Novel HIV-1 co-receptor-CCR5 promoter mutations in simians: identification of two highly polymorphic regions with extensive deletions.

Abstract

Both HIV and SIV use chemokine receptors to gain entry into a susceptible cell. Chemokine receptor-CCR5 is the major co-receptor for the macrophage tropic viruses (R5 viruses), which are mainly involved in the establishment of early infection [1,2]. Individuals possessing a 32 base pair deletion in the CCR5 coding region corresponding to the second extracellular region are strongly protected against HIV-1 infection [3]. This protection is not absolute, as X4 viruses have been isolated from individuals who were homozygous for the Δ32 allele of CCR5 [4]. This mutation is quite common in Caucasian individuals with European heritage [3,5,6] but extremely rare in Asia and Africa [7]. We reported a normal Indian who was heterozygous for the Δ32 allele of the CCR5 gene [8,9]. The promoter region of the CCR5 gene has been worked out in great detail [10,11]. It is quite polymorphic and mutations affecting the progression [11,12] or transmission [13] have been reported. Earlier it was shown that the levels of expression of CCR5 on a susceptible cell determine the ability of the virus to initiate infection successfully [14]. Because infection of different species of monkeys by either SIV or HIV results in widely varying disease patterns and pathogenesis, we sought to characterize approximately 410 bases of the promoter region of three species of monkeys, baboon (Papio anubis), bonnet (Macaca radiata) and rhesus (Macaca mulatta) and aligned them with the published sequence from humans [10] (Fig. 1).Fig. 1.: Sequence analysis of the CCR5 promoter region. Genomic DNA was isolated from the peripheral blood as described by us before [8], and approximately 410 bases from the promoter region were amplified using the primers described by Martin et al. [12]. WT represents the wild-type and MT the mutant version in humans. L/B/R indicates identical sequences in langur (L), baboon (B), and rhesus (R) group of monkeys and ↑ represents deletions. Point mutations are written in bold letters and are underlined. For nucleotide positions refer to Mummidi et al. [10].Several unique features could be observed. With few exceptions, all three monkeys showed almost similar changes, with one mutation showing resemblance at position 675 with humans. Several deletions were observed. In the ISRE binding site (207–221), a C (213) was deleted in all the monkeys besides an A–G transition in langur and rhesus only at position 221. A pentanucleotide – AACAA – deletion (225–229: for nucleotide positions refer to ref. [10]), a trinucleotide – GGG (263–265) deletion that overlaps with the PuF binding site, a dinucleotide – TG (718–719) and single nucleotide G (801) deletion was observed in all the three species of monkeys. There were a number of regions in which more than one and sometimes four bases were substituted. At the end of the CTF/NF-1 binding site (248), sequence CATC was changed to TTCA in all three species of monkeys. Similarly, in the c-myb binding site, GG was changed to AT and TT was changed to GA. It is very likely that such a change would effect the binding of this factor. Similarly, a deletion of GGG from the PuF site is expected to affect the binding of this factor. A change from TTAA in humans to AATT in all three monkeys were observed at position 714–717. Single base change was observed throughout the promoter region and sometimes the changes were specific for a particular species of monkeys. These changes were not caused by polymerase chain reaction-generated mistakes because two different clones from the same species of monkeys were analysed. Nucleotide G–A at positions 237 and 242, G–A at 291 was observed in langur only. On the contrary, Oct-1, TBP, TATA box and other regions were retained in the monkeys, indicating their importance in expression and regulation. On the basis of the clustering of mutations or deletions, we have identified region A (spanning 225–265) and region B (712–719) that are highly polymorphic and show extensive deletions. It is likely that some of these mutations may have created new binding sites that could potentially modulate the expression of the CCR5 gene. The fact that the same kind of mutation was observed among all three species of monkeys suggests that similar selective pressure must have acted on this gene. These deletions and mutations have the potential to change the structure of the chromosome and influence the binding of cellular factors for transcription. They pose interesting questions regarding the evolution and maintenance of these mutations in these species of monkeys. It is tempting to speculate that human CCR5 promoter has acquired new sequences during evolution to achieve a greater and more complex level of regulation. Acknowledgements The authors would like to thank Stephen J. O'Brien, Laboratory of Genomic Diversity, NCI, Fredrick, MD, USA for carrying out CCR5 genotyping, and Sandip K. Basu for support and encouragement. Ganapathy K. Shanmugasundaram Nandini Ramamoorti Akhil C. Banerjea