MIP-1α promoter polymorphism in humans and monkeys: identification of two polymorphic regions characterized by the insertion of unique sequences in monkeys

HIV-1, HIV-2 and SIV use chemokine receptors to gain entry and initiate infection. Although many chemokine receptors have been identified that act as HIV-1 entry co-factors, CCR5 chemokine receptor is primarily used for establishing the infection, governing transmission and tropism [1,2]. During the course of the disease, variants that can use other co-receptors appear, prominent among them being the X4 viruses that use CXCR4 chemokine receptors mainly present on human T lymphocytes. The promoter regions of the chemokine receptor CCR5 have been found to be highly polymorphic in humans [3] and monkeys [4,5], and mutations affecting the progression of HIV in humans have been described [6]. Beta-chemokines (regulated upon activation: normal T cell expressed/secreted; RANTES, MIP-1α and β) are potent inhibitors of infection by R5 viruses and stromal cell-derived factor 1 (SDF-1) (α-chemokine) can block infection by X4 viruses [1]. Recently, mutations in the chemokine RANTES promoter have been described in humans that affect the progression of HIV [7]. We recently reported the presence of novel mutations in the SDF-1 gene [8] and in RANTES promoter [9] regions of monkeys. We sought to characterize 134 bases from the promoter region of Mip-1α that constitutes the basal minimal promoter [10,11], characterized earlier from five normal humans and three species (langur, Prebytis entelus, rhesus, Macaca mulatta and baboon, Papio anubis) of monkeys. The promoter region was amplified by using the following set of primers that spanned the region −18 to −152 [11]. Forward primer: 5'–TCCTGAGCCCCTGTGGTC ACCAGGG and Reverse primer: 5'–CTCTCCTCTTTATAGGCA GCCCTG The conditions for carrying out polymerase chain reaction was same as that described earlier [12]. Amplified product was cloned into a T-tailed vector (pGEM-T-Ez, Promega Biotech, WI, USA), and the recombinant clones were sequenced as described earlier [8,9]. Sequences generated for humans and monkeys were aligned with the published sequence [11], and the results are shown in Fig. 1. Two highly polymorpic regions (A and B) were identified in langur and rhesus monkeys only, the baboon shows no polymorphism at these two regions that are identical to published human sequences. Interestingly, at the −126 position, the baboon shows point mutation that is common with the other two species of monkeys (A to G transition) but at positions −120 and −89, it showed sequences identical to humans. An insertion of a tetranucleotide AAGG in place of a G nucleotide (position −22) (region B) was observed downstream of a conserved TATA box. The insertion of four nucleotides so close to the TATA binding protein has the potential to change the secondary structure of the chromosome that is very likely to affect the efficiency of the Pol II-mediated transcription. The other polymorphic region A was present at the −108 and −109 positions, where TT was substituted by ACTC in the same two species of monkeys (rhesus and langur). This region overlaps the ICK-1 element, which plays an important regulatory role in transcription as four nuclear factors bind in this region [11]. There are three single nucleotide polymorphisms (SNP) (−89, −120 and −126) in the monkeys. Most importantly, we observed A to G transition in one out of five normal unrelated healthy Indians and in rhesus and baboon monkeys at position −126. Langur monkeys do not show any polymorphism at this region and are identical to humans. At least two monkeys from each species were analysed to rule out polymerase chain reaction-generated mistakes.Fig. 1.: Comparison of theMip-1α promoter sequences in humans and different species of monkeys. Genomic DNA was isolated from the peripheral blood as described before[12], and approximately 134 bases of the promoter region was amplified by polymerase chain reaction using the primers described in the text. For nucleotide positions refer to Ritteret al.[11]. Various transcription factor binding sites are shown at the top of the sequence. Point mutation (A to G) at position −126 was observed in one out of five humans only, the rest showed no mutation. * Shows the substitution of ACTC in place of TT (position −108 and −109) and ** represents the substitution of G with AAGG at position −22, which is just downstream of the conserved TATA box. Sequences from −35 to −74 are not shown as it is identical in humans and all the monkeys. Bab.-Hu.Wt. & Mt. indicate identical sequences among wild-type human (from published sequences), mutant human and baboon monkeys. Rhe.-Lan. indicate identical sequences of rhesus and langur monkeys.In summary, we have, for the first time, identified a new kind of mutations (insertions) in the chemokine MIP-1α gene promoter, both in monkeys and humans, the product of which is a chemokine that is known to inhibit the infection of R5 viruses. This insertion of sequences has the potential to create new or modify existing transcription factor binding sites. It was remarkable that the two polymorphic regions that were observed in langur and rhesus monkeys were not found in baboons. Common mutations in some humans at position −126 and monkeys strongly suggests that similar selective pressures must have acted on this gene. These observations raise interesting evolutionary questions and may also help understand why different species of monkeys produce widely varying outcomes to the disease when infected with HIV-1, HIV-2 or SIV. Acknowledgements The authors would like to thank Dr Sandip K. Basu for help and support, and are grateful to Dr (Mrs) Manju Sharma, Secretary, Department of Biotechnology, Government of India, and Professor V. Ramalingaswamy, National Research Professor, AIIMS, New Delhi, for their constant support and encouragement. Ritu Goila Felix Kumar Akhil C. Banerjea