Eloïse THIERRY - Academia.edu (original) (raw)

Papers by Eloïse THIERRY

The Journal of antimicrobial chemotherapy, 2015

Strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) are now commonly used to ... more Strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) are now commonly used to inhibit HIV-1 integration. To date, three main pathways conferring raltegravir/elvitegravir resistance, involving residues Y143, Q148 and N155, have been described. However, no pathway has been clearly described for dolutegravir resistance. The aim of this study was to characterize the susceptibility of two mutations, F121Y and G118R, originally described in patients failing raltegravir-containing regimens, to dolutegravir and raltegravir, and then to compare the resistance of these mutations with that of other well-known mutations involved in raltegravir resistance. Both the F121Y and G118R mutations were introduced by site-directed mutagenesis into the pNL4.3 backbone and studied in cell-based and in vitro assays. The effects of the mutations were characterized at the different steps of infection by quantitative PCR. Results obtained with in vitro and ex vivo assays consistently showed...

The Journal of antimicrobial chemotherapy, 2015

Strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) are now commonly used to ... more Strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) are now commonly used to inhibit HIV-1 integration. To date, three main pathways conferring raltegravir/elvitegravir resistance, involving residues Y143, Q148 and N155, have been described. However, no pathway has been clearly described for dolutegravir resistance. The aim of this study was to characterize the susceptibility of two mutations, F121Y and G118R, originally described in patients failing raltegravir-containing regimens, to dolutegravir and raltegravir, and then to compare the resistance of these mutations with that of other well-known mutations involved in raltegravir resistance. Both the F121Y and G118R mutations were introduced by site-directed mutagenesis into the pNL4.3 backbone and studied in cell-based and in vitro assays. The effects of the mutations were characterized at the different steps of infection by quantitative PCR. Results obtained with in vitro and ex vivo assays consistently showed...

Additional file 1: Figure S1. Structure of the native and tailless mononucleosomes used in the wo... more Additional file 1: Figure S1. Structure of the native and tailless mononucleosomes used in the work. The globular structure of the nucleosomes was analyzed by loading 250 ng of native or tailless MN on 0.8% native agarose gel run 4 h at 50 V and 4 °C then stained with SYBR®Safe 20 min. Assembled MNs migrate between 600 and 700 bp and the naked 601 DNA fragment at 147 bp. Figure S2. A. Effect of LEDGF/p75 on HIV-1 integration in vitro. Integration assay was performed as done in Fig. 2 using naked 601 DNA coupled to magnetic beads and increasing concentration of LEDGF in the presence or absence of PEG and DMSO. B. Integration activity catalyzed by IN and IN/LEDGF complex on native or tail less nucleosomes in the absence of PEG and DMSO. Integration assay was performed as done in Fig. 2 using native or tail less nucleosomes coupled to magnetic beads and either IN or IN/LEDGF complex. All values are shown as the mean ± standard deviation (error bars) of three independent sets of experim...

Journal of Biological Chemistry

Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catal... more Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catalyzed by the retroviral integrase. To date, the only substrate described to be involved in this critical reaction is the linear viral DNA produced in reverse transcription. However, during HIV-1 infection, two-long terminal repeat DNA circles (2-LTRcs) are also generated through the ligation of the viral DNA ends by the host cell's nonhomologous DNA end-joining pathway. These DNAs contain all the genetic information required for viral replication, but their role in HIV-1's life cycle remains unknown. We previously showed that both linear and circular DNA fragments containing the 2-LTR palindrome junction can be efficiently cleaved in vitro by recombinant integrases, leading to the formation of linear 3′-processed–like DNA. In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circula...

L’integration est une etape cruciale dans la replication du VIH-1, assurant la stabilite et l’exp... more L’integration est une etape cruciale dans la replication du VIH-1, assurant la stabilite et l’expression de son genome. Elle est donc la cible d’inhibiteurs de 1ere et 2nde generation, tels que le Dolutegravir (DTG). L’emergence de voies de resistance aux inhibiteurs de 1ere generation a mis en evidence leur faible barriere genetique a la resistance. Au contraire, aucune voie de resistance specifique au DTG n’a encore emerge chez les patients, illustrant sa plus haute barriere genetique. Des mutations de resistance aux inhibiteurs de 1ere generation lui conferent cependant de la resistance. Mes travaux de these ont permis la caracterisation de mutations conferant differents niveaux de resistance au DTG, notamment par des methodes exploitant ses proprietes de fluorescence.D’autre part, nous avons etudie les roles de l’ADN viral non integre circulaire du VIH-1, formes minoritaires lors d’une infection integrative mais accumulees en cas d’inhibition de l’integration. Nos resultats indi...

Scientific reports, Jan 25, 2017

FDA-approved integrase strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) ef... more FDA-approved integrase strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) efficiently inhibit HIV-1 replication. Here, we present fluorescence properties of these inhibitors. Dolutegravir displays an excitation mode particularly dependent on Mgchelation, allowing to directly probe its Mg-dependent binding to the prototype foamy virus (PFV) integrase. Dolutegravir-binding studied by both its fluorescence anisotropy and subsequent emission enhancement, strictly requires a preformed integrase/DNA complex, the ten terminal base pairs from the 3'-end of the DNA reactive strand being crucial to optimize dolutegravir-binding in the context of the ternary complex. From the protein side, mutation of any catalytic residue fully abolishes dolutegravir-binding. We also compared dolutegravir-binding to PFV F190Y, G187R and S217K mutants, corresponding to HIV-1 F121Y, G118R and G140S/Q148K mutations that confer low-to-high resistance levels against raltegravir/dolutegravi...

Retrovirology, Jan 28, 2017

Stable insertion of the retroviral DNA genome into host chromatin requires the functional associa... more Stable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein-protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome. Since histone tails are candidates for interactions with the incoming intasomes we have investigated whether they could participate in modulating the nucleosomal integration process. We show here that histone tails are required for an optimal association between HIV-1 integrase (IN) and the nucleosome for efficient integration. We also demonstrate direct interactions between IN and the amino-terminal tail of human histone H4 in vitro. Structure/function studies enabled us to identify amino acids in the carboxy-terminal domain of IN that are important for this interaction. Analysis of the nucleosome-binding properties of catalytically active mutated I...

Thermal Science, 2016

This work proposes an estimation of the possible heat recovery of self-heating compost piles for ... more This work proposes an estimation of the possible heat recovery of self-heating compost piles for building applications. The energy released during the aerobic composting of lignin and cellulose-based materials is computed by solving an inverse problem. The method consists first in an experimental phase with measurement of the temperature within the heap, then a numerical procedure allows for the inverse identification of the heat production due to the chemical reaction of composting. The simulation results show a good accordance with the experiments for the chosen source-term model. Comparing the results to the theoretical values for the energy released by aerobic composting provides an estimate for the efficiency of the reaction. The reached temperatures and recovered energy fit with the order of magnitude of building needs.

Frontiers in Microbiology, 2017

Integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL), elvitegravir, or dolute... more Integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL), elvitegravir, or dolutegravir (DTG), are efficient antiretroviral agents used in HIV treatment in order to inhibit retroviral integration. By contrast to RAL treatments leading to well-identified mutation resistance pathways at the integrase level, recent clinical studies report several cases of patients failing DTG treatment without clearly identified resistance mutation in the integrase gene raising questions for the mechanism behind the resistance. These compounds, by impairing the integration of HIV-1 viral DNA into the host DNA, lead to an accumulation of unintegrated circular viral DNA forms. This viral DNA could be at the origin of the INSTI resistance by two different ways. The first one, sustained by a recent report, involves 2-long terminal repeat circles integration and the second one involves expression of accumulated unintegrated viral DNA leading to a basal production of viral particles maintaining the viral information.

Scientific Reports, 2016

Integration of HIV-1 linear DNA into host chromatin is required for high levels of viral expressi... more Integration of HIV-1 linear DNA into host chromatin is required for high levels of viral expression, and constitutes a key therapeutic target. Unintegrated viral DNA (uDNA) can support only limited transcription but may contribute to viral propagation, persistence and/or treatment escape under specific situations. The molecular mechanisms involved in the differential expression of HIV uDNA vs integrated genome (iDNA) remain to be elucidated. Here, we demonstrate, for the first time, that the expression of HIV uDNA is mainly supported by 1-LTR circles, and regulated in the opposite way, relatively to iDNA, following NF-κB pathway modulation. Upon treatment activating the NF-κB pathway, NF-κB p65 and AP-1 (cFos/cJun) binding to HIV LTR iDNA correlates with increased iDNA expression, while uDNA expression decreases. On the contrary, inhibition of the NF-κB pathway promotes the expression of circular uDNA, and correlates with Bcl-3 and AP-1 binding to its LTR region. Finally, this study identifies NF-κB subunits and Bcl-3 as transcription factors binding the HIV promoter differently depending on viral genome topology, and opens new insights on the potential roles of episomal genomes during the HIV-1 latency and persistence. Integration of the HIV genome is an essential step of the retroviral cycle, supporting massive production of viral particles. Strikingly, integrated viral DNAs (iDNAs) only represent a minor part of reverse-transcribed genomes, that remains mainly under unintegrated viral forms (uDNAs) at early times post-infection as well as during untreated chronic infection 1,2. Unintegrated HIV genomes mainly include linear DNAs (DNA L) that are quickly degraded, and circular DNAs containing one or two long terminal repeats (1-LTRc and 2-LTRc, respectively) 3,4. Conversely, 1-LTRc and 2-LTRc episomal DNAs remain intrinsically stable and only diminish through cell death or division (e.g. following T cell activation) 4,5. Several studies have demonstrated the stability of uDNAs in non-dividing primary macrophages and resting CD4 T cells 6-9. This stability is supported by clinical trials highlighting the high levels and persistence of 2-LTRc in HIV-1 controllers 10,11. Until recently, uDNAs were considered as "dead-end products" of reverse transcription. However, several reports have now established that circular uDNAs can support low levels of HIV expression 7,8,12-15 , can constitute a reserve substrate for de novo integration 16 , and be a source of infectious virus 17-19 (for a review 20). Therefore, HIV uDNAs should be considered as potential reserve genomes that could be involved in persistence and treatment escape. Although expression of uDNAs can be several orders of magnitude lower than that of an integrated provirus 13,15 , it can lead to the expression of accessory proteins such as Nef and Tat 14,15. Importantly, this low level of Nef expression is sufficient to down-regulate CD4 expression on host cell surfaces and to induce T cell activation 7 , highlighting the importance of uDNA expression on HIV-host interaction. LTR-mediated expression from HIV iDNA is well documented. Host transcription factor mobilization and chromatin decondensation are required for robust HIV transcription, so that HIV post-integration latency is considered as a transcription factor restriction phenomenon 21,22. Notably, HIV-1 LTR contains binding sites for several inducible transcription factors, including NFκ B, NFAT or AP-1 (c-Jun/cFos family members) (for reviews 21,23,24). HIV-1 transcription is thus tightly coupled to cell type and activation status. After appropriate stimulation of

Legend to supplementary figures. Supplementary figure S1 Titers of pTrip PGK-Pac/CMV-Gfp vector s... more Legend to supplementary figures. Supplementary figure S1 Titers of pTrip PGK-Pac/CMV-Gfp vector stocks with different IN, assessed as measurements of p24/ul, vRNAc and TU/ul. Thirty stocks of vectors were analyzed to determine their content in viral capsid protein p24, in viral RNA copies (vRNAc) and transducing units (TU) per volume of supernatant. (a) The content of p24/ul of supernatant does not vary between stocks of vectors carrying different IN. (b) Although fluctuating, the content of vRNAc/ul was not statistically different in the group of vectors analyzed. (c) No significant differences in TU/ul are found between the different stocks of vectors. (d) No statistical differences are found between groups of vectors when comparing values of the ratio TU ml-1 /p24 ml-1. (e) Significant differences are measured when comparing the ratio TU/vRNAc, with higher values for D167H vectors as compared to any of all other vector types. Error bars represent mean+SEM. Statistics, One way ANOVA and Tuckey pot hoc test; (NS) p > 0.05; (*) p< 0.05; (**) p< 0.01; (***) p < 0.001. Supplementary figure S2 Analysis of pTrip PGK-Pac/CMV-Gfp transduction efficiencies in HEK-293T cells with vectors bearing different integrases with respect to time and vector input. (a) Percentage of GFP+ cells transduced with 10 vRNAc of vectors Q168A, D64V, N and LQ at different times after transduction as assessed with FACS. (b) Comparison of GFP MFI, at day 21 after transduction, in HEK-293T cells incubated with increasing M.O.I. of pTrip PGK-Pac/CMV-Gfp vectors carrying different IN mutations. At M.O.I. 300 and 900, MFI is higher in cells transduced with D167H as compared to that transduced with WT while MFI remains steady with vectors Q168A, D64V, N and LQ. At M.O.I. of 2700 vRNAc, MFI of vectors WT and D167H are equivalent sugesting toxicity associated to overtransduction with vector D167H. (c) GFP expression in HEK 293T cells transduced M.O.I. of 300 vRNAc with vectors Q168A, D64V, N and LQ at different time points after transduction; note that at day 7 after transduction all IDLV vectors allowed transducing nearly 100 % of the cells. Experiments (a) were done 3 times in duplicate with different stocks of vectors. Experiments (b) and (c) were done twice in duplicate. Error bars represent mean+SEM. Statistics: in all experiments two way ANOVA and Bonferroni's post-hoc test; (NS) p > 0.05; (*) p< 0.05; (**) p< 0.01; (***)p < 0.001. Supplementary figure S3 Comparison of transduction efficiency and integration rate of different IDLV in HEK-293T cells after transduction with increasing input of vector pTrip PGK-Pac/CMV-Gfp. (a) Percentage of GFP+ cells 4 days after transduction with 100, 300, 900 or 2700 vRNAc/cell of vectors carrying IN Q168A, D64V, N or LQ. (b) Number of puromycin resistant colonies counted 2 weeks after transduction with 100, 300, 900 or 2700 vRNAC/cell of vectors carrying IN Q168A, D64V, N or LQ. Experiments were done twice in duplicate. Non-transduced cells all died in puromycin medium. Error bars represent mean+SEM. Statistics: two way ANOVA and Bonferroni posttest, (NS) p > 0.05; (*) p< 0.005; (**) p< 0.01; (***) p < 0.001.

Journal of Antimicrobial Chemotherapy, 2015

Objectives: HIV-1 integration can be efficiently inhibited by strand-transfer inhibitors such as ... more Objectives: HIV-1 integration can be efficiently inhibited by strand-transfer inhibitors such as raltegravir, elvitegravir or dolutegravir. Three pathways conferring raltegravir/elvitegravir cross-resistance (involving integrase residues Q148, N155 and Y143) were identified. Dolutegravir, belonging to the second generation of strand-transfer compounds, inhibits the Y143 and N155 pathways, but is less efficient at inhibiting the Q148 pathway. The aim of this study was to characterize the combination of two pathways involved in raltegravir resistance described in one patient failing a dolutegravir regimen for their propensity to confer dolutegravir resistance. Methods: In this study, a patient first failing a regimen including raltegravir was treated with dolutegravir and showed an increase in viruses carrying a combination of two pathways (N155 and Q148). Impacts of these mutations on integrase activity and resistance to strand-transfer inhibitors were characterized using both in vitro and virological assays. Results: Our data showed that the combination of N155H, G140S and Q148H mutations led to strong resistance to dolutegravir. Conclusions: Combination of N155H, G140S and Q148H mutations originating from two distinct resistance pathways to raltegravir or elvitegravir led to a high level of dolutegravir resistance. Due to its high genetic barrier of resistance, it would be reasonable to use dolutegravir in first-line therapy before emergence of raltegravir or elvitegravir resistance.

Chemistry & Biology, 2015

The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro... more The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high #

Retrovirology, Jan 12, 2015

Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase... more Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation. Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cyc...

Journal of Antimicrobial Chemotherapy, 2015

Molecular Therapy—Nucleic Acids, 2014

HIV-1 derived vectors are among the most efficient for gene transduction in mammalian tissues. As... more HIV-1 derived vectors are among the most efficient for gene transduction in mammalian tissues. As the parent virus, they carry out vector genome insertion into the host cell chromatin. Consequently, their preferential integration in transcribed genes raises several conceptual and safety issues. To address part of these questions, HIV-derived vectors have been engineered to be nonintegrating. This was mainly achieved by mutating HIV-1 integrase at functional hotspots of the enzyme enabling the development of streamlined nuclear DNA circles functional for transgene expression. Few integrase mutant vectors have been successfully tested so far for gene transfer. They are cleared with time in mitotic cells, but stable within nondividing retina cells or neurons. Here, we compared six HIV vectors carrying different integrases, either wild type or with different mutations (D64V, D167H, Q168A, K186Q+Q214L+Q216L, and RRK262-264AAH) shown to modify integrase enzymatic activity, oligomerization, or interaction with key cellular cofactor of HIV DNA integration as LEDGF/p75 or TNPO3. We show that these mutations differently affect the transduction efficiency as well as rates and patterns of integration of HIV-derived vectors suggesting their different processing in the nucleus. Surprisingly and most interestingly, we report that an integrase carrying the D167H substitution improves vector transduction efficiency and integration in both HEK-293T and primary CD34+ cells.

Retrovirology, 2015

Background: Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by t... more Background: Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation. Results: Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication. Conclusions: Our data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.

The Journal of antimicrobial chemotherapy, 2015

Strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) are now commonly used to ... more Strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) are now commonly used to inhibit HIV-1 integration. To date, three main pathways conferring raltegravir/elvitegravir resistance, involving residues Y143, Q148 and N155, have been described. However, no pathway has been clearly described for dolutegravir resistance. The aim of this study was to characterize the susceptibility of two mutations, F121Y and G118R, originally described in patients failing raltegravir-containing regimens, to dolutegravir and raltegravir, and then to compare the resistance of these mutations with that of other well-known mutations involved in raltegravir resistance. Both the F121Y and G118R mutations were introduced by site-directed mutagenesis into the pNL4.3 backbone and studied in cell-based and in vitro assays. The effects of the mutations were characterized at the different steps of infection by quantitative PCR. Results obtained with in vitro and ex vivo assays consistently showed...

The Journal of antimicrobial chemotherapy, 2015

Strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) are now commonly used to ... more Strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) are now commonly used to inhibit HIV-1 integration. To date, three main pathways conferring raltegravir/elvitegravir resistance, involving residues Y143, Q148 and N155, have been described. However, no pathway has been clearly described for dolutegravir resistance. The aim of this study was to characterize the susceptibility of two mutations, F121Y and G118R, originally described in patients failing raltegravir-containing regimens, to dolutegravir and raltegravir, and then to compare the resistance of these mutations with that of other well-known mutations involved in raltegravir resistance. Both the F121Y and G118R mutations were introduced by site-directed mutagenesis into the pNL4.3 backbone and studied in cell-based and in vitro assays. The effects of the mutations were characterized at the different steps of infection by quantitative PCR. Results obtained with in vitro and ex vivo assays consistently showed...

Additional file 1: Figure S1. Structure of the native and tailless mononucleosomes used in the wo... more Additional file 1: Figure S1. Structure of the native and tailless mononucleosomes used in the work. The globular structure of the nucleosomes was analyzed by loading 250 ng of native or tailless MN on 0.8% native agarose gel run 4 h at 50 V and 4 °C then stained with SYBR®Safe 20 min. Assembled MNs migrate between 600 and 700 bp and the naked 601 DNA fragment at 147 bp. Figure S2. A. Effect of LEDGF/p75 on HIV-1 integration in vitro. Integration assay was performed as done in Fig. 2 using naked 601 DNA coupled to magnetic beads and increasing concentration of LEDGF in the presence or absence of PEG and DMSO. B. Integration activity catalyzed by IN and IN/LEDGF complex on native or tail less nucleosomes in the absence of PEG and DMSO. Integration assay was performed as done in Fig. 2 using native or tail less nucleosomes coupled to magnetic beads and either IN or IN/LEDGF complex. All values are shown as the mean ± standard deviation (error bars) of three independent sets of experim...

Journal of Biological Chemistry

Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catal... more Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catalyzed by the retroviral integrase. To date, the only substrate described to be involved in this critical reaction is the linear viral DNA produced in reverse transcription. However, during HIV-1 infection, two-long terminal repeat DNA circles (2-LTRcs) are also generated through the ligation of the viral DNA ends by the host cell's nonhomologous DNA end-joining pathway. These DNAs contain all the genetic information required for viral replication, but their role in HIV-1's life cycle remains unknown. We previously showed that both linear and circular DNA fragments containing the 2-LTR palindrome junction can be efficiently cleaved in vitro by recombinant integrases, leading to the formation of linear 3′-processed–like DNA. In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circula...

L’integration est une etape cruciale dans la replication du VIH-1, assurant la stabilite et l’exp... more L’integration est une etape cruciale dans la replication du VIH-1, assurant la stabilite et l’expression de son genome. Elle est donc la cible d’inhibiteurs de 1ere et 2nde generation, tels que le Dolutegravir (DTG). L’emergence de voies de resistance aux inhibiteurs de 1ere generation a mis en evidence leur faible barriere genetique a la resistance. Au contraire, aucune voie de resistance specifique au DTG n’a encore emerge chez les patients, illustrant sa plus haute barriere genetique. Des mutations de resistance aux inhibiteurs de 1ere generation lui conferent cependant de la resistance. Mes travaux de these ont permis la caracterisation de mutations conferant differents niveaux de resistance au DTG, notamment par des methodes exploitant ses proprietes de fluorescence.D’autre part, nous avons etudie les roles de l’ADN viral non integre circulaire du VIH-1, formes minoritaires lors d’une infection integrative mais accumulees en cas d’inhibition de l’integration. Nos resultats indi...

Scientific reports, Jan 25, 2017

FDA-approved integrase strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) ef... more FDA-approved integrase strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) efficiently inhibit HIV-1 replication. Here, we present fluorescence properties of these inhibitors. Dolutegravir displays an excitation mode particularly dependent on Mgchelation, allowing to directly probe its Mg-dependent binding to the prototype foamy virus (PFV) integrase. Dolutegravir-binding studied by both its fluorescence anisotropy and subsequent emission enhancement, strictly requires a preformed integrase/DNA complex, the ten terminal base pairs from the 3'-end of the DNA reactive strand being crucial to optimize dolutegravir-binding in the context of the ternary complex. From the protein side, mutation of any catalytic residue fully abolishes dolutegravir-binding. We also compared dolutegravir-binding to PFV F190Y, G187R and S217K mutants, corresponding to HIV-1 F121Y, G118R and G140S/Q148K mutations that confer low-to-high resistance levels against raltegravir/dolutegravi...

Retrovirology, Jan 28, 2017

Stable insertion of the retroviral DNA genome into host chromatin requires the functional associa... more Stable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein-protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome. Since histone tails are candidates for interactions with the incoming intasomes we have investigated whether they could participate in modulating the nucleosomal integration process. We show here that histone tails are required for an optimal association between HIV-1 integrase (IN) and the nucleosome for efficient integration. We also demonstrate direct interactions between IN and the amino-terminal tail of human histone H4 in vitro. Structure/function studies enabled us to identify amino acids in the carboxy-terminal domain of IN that are important for this interaction. Analysis of the nucleosome-binding properties of catalytically active mutated I...

Thermal Science, 2016

This work proposes an estimation of the possible heat recovery of self-heating compost piles for ... more This work proposes an estimation of the possible heat recovery of self-heating compost piles for building applications. The energy released during the aerobic composting of lignin and cellulose-based materials is computed by solving an inverse problem. The method consists first in an experimental phase with measurement of the temperature within the heap, then a numerical procedure allows for the inverse identification of the heat production due to the chemical reaction of composting. The simulation results show a good accordance with the experiments for the chosen source-term model. Comparing the results to the theoretical values for the energy released by aerobic composting provides an estimate for the efficiency of the reaction. The reached temperatures and recovered energy fit with the order of magnitude of building needs.

Frontiers in Microbiology, 2017

Integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL), elvitegravir, or dolute... more Integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL), elvitegravir, or dolutegravir (DTG), are efficient antiretroviral agents used in HIV treatment in order to inhibit retroviral integration. By contrast to RAL treatments leading to well-identified mutation resistance pathways at the integrase level, recent clinical studies report several cases of patients failing DTG treatment without clearly identified resistance mutation in the integrase gene raising questions for the mechanism behind the resistance. These compounds, by impairing the integration of HIV-1 viral DNA into the host DNA, lead to an accumulation of unintegrated circular viral DNA forms. This viral DNA could be at the origin of the INSTI resistance by two different ways. The first one, sustained by a recent report, involves 2-long terminal repeat circles integration and the second one involves expression of accumulated unintegrated viral DNA leading to a basal production of viral particles maintaining the viral information.

Scientific Reports, 2016

Integration of HIV-1 linear DNA into host chromatin is required for high levels of viral expressi... more Integration of HIV-1 linear DNA into host chromatin is required for high levels of viral expression, and constitutes a key therapeutic target. Unintegrated viral DNA (uDNA) can support only limited transcription but may contribute to viral propagation, persistence and/or treatment escape under specific situations. The molecular mechanisms involved in the differential expression of HIV uDNA vs integrated genome (iDNA) remain to be elucidated. Here, we demonstrate, for the first time, that the expression of HIV uDNA is mainly supported by 1-LTR circles, and regulated in the opposite way, relatively to iDNA, following NF-κB pathway modulation. Upon treatment activating the NF-κB pathway, NF-κB p65 and AP-1 (cFos/cJun) binding to HIV LTR iDNA correlates with increased iDNA expression, while uDNA expression decreases. On the contrary, inhibition of the NF-κB pathway promotes the expression of circular uDNA, and correlates with Bcl-3 and AP-1 binding to its LTR region. Finally, this study identifies NF-κB subunits and Bcl-3 as transcription factors binding the HIV promoter differently depending on viral genome topology, and opens new insights on the potential roles of episomal genomes during the HIV-1 latency and persistence. Integration of the HIV genome is an essential step of the retroviral cycle, supporting massive production of viral particles. Strikingly, integrated viral DNAs (iDNAs) only represent a minor part of reverse-transcribed genomes, that remains mainly under unintegrated viral forms (uDNAs) at early times post-infection as well as during untreated chronic infection 1,2. Unintegrated HIV genomes mainly include linear DNAs (DNA L) that are quickly degraded, and circular DNAs containing one or two long terminal repeats (1-LTRc and 2-LTRc, respectively) 3,4. Conversely, 1-LTRc and 2-LTRc episomal DNAs remain intrinsically stable and only diminish through cell death or division (e.g. following T cell activation) 4,5. Several studies have demonstrated the stability of uDNAs in non-dividing primary macrophages and resting CD4 T cells 6-9. This stability is supported by clinical trials highlighting the high levels and persistence of 2-LTRc in HIV-1 controllers 10,11. Until recently, uDNAs were considered as "dead-end products" of reverse transcription. However, several reports have now established that circular uDNAs can support low levels of HIV expression 7,8,12-15 , can constitute a reserve substrate for de novo integration 16 , and be a source of infectious virus 17-19 (for a review 20). Therefore, HIV uDNAs should be considered as potential reserve genomes that could be involved in persistence and treatment escape. Although expression of uDNAs can be several orders of magnitude lower than that of an integrated provirus 13,15 , it can lead to the expression of accessory proteins such as Nef and Tat 14,15. Importantly, this low level of Nef expression is sufficient to down-regulate CD4 expression on host cell surfaces and to induce T cell activation 7 , highlighting the importance of uDNA expression on HIV-host interaction. LTR-mediated expression from HIV iDNA is well documented. Host transcription factor mobilization and chromatin decondensation are required for robust HIV transcription, so that HIV post-integration latency is considered as a transcription factor restriction phenomenon 21,22. Notably, HIV-1 LTR contains binding sites for several inducible transcription factors, including NFκ B, NFAT or AP-1 (c-Jun/cFos family members) (for reviews 21,23,24). HIV-1 transcription is thus tightly coupled to cell type and activation status. After appropriate stimulation of

Legend to supplementary figures. Supplementary figure S1 Titers of pTrip PGK-Pac/CMV-Gfp vector s... more Legend to supplementary figures. Supplementary figure S1 Titers of pTrip PGK-Pac/CMV-Gfp vector stocks with different IN, assessed as measurements of p24/ul, vRNAc and TU/ul. Thirty stocks of vectors were analyzed to determine their content in viral capsid protein p24, in viral RNA copies (vRNAc) and transducing units (TU) per volume of supernatant. (a) The content of p24/ul of supernatant does not vary between stocks of vectors carrying different IN. (b) Although fluctuating, the content of vRNAc/ul was not statistically different in the group of vectors analyzed. (c) No significant differences in TU/ul are found between the different stocks of vectors. (d) No statistical differences are found between groups of vectors when comparing values of the ratio TU ml-1 /p24 ml-1. (e) Significant differences are measured when comparing the ratio TU/vRNAc, with higher values for D167H vectors as compared to any of all other vector types. Error bars represent mean+SEM. Statistics, One way ANOVA and Tuckey pot hoc test; (NS) p > 0.05; (*) p< 0.05; (**) p< 0.01; (***) p < 0.001. Supplementary figure S2 Analysis of pTrip PGK-Pac/CMV-Gfp transduction efficiencies in HEK-293T cells with vectors bearing different integrases with respect to time and vector input. (a) Percentage of GFP+ cells transduced with 10 vRNAc of vectors Q168A, D64V, N and LQ at different times after transduction as assessed with FACS. (b) Comparison of GFP MFI, at day 21 after transduction, in HEK-293T cells incubated with increasing M.O.I. of pTrip PGK-Pac/CMV-Gfp vectors carrying different IN mutations. At M.O.I. 300 and 900, MFI is higher in cells transduced with D167H as compared to that transduced with WT while MFI remains steady with vectors Q168A, D64V, N and LQ. At M.O.I. of 2700 vRNAc, MFI of vectors WT and D167H are equivalent sugesting toxicity associated to overtransduction with vector D167H. (c) GFP expression in HEK 293T cells transduced M.O.I. of 300 vRNAc with vectors Q168A, D64V, N and LQ at different time points after transduction; note that at day 7 after transduction all IDLV vectors allowed transducing nearly 100 % of the cells. Experiments (a) were done 3 times in duplicate with different stocks of vectors. Experiments (b) and (c) were done twice in duplicate. Error bars represent mean+SEM. Statistics: in all experiments two way ANOVA and Bonferroni's post-hoc test; (NS) p > 0.05; (*) p< 0.05; (**) p< 0.01; (***)p < 0.001. Supplementary figure S3 Comparison of transduction efficiency and integration rate of different IDLV in HEK-293T cells after transduction with increasing input of vector pTrip PGK-Pac/CMV-Gfp. (a) Percentage of GFP+ cells 4 days after transduction with 100, 300, 900 or 2700 vRNAc/cell of vectors carrying IN Q168A, D64V, N or LQ. (b) Number of puromycin resistant colonies counted 2 weeks after transduction with 100, 300, 900 or 2700 vRNAC/cell of vectors carrying IN Q168A, D64V, N or LQ. Experiments were done twice in duplicate. Non-transduced cells all died in puromycin medium. Error bars represent mean+SEM. Statistics: two way ANOVA and Bonferroni posttest, (NS) p > 0.05; (*) p< 0.005; (**) p< 0.01; (***) p < 0.001.

Journal of Antimicrobial Chemotherapy, 2015

Objectives: HIV-1 integration can be efficiently inhibited by strand-transfer inhibitors such as ... more Objectives: HIV-1 integration can be efficiently inhibited by strand-transfer inhibitors such as raltegravir, elvitegravir or dolutegravir. Three pathways conferring raltegravir/elvitegravir cross-resistance (involving integrase residues Q148, N155 and Y143) were identified. Dolutegravir, belonging to the second generation of strand-transfer compounds, inhibits the Y143 and N155 pathways, but is less efficient at inhibiting the Q148 pathway. The aim of this study was to characterize the combination of two pathways involved in raltegravir resistance described in one patient failing a dolutegravir regimen for their propensity to confer dolutegravir resistance. Methods: In this study, a patient first failing a regimen including raltegravir was treated with dolutegravir and showed an increase in viruses carrying a combination of two pathways (N155 and Q148). Impacts of these mutations on integrase activity and resistance to strand-transfer inhibitors were characterized using both in vitro and virological assays. Results: Our data showed that the combination of N155H, G140S and Q148H mutations led to strong resistance to dolutegravir. Conclusions: Combination of N155H, G140S and Q148H mutations originating from two distinct resistance pathways to raltegravir or elvitegravir led to a high level of dolutegravir resistance. Due to its high genetic barrier of resistance, it would be reasonable to use dolutegravir in first-line therapy before emergence of raltegravir or elvitegravir resistance.

Chemistry & Biology, 2015

The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro... more The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high #

Retrovirology, Jan 12, 2015

Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase... more Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation. Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cyc...

Journal of Antimicrobial Chemotherapy, 2015

Molecular Therapy—Nucleic Acids, 2014

HIV-1 derived vectors are among the most efficient for gene transduction in mammalian tissues. As... more HIV-1 derived vectors are among the most efficient for gene transduction in mammalian tissues. As the parent virus, they carry out vector genome insertion into the host cell chromatin. Consequently, their preferential integration in transcribed genes raises several conceptual and safety issues. To address part of these questions, HIV-derived vectors have been engineered to be nonintegrating. This was mainly achieved by mutating HIV-1 integrase at functional hotspots of the enzyme enabling the development of streamlined nuclear DNA circles functional for transgene expression. Few integrase mutant vectors have been successfully tested so far for gene transfer. They are cleared with time in mitotic cells, but stable within nondividing retina cells or neurons. Here, we compared six HIV vectors carrying different integrases, either wild type or with different mutations (D64V, D167H, Q168A, K186Q+Q214L+Q216L, and RRK262-264AAH) shown to modify integrase enzymatic activity, oligomerization, or interaction with key cellular cofactor of HIV DNA integration as LEDGF/p75 or TNPO3. We show that these mutations differently affect the transduction efficiency as well as rates and patterns of integration of HIV-derived vectors suggesting their different processing in the nucleus. Surprisingly and most interestingly, we report that an integrase carrying the D167H substitution improves vector transduction efficiency and integration in both HEK-293T and primary CD34+ cells.

Retrovirology, 2015

Background: Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by t... more Background: Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation. Results: Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication. Conclusions: Our data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.