Laboratory of Virology, National Institute of Immunology, JNU Campus, New Delhi-110067, India.
Sponsorship: This work was supported by a grant from the Department of Biotechnology, Government of India, to National Institute of Immunology, New Delhi, and to the corresponding author (A.C.B.).
Received: 3 October 2000; accepted: 6 February 2001.
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  and monkeys [4,5], and mutations affecting the progression of HIV in humans have been described . 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 . Recently, mutations in the chemokine RANTES promoter have been described in humans that affect the progression of HIV . We recently reported the presence of novel mutations in the SDF-1 gene  and in RANTES promoter  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 .
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 . 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 , 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 . 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.
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.
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.
Akhil C. Banerjea
1. Berger EA, Murphy PM, Farber JM. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol 1999, 17: 657 –700.
2. Edinger AL, Clements EJ, Doms RW. Chemokine and orphan receptors in HIV-2 and SIV tropism and pathogenesis. Virology 1999, 260: 211 –221.
3. Martin PM, Dean M, Smith MW. et al
. Genetic acceleration of AIDS progression by a promoter variant of CCR5. Science 1998, 282: 1907 –1910.
4. Shanmugasundaram GK, Ramamoorti N, Banerjea AC. Novel HIV-1 coreceptor-CCR5 promoter mutations in simians: identification of two highly polymorphic regions with extensive deletions. AIDS 2000, 14: 2201 –2202.
5. Mummidi S, Bamshad M, Ahuja SS. et al
. Evolution of human and nonhuman primate CC chemokine receptor 5 gene and mRNA: potential roles for haplotype and mRNA diversity, differential haplotype-specific trancriptional activity and altered transcription factor binding to polymorphic nucleotides in the pathogenesis of HIV-1 and SIV. J Biol Chem 2000, 275: 18946 –18961.
6. McDermott DH, Zimmerman PA, Guignard F. et al
. CCR5 promoter polymorphism and HIV-1 disease progression. Lancet 1998, 352: 866 –870.
7. Liu H, Chao D, Nakayama EE. et al
. Polymorphism in RANTES promoter affects HIV-1 disease progression. Proc Natl Acad Sci U S A 1999, 96: 4581 –4585.
8. Ramamoorti N, Banerjea AC. Novel SDF-1 genemutations in simians: presence of GG to AA transition in the 3’ untranslated region. AIDS 2000, 14: 1279 –1281.
9. Ramamoorti N, Goila R, Banerjea AC. Extensive polymorphism in regulated upon activation normal T cell expressed/secreted promoter of bonnet, baboon and rhesus monkeys. AIDS 2000, 14: 2401 –2403.
10. Nakao M, Nomiyama H, Shimada K. Structures of human genes coding for cytokine LD78 and their expression. Mol Cell Biol 1990, 10: 3646 –3658.
11. Ritter LM, Bryans M, Abdo O, Sharma V, Wilkie NM. MIP-1
αnuclear protein (MNP), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the humanMIP-1α gene.
Mol Cell Biol 1995, 15: 3110 –3118.
12. Husain S, Goila R, Shahi S, Banerjea AC. First report of a healthy Indian heterozygous for Δ32 mutant of HIV-1 coreceptor-CCR5 gene. Gene 1998, 207: 141 –147.