Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 dna initiate de novo integration
- Sylvain Thierry†1,
- Soundasse Munir†1,
- Eloïse Thierry1,
- Frédéric Subra1,
- Hervé Leh1,
- Alessia Zamborlini2,
- Dyana Saenz5,
- David N Levy4,
- Paul Lesbats3,
- Ali Saïb2,
- Vincent Parissi3,
- Eric Poeschla5,
- Eric Deprez1 and
- Olivier Delelis1Email author
© Thierry et al.; licensee BioMed Central. 2015
Received: 16 December 2014
Accepted: 25 February 2015
Published: 12 March 2015
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 cycles of viral replication.
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.
Integration of the human immunodeficiency virus (HIV-1) DNA into the host cell genome is a key step in the cycle of infectious retroviral particle synthesis [1,2]. Latent HIV-1 reservoirs, such as quiescent memory CD4+ T lymphocytes, constitute the major obstacle to virus eradication during long-term antiretroviral treatment . Post-integration latency probably plays the dominant role in HIV-1 persistence, but pre-integration latency, which involves unintegrated viral DNA, may also be relevant in vivo during quiescent CD4+ T cell infection, in which the virus persists as unintegrated viral DNA that is partially transcribed before cell activation [4-6]. In infected cells, including resting CD4+ T cells, unintegrated viral genomes consist of the linear form (the substrate molecule for integration generated from the reverse transcription process), circular forms resulting from autointegration and circular forms harboring one or two long terminal repeats (LTRs) (1-LTR circles: 1-LTRc and 2-LTR circles: 2-LTRc; respectively). 1-LTRc can be produced during reverse transcription as well as by homologous recombination and 2-LTRc are produced by the non-homologous end joining (NHEJ) pathway involving the ligase 4 protein [7,8]. Circularization of 2-LTRc occurs as a protective host response to the presence of linear double stranded DNA . However, the nature and biological significance of the diverse forms of unintegrated molecules remain unclear in terms of their possible use as templates for transcription or as substrates for integration .
Regarding their relative abundance, viral DNA forms can be ranked: unintegrated linear DNA (DNAL) > integrated provirus (DNAi) > 1-LTRc > 2-LTRc . It is important to note that the repartition of viral genomes is dynamic during the course of infection and is dependent of viral conditions of infections such as mutations in the viral proteins or addition of compounds targeting viral or cellular proteins. For example, raltegravir (RAL), belonging to the INSTI (INtegrase Strand Transfer Inhibitor) family, specifically impairs the strand transfer reaction and greatly alters the relative abundance of viral DNA species . In its presence, 2-LTRc accumulate strongly due to integration inhibition, producing the same effect as integrase-disabling catalytic center mutations such as D116A . It was shown that 2-LTRc represent persisting forms of unintegrated HIV-1 DNAs in non-dividing cells or in primary CD4+ T cells and are notably highly stable if cells remain growth-arrested [12-14]. They are readily detected in vivo during the natural history of HIV-1 disease in the absence of antiviral therapy and recent evidence shows they are increased in long-term elite suppressors . These 2-LTRc have long been considered to be dead-end side products that do not serve as precursors to retroviral integration [16,17]. Such conclusions were drawn from experiments performed under standard condition of infection where 2-LTRc do not accumulate. Unexpectedly, integrase (IN) proteins of HIV-1 and spumaretroviruses can actually cleave the 2-LTR circle junction (which has palindromic features) and, moreover, the enzyme does so in a manner that reproduces the canonical viral CA-3’ terminus, which is needed for proper chromosomal integration and which is normally produced by IN 3’-processing of the linear cDNA [18-21]. Therefore, in the present study, we re-addressed the 2-LTRc status by investigating the consequences in cells of reversing prolonged RAL-induced HIV-1 integration blocks. We show that RAL inhibition is reversible and identify a role of 2-LTRc in the resumption of viral integration. We demonstrate that, after RAL removal, a decrease in the 2-LTRc amount leading to a linear intermediate that is subsequently followed by new integration events.
Raltegravir action is reversible in the virological context
Nature of the viral genome accounting for RAL reversibility and resumption of viral replication
Two hypotheses can be formulated to explain the observed resumption of viral replication after RAL removal. First, small amounts of DNAi that are undetectable by real-time PCR may have been present, or, second, de novo integration may have initiated from accumulated unintegrated DNA. We investigated the possible role of undetectable DNAi in resumption of viral replication, by adding RAL to MT4 cells from days 1–3 (d1-3) instead of days 0–3 (d0-3), thus allowing a 24 hour window for integration to occur. For this d1-3 RAL treatment condition (Figure 2B, open circles), three parallel cultures were also set up at the time of drug removal, on d3, by diluting infected cells with uninfected cells at ratios of 1:10, 1:100 and 1:1000. As expected, viral replication was dose-dependent on the presence of DNAi. The kinetics of viral replication (indicated by the measure of total viral DNA amount) in cells treated with RAL from d0-3 (black diamonds) was more rapid than those of the 100- and 1000-fold dilution (d1-3 RAL) cultures (Figure 2B). Interestingly, this was the case even though no DNAi was detectable on d3 in the d0-3 RAL culture, whereas DNAi was easily detectable at d3 in the d1-3 RAL culture, regardless of the dilution factor (even in the 1:1000 dilution culture) (Figure 2C). Thus, undetectable DNAi generated up to d3 in the d0-3 RAL culture cannot account for the kinetics of replication observed after RAL removal. These results revealed that when RAL blockade is relieved after 3 days, the source of resumed HIV-1 replication is unintegrated DNA which is further used for de novo integration. These data exclude a major role of undetected integrated DNA in viral resumption after RAL removal.
Indeed, RAL removal was associated with a significant increase in integration events by d5 in d0-3 RAL blockade cultures (Figure 2C-D) and a production of infectious viral particles (Additional file 1: Figure S4). Integration events detected on d5 occurred whether or not drugs that prevent successive infection rounds (SAQ, T-20 or AZT) were added at the time of RAL removal on d3. Thus, they fully reflected de novo integration arising from pre-accumulated unintegrated viral DNA originating from infection at d0 and still present at d3. A last condition was performed adding RAL at d0 and AZT at d1 (AZT was maintained until d7), allowing reverse transcription to occur but preventing a weak replication from unintegrated viral DNA as highlighted by Trinite and colleagues . In this condition, when RAL was removed at d3, the amount of DNAi at d5 was similar to the one quantified in the condition without AZT, excluding a major role of the replication from unintegrated viral DNA in the detection of new integration events after RAL removal (Figure 2D). In contrast, the further increase in DNAi on d7 (compared to d5) in the absence of SAQ, T-20 or AZT reflected subsequent rounds of infection (Figure 2D). Newly integration events are thus compatible with synthesis of new viral progeny highlighting that integration from pre-accumulated unintegrated viral DNA is biologically relevant.
Newly integration events after RAL removal result from a DNAL intermediate generated from 2-LTRc
To gain insight into the mechanisms of RAL reversal, we quantified in an exhaustive manner all viral DNA forms: 2-LTRc, 1-LTRc, DNAi and DNAL based on previous reports . More particularly, DNAL was quantified using a linker-mediated PCR approach . Briefly, a linker compatible to the 3’-processed end of the linear DNA, was used. This linker was able to ligate to both unprocessed and 3’-processed DNA. After ligation, the DNA was purified and quantified using quantitative PCR with primers hybridizing in the linker and in the LTR. RAL treatment led to a strong circular viral forms accumulation (1-LTRc and 2-LTRc) where the 2-LTRc representativeness reached 45% of total viral DNA at d3 post-infection (Figure 3B, middle panel, black column). No DNAi was detected when cells were treated with RAL as previously described (Figure 3B, middle panel, white column). At d3 post-infection, when RAL is removed, DNAL represented 100 copies i.e. 0.2% of total viral DNA (Figure 3B, middle and bottom panels). Most importantly, from d3 to d5 post-infection, after RAL removal, we consistently observed a transient and significant increase in DNAL reaching 20% of total viral DNA concomitant with a decrease of 2-LTRc and increase of DNAi at d5 post-infection (Figure 3B, bottom panel). The anti-correlation between the decrease of 2-LTRc and DNAi was also observed at higher m.o.i using RAL or Elvitegravir (EVG), another strand-transfer inhibitor (Additional file 1: Figure S5B and C). When RAL was maintained, no DNAL or DNAi was detected at d4 or d5 post-infection (Figure 3B, middle panel) and no decrease of 2-LTRc percentage is observed. Since RAL does not influence cell division, the fact that the representativeness of 2-LTRc was not changed when RAL was maintained highlights that the 2-LTRc decrease observed when RAL was removed is not due to cell division. Furthermore, when RAL was removed, the increase in the amount of DNAL was only transient since DNAL further decreased concomitant to the observed increase in DNAi. To note, the 1-LTRc amount did not vary significantly between d3 and d5 post-infection (when RAL was maintained or after RAL removal) suggesting that 1-LTRc did not play important role in the observation of neo-integration events.
Taken together, these data demonstrate that RAL removal led to a decrease of 2-LTRc leading to an increase of DNAL that is integrated into the host cell genome. Quantifications of viral DNA genomes at d3, d4 and d5 demonstrate that, even if unintegrated DNA (linear and circular) are diluted due to cell division (from d3 to d5), amount of linear DNA (0.2% of the viral genome i.e. 100 copies) at d3 post-infection does not account for the amount of integrated viral DNA at d5 (18% of the viral genome i.e. 2,737 copies) (Figure 3B). In combination with the reciprocal correlation between 2-LTRc and DNAi, the re-appearance of DNAL at d4 after RAL removal and its further decrease (at d5) when DNAi increases suggests that the resumption of viral replication originates from integration of newly generated DNAL derived from 2-LTRc. A major role of DNAL, provided from the initial infection (d0) after reverse transcription, in the recovery of integration events can then be excluded.
Experiments were also performed with CD4+ primary cells in the same conditions as described for MT4 cells except that the m.o.i. was increased. Again, RAL reversibility was observed when RAL was removed at 48 or 72 hours post-infection (Additional file 1: Figure S7A): Upon RAL removal, HMFS increased demonstrating eGFP expression from newly integrated viral DNA. Quantitative PCR demonstrates that RAL removal, as described for MT4, results in a decrease in 2-LTRc (Additional file 1: Figure S7B, middle panel) correlated by detection of new integration events (Additional file 1: Figure S7B, upper panel). Indeed, integration recovery was accompanied by a 2-fold decrease in 2-LTRc, consistent with an interpretation that 2-LTRc is used as precursors for integration (Additional file 1: Figure S7B, lower panel). These data indicate that RAL action is also reversible in primary cells infection.
The linearization of 2-LTRc requires the integrity of catalytic activity of IN
These results indicate that when integration is inhibited without blocking IN catalytic competence (i.e. in the absence of LEDGF), the inhibition is not necessarily associated with 2-LTRc accumulation since IN is still competent to cleave 2-LTRc (see model in Figure 4B). Accordingly, inhibiting the catalytic activity of IN (RAL treatment or D116N infection) leads to accumulation of 2-LTRc due to the inability of IN to cleave these circular DNA forms. Our data imply that IN plays a key role in controlling the balance between the amounts of DNAL and 2-LTRc through direct effects on 2-LTRc - > DNAL conversion. In this context, RAL removal leads to 2-LTRC cleavage, which in turn produces new DNAL that can integrate and support resumption of viral replication. Taken together with the above-mentioned observation of new DNAL forms after RAL removal, our data indicate that IN catalytic activity is directly involved in the 2-LTRc - > DNAL conversion.
Integrase is found at the palindromic junction of 2-LTRc
The pre-integration complex (PIC) is able to cleave and integrate 2-LTRc
2-LTRc accumulate in HIV-1-infected cells in vitro and in vivo under a variety of conditions, including but not limited to the potent disruption of integrase catalysis caused by RAL. It is generally described that formation of these circular genomes prevents generation of apoptotic signals originating from DNAL extremities. We propose that HIV-1 may utilize these ligated genomes rather than consign them to uselessness. The first one is transcription of the circular forms that can be effective in some circumstances [23,32]. However, in this study, we exclude the role of unintegrated viral DNA in viral transcription leading to viral production. Indeed, we do not observe viral replication when RAL is maintained or when a D116N virus is used. The second strategy, supported by the present data, is to cleave the ligated circle such that it can be chromosomally integrated in the host DNA and therefore represents the main way to account for RAL reversibility. Patients taking integrase inhibitors as part of therapy are unlikely to stop treatment. Other studies are needed to highlight a role of 2-LTR circles after RAL removal in the viral resumption. However, our data reinforce the fact that RAL must be maintained in the treatment and not interrupted. This reversibility may be responsible for the observed failure of intermittent antiretroviral treatments occurring only when RAL was included in combination with other drugs, without RAL resistance mutation detected in integrase . Indeed, HIV “blips” (intermittent episodes of detectable low levels of HIV viremia) and virological failure were observed, for instance, when the NRTI pair (tenofovir + emtricitabine) was combined with RAL, this failure not being observed with any other drug cocktails in the absence of RAL. Our study suggests that this virological failure may be due to de novo integration occurring after treatment interruption, probably from accumulated 2-LTRc. It is a difficult task to estimate the exact impact of 2-LTRc as a substrate for integration under standard infection, i.e. WT infection in the absence of any anti-integrase drugs, due to their low representativeness compared to other viral DNA forms, especially DNAL which represents the main DNA substrate form for integration. Here, we demonstrate that, under conditions where 2-LTRc accumulate in the infected cell, 2-LTRc constitutes a back-up molecule leading to DNAL after IN-dependent cleavage at the palindromic junction. Such a cleavage is compatible with the integration of the HIV-1 genome into the host cell genome as well as with productive infection. Although DNAL remains the substrate for integration, our data highlight that 2-LTRc should not be considered as a dead-end DNA product but, in contrast, could play a crucial role for viral resurgence in several circumstances such as pre-integration latency or RAL interruption.
Our data demonstrate that RAL action is reversible and that unintegrated viral DNA can rescue viral replication after their integration in the host cell genome. Our results 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.
Cells and viruses
HIV-1 stocks were prepared by transfecting 293 T cells with the various HIV-1 molecular clones derived from pNL4-3 (Additional file 1: Figure S2) . Δenv viruses NLENG1-ES-IRES-WT and NLENG1-ES-IRES-D116N encode the WT integrase and catalytically inactive mutant D116N, respectively. Pseudotyping of Δenv viruses was performed by cotransfection of 293 T cells with a VSV-G plasmid. Virus preparations were treated with DNase I (Takara) in the presence of 10 mM MgCl2 at 37°C for 30 min and then untracentrifugation was performed (17,000 g for 1 hour). Aliquots were stored at-80°C. MT4 cells were culture in RPMI 1640. 293 T and HeLa-P4 cells were cultured in Dubelcco’s modified Eagle medium. All mediums were supplemented with 10% fetal calf serum, 100 units penicillin/ml and 100 μg streptomycin/ml (Invitrogen). PBMC were isolated from blood samples using Ficoll-Hypaque gradient centrifugation.
HIV infectivity assay
The single-cycle titers of the virus on HeLa P4 cells were determined on HeLa CD4 LTR-LacZ cells in which the expression of β-galactosidase is inducible by the HIV transactivator protein Tat. Flow cytometry analysis was performed on a FACSCalibur flow cytometer.
40 ng of p24gag antigen per 106 cells was used for infection. 3 hours after infection, cells were washed three times with PBS. Infections of PBMC were performed with 1 μg of p24gag antigen per 106 cells. When required, cells were treated in the presence of the RAL integrase inhibitor, with or without additional drugs (AZT, T-20, SAQ or Efavirenz).
Quantifications of total HIV-1 DNA, 1-LTR circles (1-LTRc), 2-LTR circles (2-LTRc), linear DNA (DNAL) and integrated HIV-1 DNA (DNAi)
All DNA quantifications were performed by real-time PCR on a Light Cycler instrument. For each quantification, an equivalent of 200,000 cells was added in the PCR reaction. The sequences of the primers and probes used for real-time PCR for total HIV-1, 2-LTRc and integrated viral DNA quantifications have been described previously . 1-LTRc and DNAL were quantified according to . Copy numbers of total HIV-1 DNA, 2-LTRc and DNAL were determined from calibration curves obtained by amplifying pre-determined quantities of cloned DNA with matching sequences ranging from 10 to 105 copies. DNAi quantification was performed by real-time Alu-LTR nested PCR, as previously described . DNAi copy number was determined from a calibration curve obtained by concomitant two-stage PCR amplification of serial dilutions of a DNAi standard (from HeLa R7 Neo) mixed with uninfected cell DNA to yield 50,000 cell equivalents. The number of cell equivalents in sample DNA was calculated by amplifying the β-globin gene.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed as previously described  at 24 and 72 hours post-infection. Briefly, 107 infected cells were treated with 1% formaldehyde for 10 min at 37°C. Subsequent procedures were performed on ice with protease inhibitors. Cross-linked cells were harvested, washed with PBS, and lysed in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH = 8.1) for 10 min at 4°C. Chromatin was sonicated (six 10 s pulses at an amplitude of 30%). After centrifugation (14,000 g, 10 min, 4°C), the supernatant was diluted 10-fold with ChIP dilution buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, pH = 8.1, 167 mM NaCl). Diluted extracts were precleared with salmon sperm DNA-protein A-agarose beads (ChIP assay kit, Upstate). One tenth of the diluted extract was kept for quantitative PCR (input). Remaining extracts were incubated for 16 h at 4°C with 1 μg/ml of the specific antibody (from Upstate Biotechnology (anti-histone H3-06755) or from Santa Cruz (anti-HIV-1-integrase 1A1 sc-52418)) and then for 1 hour with salmon sperm DNA-protein A-agarose beads. Following extensive washing, bound DNA fragments were eluted. DNA was recovered by incubation for 4 hours at 65°C in elution buffer supplemented with 200 mM NaCl and incubated with proteinase-K (20 μg/ml) for 1 hour at 45°C. DNA was extracted before PCR quantification. The immunoprecipitated and input DNA were subjected to PCR quantification. Results are expressed as the fraction of immunoprecipitated DNA for each set of conditions.
3×106 cells were lysed in RIPA buffer with protease inhibitors. 50 μg of protein were loaded on SDS-PAGE, transferred overnight to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in TBS-10% milk, incubated overnight at 4°C with primary antibody diluted in TBS-5% milk-0.05% Tween 20 (anti-integrase sc69721, Santa Cruz). Membranes were washed in TBS-0.1% Tween-20 and incubated for 1 h at room temperature with secondary antibody diluted in TBS-5% milk-0.05% Tween 20. Detection was performed by chemiluminescence (ECL).
Extract preparation were prepared as described previously with some modifications to allow the recovery of 2-LTRc complexed with viral and cellular proteins . MT4 cells (2×107) were infected with 20 μg of p24Gag antigen of NLENG1-ES-IRES-D116N or NLENG1-ES-IRES-WT +/− 500 nM RAL in a total volume of 500 μl for 3 h at 37°C. Cells were then washed three times in 20 ml of PBS and resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics at a final concentration of 2 × 105 cells/ml. 72 hours post infection cells were harvested, washed twice with 25 ml of PBS and lysed in 3 cell pellet volumes of lysis buffer (20 mM Tris–HCl pH 8.0, 0.3 M KCl, 5 mM MgCl2, 10% (v/v) glycerol, 0.1% tween 20, 1 mM PMSF protease inhibitor cocktail from Sigma and RAL (500 nM) when required). Cell lysis was completed by two successive rounds of freeze-thaw, then incubated for 30 min at 4°C on rotating wheel. Two successive centrifugation steps at 16,000 g for 30 min at 4°C allowed complete removal of insoluble materials. The collected supernatant corresponding to soluble proteins within the cells was called whole cell extracts (WCE) and passed through 25G gauge needle attached on a 1 ml syringe. An aliquot was harvested and DNA was extracted as previously described. The remaining part was submitted to dialysis 3 and 6 hours at 37°C in a buffer allowing reaction (20 mM HEPES-KOH pH 7.4, 150 mM KCl, 1 mM MgCl2, 4% glycerol, and 1 mM DTT added just before starting the reaction). DNA was then submitted to proteinase K digestion (0.5 mg/ml) for 1 hour at 56°C and extracted with phenol:chloroform:isoamylalcohol 25:24:1. As the protocol does not allow a complete removal of the cellular genome, DNAi was quantified from the host cell genome. The remaining genomic DNA after PIC purification, quantified by quantitative PCR, represent 10% of the initial cellular DNA amount.
The work was performed supported by the Centre National de la Recherche Scientifique (CNRS) and the French National Research Agency against AIDS (ANRS). We thank Benjamin Trinité (University College of Dentistry, USA) for fruitful discussions.
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