Eric Deprez

Relationship between the oligomeric status of HIV-1 integrase on DNA and enzymatic activity

The 3′-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3′-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN·DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3′-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3′-processing, with a dimer of dimers responsible for the subsequent full integration.

The G140S mutation in HIV integrases from raltegravir-resistant patients rescues catalytic defect due to the resistance Q148H mutation

Raltegravir (MK-0518) is the first integrase (IN) inhibitor to be approved by the US FDA and is currently used in clinical treatment of viruses resistant to other antiretroviral compounds. Virological failure of Raltegravir treatment is associated with mutations in the IN gene following two main distinct genetic pathways involving either the N155 or Q148 residue. Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway. Here, we investigated the viral DNA kinetics for mutants identified in Raltegravir-resistant patients. We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation. We also characterized the corresponding recombinant INs properties. Enzymatic performances closely parallel ex vivo studies. The Q148H mutation ‘freezes’ IN into a catalytically inactive state. By contrast, the conformational transition converting the inactive form into an active form is rescued by the G140S/Q148H double mutation. In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context. Altogether, these results account for the predominance of G140S/Q148H mutants in clinical trials using Raltegravir.

Styrylquinolines, Integrase Inhibitors Acting Prior to Integration: a New Mechanism of Action for Anti-Integrase Agents

ABSTRACT We have previously shown that styrylquinolines (SQLs) are integrase inhibitors in vitro. They compete with the long terminal repeat substrate for integrase. Here, we describe the cellular mode of action of these molecules. We show that SQLs do not interfere with virus entry. In fact, concentrations of up to 20 times the 50% inhibitory concentration did not inhibit cell-to-cell fusion or affect the interaction between GP120 and CD4 in vitro. Moreover, the pseudotype of the retrovirus envelope did not affect drug activity. Quantitative reverse transcription PCR experiments showed that SQLs do not inhibit the entry of the genomic RNA. In contrast, the treatment of human immunodeficiency virus type 1-infected cells with SQLs reduced the amount of the late cDNA, suggesting for the first time that integrase targeting molecules may affect the accumulation of DNA during reverse transcription. The cellular target of SQLs was confirmed by the appearance of mutations in the integrase gene when viruses were grown in the presence of increasing concentrations of SQLs. Finally, these mutations led to SQL-resistant viruses when introduced into the wild-type sequence. In contrast, SQLs were fully active against reverse transcriptase inhibitor- and diketo acid-resistant viruses, positioning SQLs as a second group of anti-integrase compounds.

Impact of Y143 HIV-1 Integrase Mutations on Resistance to Raltegravir In Vitro and In Vivo

ABSTRACT Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drugs activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC50) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.

Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage

BackgroundLEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction.ResultsWe describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket.ConclusionMut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place.

Probing of HIV-1 integrase/DNA interactions using novel analogs of viral DNA

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2′-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3′-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7–9 affected 3′-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5–8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.

Kinetic study of the HIV-1 DNA 3'-end processing.

The 3′‐processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)‐1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady‐state Michaelis–Menten formalism is often applied to calculate characteristic equilibrium/kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a single‐turnover mode and is intrinsically very slow (single‐turnover rate constant = 0.004 min−1), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN–DNA product complexes were very stable over the time‐course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time‐course of product formation approximates to a straight line for 90 min (apparent initial velocity), but we show that this linear phase is due to the slow single‐turnover rate constant and does not indicate steady‐state multiple turnover. Finally, our data ruled out the possibility that there were large amounts of inactive proteins or dead‐end complexes in the assay. Most of complexes initially formed were active although dramatically slow.

Quantitative analysis of the time-course of viral DNA forms during the HIV-1 life cycle

BackgroundHIV-1 DNA is found both integrated in the host chromosome and unintegrated in various forms: linear (DNAL) or circular (1-LTRc, 2-LTRc or products of auto-integration). Here, based on pre-established strategies, we extended and characterized in terms of sensitivity two methodologies for quantifying 1-LTRc and DNAL, respectively, the latter being able to discriminate between unprocessed or 3′-processed DNA.ResultsQuantifying different types of viral DNA genome individually provides new information about the dynamics of all viral DNA forms and their interplay. For DNAL, we found that the 3′-processing reaction was efficient during the early stage of the replication cycle. Moreover, strand-transfer inhibitors (Dolutegravir, Elvitegravir, Raltegravir) affected 3′-processing differently. The comparisons of 2-LTRc accumulation mediated by either strand-transfer inhibitors or catalytic mutation of integrase indicate that 3′-processing efficiency did not influence the total 2-LTRc accumulation although the nature of the LTR-LTR junction was qualitatively affected. Finally, a significant proportion of 1-LTRc was generated concomitantly with reverse transcription, although most of the 1-LTRc were produced in the nucleus.ConclusionsWe describe the fate of viral DNA forms during HIV-1 infection. Our study reveals the interplay between various forms of the viral DNA genome, the distribution of which can be affected by mutations and by inhibitors of HIV-1 viral proteins. In the latter case, the quantification of 3′-processed DNA in infected cells can be informative about the mechanisms of future integrase inhibitors directly in the cell context.

Efficient and Specific Internal Cleavage of a Retroviral Palindromic DNA Sequence by Tetrameric HIV-1 Integrase

Background HIV-1 integrase (IN) catalyses the retroviral integration process, removing two nucleotides from each long terminal repeat and inserting the processed viral DNA into the target DNA. It is widely assumed that the strand transfer step has no sequence specificity. However, recently, it has been reported by several groups that integration sites display a preference for palindromic sequences, suggesting that a symmetry in the target DNA may stabilise the tetrameric organisation of IN in the synaptic complex. Methodology/Principal Findings We assessed the ability of several palindrome-containing sequences to organise tetrameric IN and investigated the ability of IN to catalyse DNA cleavage at internal positions. Only one palindromic sequence was successfully cleaved by IN. Interestingly, this symmetrical sequence corresponded to the 2-LTR junction of retroviral DNA circles—a palindrome similar but not identical to the consensus sequence found at integration sites. This reaction depended strictly on the cognate retroviral sequence of IN and required a full-length wild-type IN. Furthermore, the oligomeric state of IN responsible for this cleavage differed from that involved in the 3′-processing reaction. Palindromic cleavage strictly required the tetrameric form, whereas 3′-processing was efficiently catalysed by a dimer. Conclusions/Significance Our findings suggest that the restriction-like cleavage of palindromic sequences may be a general physiological activity of retroviral INs and that IN tetramerisation is strongly favoured by DNA symmetry, either at the target site for the concerted integration or when the DNA contains the 2-LTR junction in the case of the palindromic internal cleavage.

Impairment of human immunodeficiency virus type-1 integrase SUMOylation correlates with an early replication defect

HIV-1 integrase (IN) orchestrates the integration of the reverse transcribed viral cDNA into the host cell genome and participates also in other steps of HIV-1 replication. Cellular and viral factors assist IN in performing its multiple functions, and post-translational modifications contribute to modulate its activities. Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO acceptor sites. Viruses harboring SUMOylation site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Because SUMOylation-defective IN mutants retained WT catalytic activity, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication.