific relationships within this family for geographical locations previously unexamined by molecular methods including habitats near the Middle Atlantic, the Aleutian Trench, and Costa Rica. Received: 10 March 1999 / Accepted: 13 September 1999     

Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans,but not in chimpanzees.

To test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution, nucleotide sequences were determined for the 1041 bp of the NADH dehydrogenase subunit 2 (ND2) gene in 20 geographically diverse humans and 20 common chimpanzees. Contingency tests of neutrality were performed using four mutational categories for the ND2 molecule: synonymous and nonsynonymous mutations in the transmembrane regions, and synonymous and nonsynonymous mutations in the surface regions. The following three topological mutational categories were also used: intraspecific tips, intraspecific interiors, and interspecific fixed differences. The analyses reveal a significantly greater number of nonsynonymous polymorphisms within human transmembrane regions than expected based on interspecific comparisons, and they are inconsistent with a neutral equilibrium model. This pattern of excess nonsynonymous polymorphism is not seen within chimpanzees. Statistical tests of neutrality, such as TAJIMA''s D test, and the D and F tests proposed by FU and LI, indicate an excess of low frequency polymorphisms in the human data, but not in the chimpanzee data. This is consistent with recent directional selection, a population bottleneck or background selection of slightly deleterious mutations in human mtDNA samples. The analyses further support the idea that mitochondrial genome evolution is governed by selective forces that have the potential to affect its use as a "neutral" marker in evolutionary and population genetic studies.     

Geographical variation in selection, from phenotypes to molecules

Molecular technologies now allow researchers to isolate quantitative trait loci (QTLs) and measure patterns of gene sequence variation within chromosomal regions containing important polymorphisms. I develop a simulation model to investigate gene sequence evolution within genomic regions that harbor QTLs. The QTLs influence a trait experiencing geographical variation in selection, which is common in nature and produces obvious differentiation at the phenotypic level. Counter to expectations, the simulations suggest that selection can substantially affect quantitative genetic variation without altering the amount and pattern of molecular variation at sites closely linked to the QTLs. Even with large samples of gene sequences, the likelihood of rejecting neutrality is often low. The exception is situations where strong selection is combined with low migration among demes, conditions that may be common in many plant species. The results have implications for gene sequence surveys and, perhaps more generally, for interpreting the apparently weak connection between levels of molecular and quantitative trait variation within species.     

Long-term balancing selection at the west nile virus resistance gene, Oas1b, maintains transspecific polymorphisms in the house mouse

Oligoadenylate synthetases (OASs) are interferon-inducible enzymes that participate in the first line of defense against a wide range of viral infection. Recent studies have determined that Oas1b, a member of the OAS gene family in the house mouse (Mus musculus), provides specific protection against flavivirus infection (e.g., West Nile virus, dengue fever virus, and yellow fever virus). We characterized the nucleotide sequence variation in coding and noncoding regions of the Oas1b gene for a large number of wild-derived strains of M. musculus and related species. Our sequence analyses determined that this gene is one of the most polymorphic genes ever described in any mammal. The level of variation in noncoding regions of Oas1b is an order of magnitude higher than the level reported for other regions of the mouse genome and is significantly different from the level of intraspecific variation expected under neutrality. Furthermore, a phylogenetic analysis of intronic sequences demonstrated that Oas1b alleles are ancient and that their divergence predates several speciation events, resulting in transspecific polymorphisms. The amino acid sequence of Oas1b is also extremely variable, with 1 out of 7 amino acid positions being polymorphic within M. musculus. Oas1b alleles are comparatively more divergent at synonymous positions than most autosomal genes and the ratio of nonsynonymous to synonymous substitution is remarkably high, suggesting that positive selection has been acting on Oas1b. The ancestry of Oas1b polymorphisms and the high level of amino acid polymorphisms strongly suggest that the allelic variation at Oas1b has been maintained in mouse populations by long-term balancing selection.     

Chemokine Coreceptor Usage by Diverse Primary Isolates of Human Immunodeficiency Virus Type 1

We tested chemokine receptor subset usage by diverse, well-characterized primary viruses isolated from peripheral blood by monitoring viral replication with CCR1, CCR2b, CCR3, CCR5, and CXCR4 U87MG.CD4 transformed cell lines and STRL33/BONZO/TYMSTR and GPR15/BOB HOS.CD4 transformed cell lines. Primary viruses were isolated from 79 men with confirmed human immunodeficiency virus type 1 (HIV-1) infection from the Chicago component of the Multicenter AIDS Cohort Study at interval time points. Thirty-five additional well-characterized primary viruses representing HIV-1 group M subtypes A, B, C, D, and E and group O and three primary simian immunodeficiency virus (SIV) isolates were also used for these studies. The restricted use of the CCR5 chemokine receptor for viral entry was associated with infection by a virus having a non-syncytium-inducing phenotype and correlated with a reduced rate of disease progression and a prolonged disease-free interval. Conversely, broadening chemokine receptor usage from CCR5 to both CCR5 and CXCR4 was associated with infection by a virus having a syncytium-inducing phenotype and correlated with a faster rate of CD4 T-cell decline and progression of disease. We also observed a greater tendency for infection with a virus having a syncytium-inducing phenotype in men heterozygous for the defective CCR5 Δ32 allele (25%) than in those men homozygous for the wild-type CCR5 allele (6%) (P = 0.03). The propensity for infection with a virus having a syncytium-inducing phenotype provides a partial explanation for the rapid disease progression among some men heterozygous for the defective CCR5 Δ32 allele. Furthermore, we did not identify any primary viruses that used CCR3 as an entry cofactor, despite this CC chemokine receptor being expressed on the cell surface at a level commensurate with or higher than that observed for primary peripheral blood mononuclear cells. Whereas isolates of primary viruses of SIV also used STRL33/BONZO/TYMSTR and GPR15/BOB, no primary isolates of HIV-1 used these particular chemokine receptor-like orphan molecules as entry cofactors, suggesting a limited contribution of these other chemokine receptors to viral evolution. Thus, despite the number of chemokine receptors implicated in viral entry, CCR5 and CXCR4 are likely to be the physiologically relevant chemokine receptors used as entry cofactors in vivo by diverse strains of primary viruses isolated from blood.     

Comparison CCR5de132 mutation in the CCR5 gene frequencies in Russians, Tuvinians, and in different groups of HIV-infected individuals

The 32-bp deletion (CCR5del32 mutation) in the CCR5 (chemokine (C-C motif) receptor 5) gene, encoding CCR5 chemokine receptor, is one of the factors determining natural resistance to human immunodeficiency virus (HIV-1) infection. In the present study, the samples of Russians (n = 107), Tuvinians (n = 50), and HIV-infected individuals were examined for the presence of CCR5del32 mutation in the CCR5 gene. The CCR5del32 allele frequency in Russians and Tuvinians constituted 7.84 and 2%, respectively. Among HIV-1 infected individuals, two groups, of macrophage-tropic HIV-1 strain- and T-cell-tropic HIV-1 strain-infected were distinguished. The CCR5del32 allele frequency in the first group (6.45%) was lower than in the second one (8.73%). Statistical treatment of the HIV-1 infected individuals typing data showed that the difference in the CCR5del32 allele frequencies between the groups of sexually (macrophage-tropic) and parenterally (T-cell-tropic) infected individuals observed was within the limit of random deviation.     

The evolutionary history of the CCR5-Delta32 HIV-resistance mutation

The CCR5 chemokine receptor is exploited by HIV-1 to gain entry into CD4+ T cells. A deletion mutation (Delta32) confers resistance against HIV by obliterating the expression of the receptor on the cell surface. Intriguingly, this allele is young in evolutionary time, yet it has reached relatively high frequencies in Europe. These properties indicate that the mutation has been under intense positive selection. HIV-1 has not exerted selection for long enough on the human population to drive the CCR5-Delta32 allele to current frequencies, fueling debate regarding the selective pressure responsible for rise of the allele. The allele exists at appreciable frequencies only in Europe, and within Europe, the frequency is higher in the north. Here we review the population genetics of the CCR5 locus, the debate over the historical selective pressure acting on CCR5-Delta32, the inferences that can potentially be drawn from the geographic distribution of CCR5-Delta32 and the role that other genetic polymorphisms play in conferring resistance against HIV. We also discuss parallel evolution that has occurred at the CCR5 locus of other primate species. Finally, we highlight the promise that therapies based on interfering with the CCR5 receptor could have in the treatment of HIV.     

Two distinct CCR5 domains can mediate coreceptor usage by human immunodeficiency virus type 1.

The chemokine receptor CCR5 is the major fusion coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). To define the structures of CCR5 that can support envelope (Env)-mediated membrane fusion, we analyzed the activity of homologs, chimeras, and mutants of human CCR5 in a sensitive gene reporter cell-cell fusion assay. Simian, but not murine, homologs of CCR5 were fully active as HIV-1 fusion coreceptors. Chimeras between CCR5 and divergent chemokine receptors demonstrated the existence of two distinct regions of CCR5 that could be utilized for Env-mediated fusion, the amino-terminal domain and the extracellular loops. Dual-tropic Env proteins were particularly sensitive to alterations in the CCR5 amino-terminal domain, suggesting that this domain may play a pivotal role in the evolution of coreceptor usage in vivo. We identified individual residues in both functional regions, Asp-11, Lys-197, and Asp-276, that contribute to coreceptor function. Deletion of a highly conserved cytoplasmic motif rendered CCR5 incapable of signaling but did not abrogate its ability to function as a coreceptor, implying the independence of fusion and G-protein-mediated chemokine receptor signaling. Finally, we developed a novel monoclonal antibody to CCR5 to assist in future studies of CCR5 expression.     

Estimates of natural selection due to protein tertiary structure inform the ancestry of biallelic loci

We consider the inference of which of two alleles is ancestral when the alleles have a single nonsynonymous difference and when natural selection acts via protein tertiary structure. Whereas the probability that an allele is ancestral under neutrality is equal to its frequency, under selection this probability depends on allele frequency and on the magnitude and direction of selection pressure. Although allele frequencies can be well estimated from intraspecific data, small fitness differences have a large evolutionary impact but can be difficult to estimate with only intraspecific data. Methods for predicting aspects of phenotype from genotype can supplement intraspecific sequence data. Recently developed statistical techniques can assess effects of phenotypes, such as protein tertiary structure on molecular evolution. While these techniques were initially designed for comparing protein-coding genes from different species, the resulting interspecific inferences can be assigned population genetic interpretations to assess the effect of selection pressure, and we use them here along with intraspecific allele frequency data to estimate the probability that an allele is ancestral. We focus on 140 nonsynonymous single nucleotide polymorphisms of humans that are in proteins with known tertiary structures. We find that our technique for employing protein tertiary structure information yields some biologically plausible results but that it does not substantially improve the inference of ancestral human allele types.     

Utilization of C-C chemokine receptor 5 by the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239.

We examined chemokine receptors for the ability to facilitate the infection of CD4-expressing cells by viruses containing the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239. Expression of either human or simian C-C chemokine receptor CCR5 allowed the SIVmac239 envelope glycoproteins to mediate virus entry and cell-to-cell fusion. Thus, distantly related immunodeficiency viruses such as SIV and the primary human immunodeficiency virus type 1 isolates can utilize CCR5 as an entry cofactor.     

DNA水平自然选择作用的检测

上个世纪60年代,Kimura提出的“中性进化”假说使经典的达尔文自然选择学说遭遇了前所未有的挑战。但新近的研究表明：在DNA水平,越来越多的证据支持“自然选择”的进化理论。这些研究成果得益于近年来大量群体和基因组DNA数据的积累,以及理论群体遗传学的发展。在DNA水平检测选择作用是否存在的方法包括两大类：种内多态性检验和种间差异度检验。前者以Tajima(1989)提出的D检验为代表,后者大都基于“中性条件下,种内与种间进化速率一致”的原理。这些方法以中性假说作为零假设,结合统计检验方法分析DNA数据,被称为“中性检验”。这些方法对于解决一些有关进化的基础理论问题和人类遗传学及生物信息学的深入研究都具有重要意义。本文介绍几个应用广泛的检测方法,以使国内的读者了解它们的基本思路和操作方法。     

Dopamine receptor-interacting protein 78 acts as a molecular chaperone for CCR5 chemokine receptor signaling complex organization

Chemokine receptors are members of the G protein-coupled receptor (GPCR) family. CCR5 and CXCR4 act as co-receptors for human immunodeficiency virus (HIV) and several efforts have been made to develop ligands to inhibit HIV infection by blocking those receptors. Removal of chemokine receptors from the cell surface using polymorphisms or other means confers some levels of immunity against HIV infection. Up to now, very limited success has been obtained using ligand therapies so we explored potential avenues to regulate chemokine receptor expression at the plasma membrane. We identified a molecular chaperone, DRiP78, that interacts with both CXCR4 and CCR5, but not the heterodimer formed by these receptors. We further characterized the effects of DRiP78 on CCR5 function. We show that the molecular chaperone inhibits CCR5 localization to the plasma membrane. We identified the interaction region on the receptor, the F(x)6LL motif, and show that upon mutation of this motif the chaperone cannot interact with the receptor. We also show that DRiP78 is involved in the assembly of CCR5 chemokine signaling complex as a homodimer, as well as with the Gαi protein. Finally, modulation of DRiP78 levels will affect receptor functions, such as cell migration in cells that endogenously express CCR5. Our results demonstrate that modulation of the functions of a chaperone can affect signal transduction at the cell surface.     

The case for selection at CCR5-Delta32

The C-C chemokine receptor 5, 32 base-pair deletion (CCR5-Delta32) allele confers strong resistance to infection by the AIDS virus HIV. Previous studies have suggested that CCR5-Delta32 arose within the past 1,000 y and rose to its present high frequency (5%-14%) in Europe as a result of strong positive selection, perhaps by such selective agents as the bubonic plague or smallpox during the Middle Ages. This hypothesis was based on several lines of evidence, including the absence of the allele outside of Europe and long-range linkage disequilibrium at the locus. We reevaluated this evidence with the benefit of much denser genetic maps and extensive control data. We find that the pattern of genetic variation at CCR5-Delta32 does not stand out as exceptional relative to other loci across the genome. Moreover using newer genetic maps, we estimated that the CCR5-Delta32 allele is likely to have arisen more than 5,000 y ago. While such results can not rule out the possibility that some selection may have occurred at C-C chemokine receptor 5 (CCR5), they imply that the pattern of genetic variation seen at CCR5-Delta32 is consistent with neutral evolution. More broadly, the results have general implications for the design of future studies to detect the signs of positive selection in the human genome.     

The role of gene conversion in determining sequence variation and divergence in the Est-5 gene family in Drosophila pseudoobscura.

Nucleotide sequences of eight Est-5A and Est-5C genes corresponding to previously sequenced Est-5B genes in Drosophila pseudoobscura were determined to compare patterns of polymorphism and divergence among members of this small gene family. The three esterase genes were also sequenced from D. persimilis and D. miranda for interspecific comparisons. The data provide evidence that gene conversion between loci contributes to polymorphism and to the homogenization of the Est5 genes. For Est-5B, which encodes one of the most highly polymorphic proteins in Drosophila, 12% of the segregating amino acid variants appear to have been introduced via gene conversion from other members of the gene family. Interlocus gene conversion can also explain high sequence similarity, especially at synonymous sites, between Est-5B and Est-5A. Tests of neutrality using interspecific comparisons show that levels of polymorphism conform to neutral expectations at each Est-5 locus. However, McDonald-Kreitman tests based on intraspecific gene comparisons indicate that positive selection on amino acids has accompanied Est-5 gene duplication and divergence in D. pseudoobscura.     

Genetic protection against hepatitis B virus conferred by CCR5Delta32: Evidence that CCR5 contributes to viral persistence

Recovery from acute hepatitis B virus (HBV) infection requires a broad, vigorous T-cell response, which is enhanced in mice when chemokine receptor 5 (CCR5) is missing. To test the hypothesis that production of a nonfunctional CCR5 (CCR5Delta32 [a functionally null allele containing a 32-bp deletion]) increases the likelihood of recovery from hepatitis B in humans, we studied 526 persons from three cohorts in which one person with HBV persistence was matched to two persons who recovered from an HBV infection. Recovery or persistence was determined prior to availability of lamivudine. We determined genotypes for CCR5Delta32 and for polymorphisms in the CCR5 promoter and in coding regions of the neighboring genes, chemokine receptor 2 (CCR2) and chemokine receptor-like 2 (CCRL2). Allele and haplotype frequencies were compared among the 190 persons with viral recovery and the 336 with persistence by use of conditional logistic regression. CCR5Delta32 reduced the risk of developing a persistent HBV infection by nearly half (odds ratio [OR], 0.53; 95% confidence interval [CI], 0.33 to 0.83; P = 0.006). This association was virtually identical in persons with and without a concomitant human immunodeficiency virus infection. Of the nine individuals who were homozygous for the deletion, eight recovered from infection (OR, 0.25; 95% CI, 0.03 to 1.99; P = 0.19). None of the other neighboring polymorphisms examined were associated with HBV outcome. These data demonstrate a protective effect of CCR5Delta32 in recovery from an HBV infection, provide genetic epidemiological evidence for a role of CCR5 in the immune response to HBV, and suggest a potential therapeutic treatment for patients persistently infected with HBV.