Human macrophage inflammatory protein (MIP 1β/CCL4) is a proinflammatory chemokine that promotes leukocyte accumulation in various inflammatory conditions by contributing to establish a gradient that directs immune cells towards infected tissue. Furthermore, by interacting with the chemokine receptor CCR5, it takes part in protective immunity against HIV-1 [1–3].
MIP-1β belongs to the superfamily of the CC chemokine characterized by the contiguous position of their first two conserved cysteines in the N-terminal part of the protein. This domain is followed by a relatively long first loop (the so-called ‘N-loop’), three antiparallel β-strands separated by short loops, and a C-terminal α-helix. Several studies of different chemokines have led to the observation that the N-terminal domain of these proteins is necessary for triggering receptor signalling, whereas their core domain contains the motifs responsible for their tight binding to the receptors [4,5]. The N-loop, which spans residues 13–19, is followed by the ‘310 turn’ that includes residues 20–24. In the latter, it has been shown that the Arg22 basic residue appears to be critical for the interaction with CCR5 through its positive charge .
Moreover, it has been shown recently that two paralogous genes, Act-2 and Lag-1, encode for MIP-1β . The two proteins share a common length and are identical at 89 out of 92 amino acids. Two amino acid differences occur in the signal peptide, whereas the third is in the mature protein.
As only few data are available on the polymorphism of this ligand, we focused our work on the Lag-1 gene and more particularly on the natural R22H variant [8,9], wondering if it could affect HIV-1 infection susceptibility and disease progression.
We thus analysed the frequency of this variant in the French SEROCO cohort composed of 1427 HIV-infected patients . In that way, we amplified selectively the Lag-1 gene with primers targeting this gene that do not interfere with Act-2 gene amplification, and performed R22H detection by allelic discrimination combined with real time polymerase chain reaction assay.
A Kaplan–Meier survival analysis revealed that HIV-1 individuals bearing this mutated allele at the homozygous or heterozygous state exhibit a worse global survival than wild-type individuals (Fig. 1, P = 0.01, with a relative risk of death of 1.35 (95% confidence interval 1.07–1.70)). This observation may be compared with CCR5 delta32 homozygous individuals, who exhibit a rapid disease course when they become HIV-1 infected . This mutation is located in the 310 turn, at a position critical for the binding to CCR5. So far, several studies have described that two clusters of basic residues including R46 and K48 in the ‘40's loop’ and Arg18, Lys19 and Arg22 are implicated in the tight binding of this chemokine to its receptor. Moreover, when the positive charge of Arg22 was replaced with a negative one, a severe decrease in CCR5 binding affinity was observed in a study [6,12]. It seems that these conserved basic residues could interact with conserved negatively charged residues within the CCR5 receptor. The change at the residue 22 of an arginine in histidine, both positively charged, may thus modify the affinity of Lag-1 for the CCR5 receptor.
In view of those results, it is essential to confirm the results on other cohorts of HIV-1-infected subjects. Further biochemical experiments on CCR5 binding affinity and several assays on this variant function then need to be done to clarify this effect after the change of residue at this position. Moreover, an in-silico approach with the Polyphen prediction web tool (http://www.bork.embl-heidelberg.de/PolyPhen) could determine a possibly damaging R22H variant that may affect protein function or structure. Furthermore, as we could not determine a clear change in HIV-1 infection susceptibility in our study, questions were raised about the alteration of the entry of HIV-1 by competition on the co-receptor with R22H. In-vitro experiments on HIV-1 infection inhibition thus need to be investigated.
Sponsorship: This work was supported by the french National Agency of AIDS Research ANRS.
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