Humans show variation in vulnerability to HIV-1 infection and especially in the clinical outcome after infection, a variability that is governed by multiple host genetic factors [1–3]. Lens epithelium-derived growth factor (LEDGF/p75) is a lentivirus-specific cellular cofactor involved in the tethering of viral preintegration complex to cellular chromatin, directly interacting with viral integrase [4,5]. The crucial role of LEDGF in HIV replication has been evidenced by mutagenesis, RNA interference, transdominant expression of protein domains and knockout studies [4–6]. Small-molecule inhibitors of LEDGF–integrase interaction that block the integration step have been successfully developed, providing further evidence of the relevance of LEDGF as a HIV cellular cofactor . Recently, a series of single-nucleotide polymorphisms (SNPs) in LEDGF were associated to HIV-1 disease progression in two cohorts of African HIV-1-positive individuals . The SNP rs12339417 was associated with slower decline of CD4+ T cells and lower messenger RNA (mRNA) levels of LEDGF. The exonic SNP rs61744944 induced a missense mutation (Q472L) in the C-terminal region of the protein but without an apparent effect on HIV replication in cell culture.
To further investigate the existence of genetic variants within LEDGF, the integrase-binding motif and flanking regions (exons 11 to 14) were analyzed by direct sequencing in a group of 92 HIV progressors and 149 long-term nonprogressors. The progressors were from the HIV Unit of Hospital Germans Trias i Pujol. Selection criterion was a CD4 cell count below 200 cells/μl for patients with 10 years or less of reported infection since first diagnosis. Samples from long term non-progressor (LTNP) patients were provided by the HIV BioBank integrated in the Spanish AIDS Research Network (RIS). Eligibility criteria were a confirmed HIV infection for over 10 years, CD4 cell counts above 500 cells/μl throughout the course of infection and viral loads below 10 000 copies/ml in the absence of antiretroviral therapy for over 10 years . All samples were of white origin. Selected regions were PCR-amplified and sequenced using an ABI Prism 3100 Genetic Analyzer and ABI PRISM BigDye Terminator v3.1 Sequencing Kit (Applied Biosystems, Madrid, Spain).
Five SNPs were found within the noncoding regions analyzed (Fig. 1b); one was previously annotated (rs61744944) and three have been described in the present work (chromosomal positions 15459889, 15459882 and 15459145). The identified SNPs were not informative enough for performing genetic association analysis due to the small observed heterozygosity (H ≤ 0.04). In addition, two missense mutations were identified in two samples belonging to the LTNP cohort (p.Ile436Ser and p.Thr473Ile) (Fig. 1). Both mutations are located in the helix-turn-helix motifs in the C-terminal region of the protein. Alignment of LEDGF proteins from different species showed that amino acids at positions 436 and 473 were conserved throughout evolution, suggesting that these might be important residues for protein function. Sequencing of the entire coding region of the LEDGF gene in these two patients did not identify any other genetic variant (data not shown).
A screening of the prevalence of c.1728C > T (p.Thr473Ile) mutation was performed in 1500 uninfected Spanish individuals obtained from the Spanish DNA Biobank (Salamanca, Spain). The c.1728C > T mutation was typed using a Custom-designed TaqMan SNP genotyping assay (Applied Biosystems) following the manufacturer's procedure and standardized protocol . Reactions were analyzed on an ABI PRISM 7000 (Applied Biosystems) and allele calling was performed using AutoCaller Software, version 1.1 (Applied Biosystems). Mutation p.Thr473Ile was not detected in any of the uninfected Spanish individuals tested, indicating that the prevalence of this genetic variant is low, at least in uninfected general population individuals.
Our results highlight the need for resequencing of candidate genes affecting disease to identify rare genetic variants that go undetected in genome-wide association studies (GWAS) [2,3], as is the case for LEDGF, emphasizing the contribution of its genetic variation to HIV disease. The premise of a GWAS is that an association can uncover the effect of a closely linked functional variant. However, the discovery of a variant that influences the probability of getting a disease can make a contribution to understanding the disease cause only if the causal functionally relevant variation can be identified . Based on this, there is a fundamental difference between the feasibility to identify the functional basis of common as compared to rare variants, which may only be identified by resequencing of candidate genes/regions .
These findings, together with the previous study by Madlala et al. suggest that genetic variation in LEDGF may influence susceptibility to HIV-1 infection and disease progression, which provides in-vivo evidence that LEDGF/p75 is an important host cofactor for HIV-1 replication. Moreover, association of LEDGF genetic variability with HIV/AIDS disease should provide further rational for the development of agents targeting the integrase–LEDGF/p75 interaction as an antiviral strategy.
Conflicts of interest
There are no conflicts of interest.
Financial support: The HIV BioBank RIS is supported by the Instituto de Salud Carlos III and Spanish MICINN (project RD06/0006/0035). This work was supported in part by MICINN projects BFU2009–06958, SAF2010–18917 and FIPSE 360783–09. E.B. is a research fellow from FIS.
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