Species-Specific Changes in the CCR5 Gene from African and Asian Nonhuman Primates

479 BOTH HUMAN IMMUNODEFICIENCY VIRUS (HIV) and simian immunodeficiency virus (SIV) use their external envelope glycoprotein in conjunction with the CD4 cell surface protein of lymphocytes and monocyte-macrophages, and one of the chemokine coreceptors (CCR1-5, CXCR4, Bob and Bonzo) to achieve viral fusion and cell entry.1±3 These chemokine receptors belong to the seven transmembrane (TM)-domain G protein-coupled family of cell surface receptors.1 Experimental studies on interspecies transmission of SIV between patas and tantalus monkeys from Central Africa suggest that although these African nonhuman primate species are susceptible to SIV infection from other species, they do not develop any SIV-related disease.4 In contrast, the Asian nonhuman primate species, free from SIV infection because of their geographic segregation, develop similar AIDS-like symptoms when experimentally infected with SIV. The underlying host genetic reasons for the differences in disease manifestation between African and and Asian monkeys remain unclear. Genetic differences in the CCR5 gene (in particular, a 32-bp deletion) have been thought to play a crucial role in resistance to HIV disease in some patients.5 Similarly, an amino acid mutation, V64I, encoded in the CCR2 gene, has also been found to confer some protection in HIV-1-infected individuals. Despite the availability of data on single amino acid mutations and deletions in the chemokine receptor genes, understanding of the natural resistance to HIV disease in some humans and to SIV disease in African primates remains incomplete. To determine molecular changes in the CCR5 gene of African and Asian nonhuman primates that might suggest the reasons for resistance to SIV disease in African monkeys, and disease susceptibility to SIV in Asian macaques, we sequenced the genes from several representative monkey species of African and Asian origin. The CCR5 genes were amplified by polymerase chain reaction (PCR) from DNA extracted from the peripheral blood mononuclear cells (PBMCs) of 11 different monkeys or apes (belonging to 7 different species) from Africa and Asia: (1) chimpanzee (Pan troglodytes, Gabon, Africa)7; (2) gorilla (Gorilla gorilla, Gabon, Africa); (3) vervet subspecies of African green monkeys (AGMs) (Cercopithecus aethiops pygerythrus, South Africa); (4) baboons (Papio aneubis, Africa); (5) two Asian pigtail macaques (Macaca nemestrina, Indonesia); (6) one stumptail macaque (Macaca arctoides, Indonesia); and (7) one rhesus macaque (Macaca mulatta, India). The PCR, sequencing, and phylogeny protocols used for the analyses of the CCR5 genes are discussed in Fig. 1A and Fig. 2. We identified 12 nucleotide changes in the CCR5 gene, relative to the human sequence, that were specific for each of the 7 nonhuman primate species analyzed (data not shown). By contrast, it has been previously shown that there is relative homogeneity of this gene in Homo sapiens.11 All 12 cysteine residues (critical in protein folding) present at positions 20, 58, 101, 178, 213, 224, 269, 290, 291, 321, 323, and 324 and all 7 TM domains in the CCR5 protein showed a tight evolutionary conservation in both human and nonhuman primates (Fig. 1A). We also observed molecular changes specific to humans and chimpanzees only; humans and AGMs only; rhesus macaques and AGMs only; and pigtail stumptail macaques and AGMs (Table 1). The CCR5 gene of Asian macaques was the most divergent, differing by 2.4±2.7% from humans and apes at the nucleotide level, and by 1.9±4.2% at the peptide level (Fig. 1A), suggesting the antiquity of macaques as a species. This was also reflected in the phylogenetic tree (Fig. 2), where all three macaque species, chosen from two different parts of Asia, formed a distinct cluster. Similarly, the vervet monkeys and baboons also formed distinct clusters