Characterization of LEDGF/p75 Genetic Variants and Association with HIV-1 Disease Progression

Messiaen, Peter; Spiegelaere, Ward De; Alcamí, José; Vervisch, Karen; Acker, Petra Van; Verhasselt, Bruno; Meuwissen, Pieter; Calonge, Esther; Gonzàlez, Núria; Gutierrez-Rodero, Félix; Rodríguez-Martín, Carmen; Sermijn, Erica; Poppe, Bruce; Vogelaers, Dirk; Verhofstede, Chris; Vandekerckhove, Linos

doi:10.1371/journal.pone.0050204

Cited by 10 publications

(16 citation statements)

References 54 publications

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“…S3 in the supplemental material. Samples were derived from two studies which were approved by the ethical committee of Ghent University Hospital (reference numbers B67020071646 [14] …”

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confidence: 99%

Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

et al. 2015

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bEpisomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation. With the persistence of a viral HIV-1 reservoir despite the use of combination antiretroviral therapy (cART) (1), there is a strong need for sensitive tools to characterize the viral reservoir and its dynamics. Recent data indicate that episomal unintegrated circular HIV-1 DNA (two-long terminal repeat [2-LTR] circles) serves as a marker for viral replication (2-4).DNA isolation procedures for 2-LTR recovery were recently compared (5). Although 2-LTR circles were recovered more efficiently by total DNA isolation than by plasmid DNA isolation in vitro, the small number of patients (n ϭ 3) assessed impeded an in vivo evaluation at the lower end of detection. This is especially important, because of the small abundance of 2-LTR circles in patients on cART (6, 7). PCR inhibition by excessive DNA load limits the input material per reaction of total genomic DNA (gDNA) (8), while plasmid DNA isolation which specifically isolates episomal DNA from the bulk chromosomal DNA allows input from larger amounts of cells.Here, two sample processing procedures were compared to quantify 2-LTR episomal HIV-1 DNA by using droplet digital PCR (ddPCR) in peripheral blood mononuclear cells (PBMCs) from a large number of HIV-1-infected patients. This comparison was made by ddPCR, which provides direct absolute quantification with a sensitivity similar to or better than that of real-time quantitative PCR (qPCR), as shown in studies assessing low levels of plasma HIV-1 RNA (9), cell-associated HIV-1 RNA (10), total viral DNA (8, 11-13), and 2-LTR circles (8, 13). In addition, absolute quantification by ddPCR provides a direct measure to assess template loss in pre-PCR processing steps.A modified plasmid DNA isolation method was optimized, using a reference plasmid (pSIF1-H1) (see Fig. S1 in the supplemental material), further referred to as pSIF, spiked in the samples before DNA isolation for normalization to cell equivalents (see Methods and Results in the supplemental material for in-depth optimization and Fig. S2 in the supplemental material for the workflow). The pSIF plasmid was quantified by detection of the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) (see Methods and Table S1 in the supplemental material), further referred to as the pSIF assay.Quantification of pSIF in plasmid DNA and RPP30 in gDNA isolates revealed a higher number of cell equivalents per ddPCR replicate in plasmid DNA than in genomic DNA isolates, i.e., 6.1-fold and 12.7-fold more cell equivalents in the dilution series and in the patient-derived samples (n ϭ 59), respectively (see Results in the supplemental material). 2-LTR quantities were normaliz...

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“…S3 in the supplemental material. Samples were derived from two studies which were approved by the ethical committee of Ghent University Hospital (reference numbers B67020071646 [14] …”

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confidence: 99%

Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

et al. 2015

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“…Our data show a trend towards an association between the T allele (472L) and LTNP status, suggesting that this variat might confer a protective effect. This SNP has not been reported to significantly affect LEDGF/p75 structure [41], IN binding affinity or HIV replication levels [40], [45]. However, data reported by Madlala and colleagues [40] suggested an association between rs61744944 T and lower viral loads in acute phase of HIV-1 infection.…”

Section: Discussionmentioning

confidence: 96%

Association of Single Nucleotide Polymorphisms in the Lens Epithelium-Derived Growth Factor (LEDGF/p75) with HIV-1 Infection Outcomes in Brazilian HIV-1+ Individuals

et al. 2014

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The lens epithelium-derived growth factor p75 (LEDGF/p75), coded by the PSIP1 gene, is an important host co-factor that interacts with HIV-1 integrase to target integration of viral cDNA into active genes. The aim of this study was to investigate the association of SNPs in the PSIP1 gene with disease outcome in HIV-1 infected patients. We performed a genetic association study in a cohort of 171 HIV-1 seropositive Brazilian individuals classified as rapid progressors (RP, n = 69), typical progressors (TP, n = 79) and long-term nonprogressors (LTNP, n = 23). The exonic SNP rs61744944 and 9 tag SNPs were genotyped. A group of 192 healthy subjects was analyzed to determine the frequency of SNPs and haplotypes in the general population. Linkage disequilibrium (LD) analyses indicated that the SNPs analyzed were not in high LD (r2<0.8). Logistic regression models suggested that patients carrying the T allele rs61744944 (472L) were more likely to develop a LTNP phenotype (OR = 4.98; p = 0.05) as compared to TP group. The same trend was observed when LTNPs were compared to the RP group (OR = 3.26). Results of haplotype analyses reinforced this association, since the OR values obtained for the haplotype carrying allele T at rs61744944 also reflected an association with LTNP status (OR = 6.05; p = 0.08 and OR = 3.44; p = 0.12 for comparisons to TP and RP, respectively). The rare missense variations Ile436Ser and Thr473Ile were not identified in the patients enrolled in this study. Gene expression analyses showed lower LEDGF/p75 mRNA levels in peripheral blood mononuclear cells obtained from HIV-1 infected individuals. However, these levels were not influenced by any of the SNPs investigated. In spite of the limited number of LTNPs, these data suggest that the PSIP1 gene could be associated with the outcome of HIV-1 infection. Further analyses of this gene may guide the identification of causative variants to help predict disease course.

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“…All identified missense mutations are located in the helixturn-helix (HTH) motifs at the C-terminal region of the protein, which is after the IBD. Although none of the mutations restricted HIV replication in vitro (29,31,32), these findings suggest that genetic variation in PSIP1 may influence susceptibility to HIV-1 infection and disease progression.…”

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confidence: 78%

“…Although the C-terminal region of LEDGF/p75 is poorly functionally characterized, several genetic variants in HIVinfected patients have been identified outside the known functional domains, variants that might be related to different susceptibilities to HIV infection and disease outcomes (28)(29)(30). Although the identified LEDGF/p75 variants support efficient HIV-1 infection ex vivo, the C-terminal amino acid positions involved are well conserved throughout the evolution, suggesting an important role for protein functionality (31,32).…”

Section: Discussionmentioning

confidence: 99%

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Zinc Finger Endonuclease Targeting PSIP1 Inhibits HIV-1 Integration

Badía

Pauls

Riveira-Muñoz

et al. 2014

Antimicrob Agents Chemother

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Genome editing using zinc finger nucleases (ZFNs) has been successfully applied to disrupt CCR5 or CXCR4 host factors and inhibit viral entry and infection. Gene therapy using ZFNs to modify the PSIP1 gene, which encodes the lens epithelium-derived growth factor (LEDGF) protein, might restrain an early step of the viral replication cycle at the integration level. ZFNs targeting the PSIP1 gene (ZFN LEDGF ) were designed to specifically recognize the sequence after the integrase binding domain (IBD) of the LEDGF/p75 protein. ZFN LEDGF successfully recognized the target region of the PSIP1 gene in TZM-bl cells by heteroduplex formation and DNA sequence analysis. Gene editing induced a frameshift of the coding region and resulted in the abolishment of LEDGF expression at the mRNA and protein levels. Functional assays revealed that infection with the HIV-1 R5 BaL or X4 NL4-3 viral strains was impaired in LEDGF/p75 knockout cells regardless of entry tropism due to a blockade in HIV-1 proviral integration into the host genome. However, residual infection was detected in the LEDGF knockout cells. Indeed, LEDGF knockout restriction was overcome at a high multiplicity of infection, suggesting alternative mechanisms for HIV-1 genome integration rather than through LEDGF/p75. However, the observed residual integration was sensitive to the integrase inhibitor raltegravir. These results demonstrate that the described ZFN LEDGF effectively targets the PSIP1 gene, which is involved in the early steps of the viral replication cycle; thus, ZFN LEDGF may become a potential antiviral agent for restricting HIV-1 integration. Moreover, LEDGF knockout cells represent a potent tool for elucidating the role of HIV integration cofactors in virus replication.

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Characterization of LEDGF/p75 Genetic Variants and Association with HIV-1 Disease Progression

Cited by 10 publications

References 54 publications

Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

Association of Single Nucleotide Polymorphisms in the Lens Epithelium-Derived Growth Factor (LEDGF/p75) with HIV-1 Infection Outcomes in Brazilian HIV-1+ Individuals

Zinc Finger Endonuclease Targeting PSIP1 Inhibits HIV-1 Integration

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