OBJECTIVES:This study aimed to gain a better understanding of the role of insertional mutations in the integrase (IN)-coding sequence of 13 HIV-1-infected people. RESULTS:Here, we present the first documentation of amino acid insertion in the IN-coding sequence of HIV-1-infected people at positions 168, 253, 255 and 260. The consequences of these mutations in terms of viral replication and resistance to INSTIs were analysed using virological and biochemical assays and 3D modelling. Analysis of viral genomes by quantitative PCR demonstrated that insertional mutations reduce reverse transcription efficiency and had different impacts on viral integration. Taken together, we showed that mutants were delayed in their replication. Virological assays using two potent strand-transfer inhibitors (INSTIs), raltegravir and dolutegravir, demonstrated that no resistance to INSTIs was observed. CONCLUSIONS:Our study has revealed that the mutations observed in people living with HIV-1 do not confer resistance to anti-integrase compounds but are detrimental to viral replication.

Multi-branched probes based on 2-hydroxymethylene 8-dimethylaminoquinoline (8-DMAQ) and triphenylamine (TPA) have been synthesized and compared under UV and femtosecond near-infrared two-photon (2P) activation conditions. While octupolar (3) and quadrupolar (2) DMAQ derivatives showed superior ε and 2P absorption cross-section (σ2) values relative to the dipolar (1) probe, the fragmentation efficiency exhibited an inverse correlation with the enhanced absorption. Compound 1 emerged as the most effective probe reported to date for the release of organic substrates under both UV and 2P activation conditions at wavelengths accessible to commercially available lasers, achieving a quantum yield (Q) of 12% with σ2 = 86 GM and uncaging cross-section δu = 10.2 GM at 735 nm.

An essential step in the life cycle of human immunodeficiency virus type 1 (HIV-1) is the packaging of tRNA3Lys during viral assembly. This step, which requires the assembly of a ternary complex consisting of GagPol, mitochondrial lysyl-tRNA synthetase, and tRNA3Lys, is essential for the production of infectious viruses. Indeed, it allows the initiation of reverse transcription in the particles. In this work, we used an in vitro assay to isolate molecules from a chemical library that disrupt the interaction between the integrase domain of GagPol and mitochondrial lysyl-tRNA synthetase. The three selected molecules also inhibit HIV-1 replication in an ex vivo assay. The number of HIV-1 particles produced in the presence of the drugs is greatly reduced, with a half-maximal inhibitory concentration obtained at a drug concentration of 5 µM. In addition, we have shown that the few particles produced in the presence of the drugs exhibit significantly reduced infectivity, due to their inability to synthesize minus-strand strong stop cDNA. In parallel, the tRNA3Lys content of the particles obtained in the presence of drugs decreased significantly. The inability to package tRNA3Lys into virions results in replication-defective particles.IMPORTANCESeveral families of inhibitors have been selected to tackle the human immunodeficiency virus (HIV). Cell entry, reverse transcriptase, integrase, or protease is the most popular targets along the viral life cycle. During the budding step of the virus, the packaging of tRNA3Lys into particles, which serves as a primer for the initiation of reverse transcription in the virus, has been shown to be essential for the production of infectious virions. In this study, we isolated molecules that inhibit viral replication by inhibiting the incorporation of tRNA3Lys into virions. The inhibitors of HIV-1 replication described in this work demonstrate that the HIV-1 tRNA packaging step can be exploited for the development of a new family of drugs with novel resistance profiles.

Triphenylamine-sensitized 8-dimethylaminoquinoline (TAQ) probes showed fair two-photon absorption and fragmentation cross sections in releasing kainate and GABA ligands. The water-soluble PEG and TEG-analogs allowed cell internalization and efficient light-gated liberation of the rhodamine reporter under UV and two-photon (NIR) irradiation conditions.

Purinergic receptors and NOD-like receptor protein 3 (NLRP3) inflammasome regulate inflammation and viral infection, but their effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain poorly understood. Here, we report that the purinergic receptor P2X7 and NLRP3 inflammasome are cellular host factors required for SARS-CoV-2 infection. Lung autopsies from patients with severe coronavirus disease 2019 (COVID-19) reveal that NLRP3 expression is increased in host cellular targets of SARS-CoV-2 including alveolar macrophages, type II pneumocytes and syncytia arising from the fusion of infected macrophages, thus suggesting a potential role of NLRP3 and associated signaling pathways to both inflammation and viral replication. In vitro studies demonstrate that NLRP3-dependent inflammasome activation is detected upon macrophage abortive infection. More importantly, a weak activation of NLRP3 inflammasome is also detected during the early steps of SARS-CoV-2 infection of epithelial cells and promotes the viral replication in these cells. Interestingly, the purinergic receptor P2X7, which is known to control NLRP3 inflammasome activation, also favors the replication of D614G and alpha SARS-CoV-2 variants. Altogether, our results reveal an unexpected relationship between the purinergic receptor P2X7, the NLRP3 inflammasome and the permissiveness to SARS-CoV-2 infection that offers novel opportunities for COVID-19 treatment.

Our work highlights the role of HIV-1 unintegrated viral DNA in viral replication. A virus, resistant to strand-transfer inhibitors, has been selected in vitro . This virus highlights a mutation in the 3'PPT region and not in the integrase gene. This mutation modifies the reverse transcription step leading to the accumulation of 1-LTR circles and not the linear DNA. This accumulation of 1-LTR circles leads to viral replication without integration of the viral genome.

A new set of photoactivatable NADPH mimics bearing a thiourea linkage between a diarylbutadiene and an adenosine moiety functionalized by O-carboxymethyl groups has been designed and synthesized in a convergent strategy. These compounds display absorption and fluorescence emission maxima in DMSO (λmax,abs = 390 nm and λmax,em = 460 nm, respectively) consistent with the previously described analogues, with good fluorescence quantum yields (ΦF = 0.35–0.36), as well as two-photon absorption (σ2 = 10.1 GM at λmax,exc = 780 nm). These molecules could be useful photosensitive tools for biological studies, especially for cellular studies of nitric oxide synthases.

Mitochondria are involved in many cellular pathways and dysfunctional mitochondria are linked to various diseases. Hence efforts have been made to design mitochondria-targeted fluorophores for monitoring the mitochondrial status. However, the factors that govern the mitochondria-targeted potential of dyes are not well-understood. In this context, we synthesized analogues of the TP-2Bzim probe belonging to the vinyltriphenylamine (TPA) class and already described for its capacity to bind nuclear DNA in fixed cells and mitochondria in live cells. These analogues (TP-1Bzim, TPn -2Bzim, TP1+ -2Bzim, TN-2Bzim) differ in the cationic charge, the number of vinylbenzimidazolium branches and the nature of the triaryl core. Using microscopy, we demonstrated that the cationic derivatives accumulate in mitochondria but do not reach mtDNA. Under depolarisation of the mitochondrial membrane, TP-2Bzim and TP1+ -2Bzim translocate to the nucleus in direct correlation with their strong DNA affinity. This reversible phenomenon emphasizes that these probes can be used to monitor ΔΨm variations.

A specific light trigger for activating endothelial Nitric Oxide-Synthase (eNOS) in real time would be of unique value to decipher cellular events associated with eNOS activation or to generate on demand cytotoxic levels of NO at specific sites for cancer research. We previously developed novel tools called nanotriggers (NT), which recognized constitutive NO-synthase, eNOS or neuronal NOS (nNOS), mainly via their 2’ phosphate group which is also present in NADPH in its binding site. Laser excitation of NT1 bound to eNOS triggered recombinant NOS activity and released NO. We recently generated new NTs carrying a 2’ or 3’ carboxylate group or two 2’ and 3’ carboxylate moieties replacing the 2’ phosphate group of NADPH. Among these new NT, only the 3’ carboxylate derivative released NO from endothelial cells upon laser activation. Here, Molecular Dynamics (MD) simulations showed that the 3’ carboxylate NT formed a folded structure with a hydrophobic hub, inducing a good stacking on FAD that likely drove efficient activation of nNOS. This NT also carried an additional small charged group which increased binding to e/nNOS; fluorescence measurements determined a 20-fold improved affinity upon binding to nNOS as compared to NT1 affinity. To gain in specificity for eNOS, we augmented a previous NT with a “hook” targeting variable residues in the NADPH site of eNOS. We discuss the potential of exploiting the chemical diversity within the NADPH site of eNOS for reversal of endothelial dysfunction in cells and for controlled generation of cytotoxic NO-derived species in cancer tissues.

Triphenylamines (TPAs) were previously shown to trigger cell death under prolonged one- or two-photon illumination. Their initial subcellular localization, before prolonged illumination, is exclusively cytoplasmic and they translocate to the nucleus upon photoactivation. However, depending on their structure, they display significant differences in terms of precise initial localization and subsequent photoinduced cell death mechanism. Here, we investigated the structural features of TPAs that influence cell death by studying a series of molecules differing by the number and chemical nature of vinyl branches. All compounds triggered cell death upon one-photon excitation, however to different extents, the nature of the electron acceptor group being determinant for the overall cell death efficiency. Photobleaching susceptibility was also an important parameter for discriminating efficient/inefficient compounds in two-photon experiments. Furthermore, the number of branches, but not their chemical nature, was crucial for determining the cellular uptake mechanism of TPAs and their intracellular fate. The uptake of all TPAs is an active endocytic process but two- and three-branch compounds are taken up via distinct endocytosis pathways, clathrin-dependent or -independent (predominantly caveolae-dependent), respectively. Two-branch TPAs preferentially target mitochondria and photoinduce both apoptosis and a proper necrotic process, whereas three-branch TPAs preferentially target late endosomes and photoinduce apoptosis only.

BACKGROUND:Nitric-oxide synthases (NOS) catalyze the formation of NO using NADPH as electron donor. We have recently designed and synthesized a new series of two-photon absorbing and photoactivatable NADPH analogues (NT). These compounds bear one or two carboxymethyl group(s) on the 2'- or/and 3'-position(s) of the ribose in the adenosine moiety, instead of a 2'-phosphate group, and differ by the nature of the electron donor in their photoactivatable chromophore (replacing the nicotinamide moiety). Here, we addressed the ability of NTs to photoinduce eNOS-dependent NO production in endothelial cells.METHODS:The cellular fate of NTs and their photoinduced effects were studied using multiphoton fluorescence imaging, cell viability assays and a BODIPY-derived NO probe for NO measurements. The eNOS dependence of photoinduced NO production was addressed using two NOS inhibitors (NS1 and L-NAME) targeting the reductase and the oxygenase domains, respectively.RESULTS:We found that, two compounds, those bearing a single carboxymethyl group on the 3'-position of the ribose, colocalize with the Golgi apparatus (the main intracellular location of eNOS) and display high intracellular two-photon brightness. Furthermore, a eNOS-dependent photooxidation was observed for these two compounds only, which is accompanied by a substantial intracellular NO production accounting for specific photocytotoxic effects.CONCLUSIONS:We show for the first time that NT photoactivation efficiently triggers electron flow at the eNOS level and increases the basal production of NO by endothelial cells.GENERAL SIGNIFICANCE:Efficient photoactivatable NADPH analogues targeting NOS could have important implications for generating apoptosis in tumor cells or modulating NO-dependent physiological processes.

Restriction factors are antiviral components of intrinsic immunity which constitute a first line of defense by blocking different steps of the human immunodeficiency virus (HIV) replication cycle. In immune cells, HIV infection is also sensed by several pattern recognition receptors (PRRs), leading to type I interferon (IFN-I) and inflammatory cytokines production that upregulate antiviral interferon-stimulated genes (ISGs). Several studies suggest a link between these two types of immunity. Indeed, restriction factors, that are generally interferon-inducible, are able to modulate immune responses. This review highlights recent knowledge of the interplay between restriction factors and immunity inducing antiviral defenses. Counteraction of this intrinsic and innate immunity by HIV viral proteins will also be discussed.

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 circularized genome can also be efficiently used as a substrate in HIV-1 integrase-mediated integration both in vitro and in eukaryotic cells. Notably, we demonstrate that the palindrome cleavage occurs via a two-step mechanism leading to a blunt-ended DNA product, followed by a classical 3-processing reaction; this cleavage leads to integrase-dependent integration, highlighted by a 5-bp duplication of the host genome. Our results suggest that 2-LTRc may constitute a reserve supply of HIV-1 genomes for proviral integration.

Photodynamic therapy (PDT) is a promising therapeutic method for several diseases, in particular for cancer. This approach uses a photosensitizer, oxygen, and an external light source to produce reactive oxygen species (ROS) at lethal doses to induce cell death. One drawback of current PDT is the use of visible light which has poor penetration in tissues. Such a limitation could be overcome by the use of novel organic compounds compatible with photoactivation under near-infrared light excitation. Triphenylamines (TPAs) are highly fluorescent compounds that are efficient to induce cell death upon visible light excitation (458 nm), but outside the biological spectral window. Interestingly, we recently showed that TPAs target cytoplasmic organelles of living cells, mainly mitochondria, and induce a high ROS production upon 2-photon excitation (in the 760-860 nm range), leading to a fast apoptosis process. However, we observed significant differences among the tested TPA compounds in terms of cell distribution and time courses of cell death-related events (apoptosis vs necrosis). In summary, TPAs represent serious candidates as photosensitizers that are compatible with 2-photon excitation to simultaneously trigger and imaging cell death although the relationship between their subcellular localization and the cell death mechanism involved is still a matter of debate.

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.

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 Mg2+ chelation, allowing to directly probe its Mg2+-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/dolutegravir. The dolutegravir-binding properties derived from fluorescence-based binding assays and drug susceptibilities in terms of catalytic activity, are well correlated. Indeed, dolutegravir-binding to wild-type and F190Y integrases are comparable while strongly compromised with G187R and S217K. Accordingly, the two latter mutants are highly resistant to dolutegravir while F190Y shows only moderate or no resistance. Intrinsic fluorescence properties of dolutegravir are thus particularly suitable for a thorough characterization of both DNA-binding properties of integrase and resistance mutations.

Background:Dolutegravir, an integrase strand-transfer inhibitor (STI), shows a high genetic barrier to resistance. Dolutegravir is reported to be effective against viruses resistant to raltegravir and elvitegravir. In this study, we report the case of a patient treated with dolutegravir monotherapy. Failure of dolutegravir treatment was observed concomitant with the appearance of N155H-K211R-E212T mutations in the integrase (IN) gene in addition to the polymorphic K156N mutation that was present at baseline in this patient.Methods:The impact of N155H-K156N-K211R-E212T mutations was studied in cell-free, culture-based assays and by molecular modelling.Results:Cell-free and culture-based assays confirm that selected mutations in the patient, in the context of the polymorphic mutation K156N present at the baseline, lead to high resistance to dolutegravir requiring that the analysis be done at timepoints longer than usual to properly reveal the results. Interestingly, the association of only N155H and K156N is sufficient for significant resistance to dolutegravir. Modelling studies showed that dolutegravir is less stable in IN/DNA complexes with respect to the WT sequence.Conclusions:Our results indicate that the stability of STI IN/DNA complexes is an important parameter that must be taken into account when evaluating dolutegravir resistance. This study confirms that a pathway including N155H can be selected in patients treated with dolutegravir with the help of the polymorphic K156N that acts as a secondary mutation that enhances the resistance to dolutegravir.

Cadmium is a toxic metal that inactivates DNA-repair proteins via multiple mechanisms, including zinc substitution. In this study, we investigated the effect of Cd(2+) on the Bloom protein (BLM), a DNA-repair helicase carrying a zinc-binding domain (ZBD) and playing a critical role to ensure genomic stability. One characteristics of BLM-deficient cells is the elevated rate of sister chromatid exchanges, a phenomenon that is also induced by Cd(2+). Here, we show that Cd(2+) strongly inhibits both ATPase and helicase activities of BLM. Cd(2+) primarily prevents BLM-DNA interaction via its binding to sulfhydryl groups of solvent-exposed cysteine residues and, concomitantly, promotes the formation of large BLM multimers/aggregates. In contrast to previously described Cd(2+) effects on other zinc-containing DNA-repair proteins, the ZBD appears to play a minor role in the Cd(2+)-mediated inhibition. While the Cd(2+)-dependent formation of inactive multimers and the defect of DNA-binding were fully reversible upon addition of EDTA, the inhibition of the DNA unwinding activity was not counteracted by EDTA, indicating another mechanism of inhibition by Cd(2+) relative to the targeting of a catalytic residue. Altogether, our results provide new clues for understanding the mechanism behind the ZBD-independent inactivation of BLM by Cd(2+) leading to accumulation of DNA double-strand breaks.

Fluorescence based technologies are widely used to study enzyme properties and to understand their mode of action. In particular, fluorescence anisotropy (including both steady-state and time-resolved approaches), fluorescence correlation spectroscopy (FCS) and resonance energy transfer (FRET) were used for monitoring the DNA-binding and enzymatic activities of retroviral integrases. This protein is involved in the integration step of the viral genome that is crucial for retroviral replication. Thanks to these methodologies, important information has been obtained regarding the oligomeric status responsible for integrase catalytic activities as well as the positioning of integrase onto its DNA substrate. We present in this chapter a steady-state fluorescent anisotropy-based assay which monitors, in the same sample, both the DNA-binding step and the subsequent reaction catalysed by integrase allowing the separation of binding and catalytic parameters. The combination of this approach with the analysis of time-resolved fluorescence anisotropy decays is also particularly suitable for studying the relationship between the overall size of integrase-DNA complexes and the catalytic activity. The characterization of catalytically competent complexes by time-resolved fluorescence anisotropy led to the identification of the dimeric form of HIV-1 integrase as the optimal active form for catalytic activity. The protein:DNA ratio determines the aggregative properties of HIV-1 integrase, and high ratios led to aggregative and inactive forms. The better solubility of prototype foamy virus (PFV-1) integrase compared to HIV-1 integrase was compatible with protein labeling using an extrinsic fluorophore, allowing the study of the integrase/DNA interaction specificity by FRET between the fluorescently-labeled PFV-1 integrase and viral DNA ends. It has also been possible to investigate, by a combined approach of FCS and time-resolved fluorescence anisotropy, the oligomeric state of PFV-1 integrase. The results show a monomer-dimer equilibrium of PFV-1 integrase in solution and that DNA promotes protein dimerization although high protein:DNA ratios led to aggregated complexes, confirming results obtained with HIV-1 integrase. The use of fluorescence-based assays for the study of the mechanism of action of anti-integrase compounds is also discussed.