RESUMO
The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3'-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.
Assuntos
Integrase de HIV , HIV-1 , Integração Viral , Integrase de HIV/metabolismo , Integrase de HIV/química , Integrase de HIV/genética , HIV-1/metabolismo , HIV-1/fisiologia , Humanos , DNA Viral/metabolismo , DNA Viral/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/químicaRESUMO
The human immunodeficiency virus-1 (HIV-1) nucleocapsid protein (NCp7) is a nucleic acid chaperone protein with two highly conserved zinc fingers. To exert its key roles in the viral cycle, NCp7 interacts with several host proteins. Among them, the human NoL12 protein (hNoL12) was previously identified in genome wide screens as a potential partner of NCp7. hNoL12 is a highly conserved 25 kDa nucleolar RNA-binding protein implicated in the 5'end processing of ribosomal RNA in the nucleolus and thus in the assembly and maturation of ribosomes. In this work, we confirmed the NCp7/hNoL12 interaction in cells by Förster resonance energy transfer visualized by Fluorescence Lifetime Imaging Microscopy and co-immunoprecipitation. The interaction between NCp7 and hNoL12 was found to strongly depend on their both binding to RNA, as shown by the loss of interaction when the cell lysates were pretreated with RNase. Deletion mutants of hNoL12 were tested for their co-immunoprecipitation with NCp7, leading to the identification of the exonuclease domain of hNoL12 as the binding domain for NCp7. Finally, the interaction with hNoL12 was found to be specific of the mature NCp7 and to require NCp7 basic residues. IMPORTANCE HIV-1 mature nucleocapsid (NCp7) results from the maturation of the Gag precursor in the viral particle and is thus mostly abundant in the first phase of the infection which ends with the genomic viral DNA integration in the cell genome. Most if not all the nucleocapsid partners identified so far are not specific of the mature form. We described here the specific interaction in the nucleolus between NCp7 and the human nucleolar protein 12, a protein implicated in ribosomal RNA maturation and DNA damage response. This interaction takes place in the cell nucleolus, a subcellular compartment where NCp7 accumulates. The absence of binding between hNoL12 and Gag makes hNoL12 one of the few known specific cellular partners of NCp7.
Assuntos
HIV-1 , Proteínas Nucleares , Proteínas do Nucleocapsídeo , Proteínas de Ligação a RNA , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Humanos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/genética , HIV-1/metabolismo , Proteínas Nucleares/metabolismo , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Dedos de Zinco , Transferência Ressonante de Energia de Fluorescência , Ligação Proteica , ImunoprecipitaçãoRESUMO
The HIV-1 Gag protein playing a key role in HIV-1 viral assembly has recently been shown to interact through its nucleocapsid domain with the ribosomal protein L7 (RPL7) that acts as a cellular co-factor promoting Gag's nucleic acid (NA) chaperone activity. To further understand how the two proteins act together, we examined their mechanism individually and in concert to promote the annealing between dTAR, the DNA version of the viral transactivation element and its complementary cTAR sequence, taken as model HIV-1 sequences. Gag alone or complexed with RPL7 was found to act as a NA chaperone that destabilizes cTAR stem-loop and promotes its annealing with dTAR through the stem ends via a two-step pathway. In contrast, RPL7 alone acts as a NA annealer that through its NA aggregating properties promotes cTAR/dTAR annealing via two parallel pathways. Remarkably, in contrast to the isolated proteins, their complex promoted efficiently the annealing of cTAR with highly stable dTAR mutants. This was confirmed by the RPL7-promoted boost of the physiologically relevant Gag-chaperoned annealing of (+)PBS RNA to the highly stable tRNALys3 primer, favoring the notion that Gag recruits RPL7 to overcome major roadblocks in viral assembly.
Assuntos
Infecções por HIV/genética , HIV-1/genética , Proteínas Ribossômicas/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Sequência de Aminoácidos/genética , Infecções por HIV/virologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Chaperonas Moleculares/genética , Conformação de Ácido Nucleico , Ácidos Nucleicos/genética , RNA Viral/genética , Montagem de Vírus/genéticaRESUMO
The human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol.
Assuntos
HIV-1 , Membrana Celular , Genômica , HIV-1/genética , Humanos , RNA Guia de Cinetoplastídeos , RNA Viral , Montagem de Vírus , Dedos de ZincoRESUMO
In the quest for the identification of the light emitter(s) responsible for the firefly bioluminescence, the study of oxyluciferin analogues with controlled chemical and electronic structures is of particular importance. In this article, we report the results of our experimental and computational investigation of the pH-dependent absorption spectra characterizing three analogues bound into the luciferase cavity, together with adenosine-monophosphate (AMP). While the analogue microscopic pKa values do not differ much from their reference values, it turns out that the AMP protonation state is analogue-dependent and never doubly-deprotonated. A careful analysis of the interactions evidences the main role of E344 glutamic acid, as well as the flexibility of the cavity which can accommodate any oxyluciferin analogue. The consideration of the absorption spectra suggests that the oxyluciferin enolate form has to be excluded from the list of the bioluminescence reaction products.
Assuntos
Indóis/química , Luciferases de Vaga-Lume/química , Pirazinas/química , Animais , Domínio Catalítico , Concentração de Íons de Hidrogênio , Luciferases de Vaga-Lume/metabolismo , Espectrometria de FluorescênciaRESUMO
Due to its essential roles in the viral replication cycle and to its highly conserved sequence, the nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 is a target of choice for inhibiting replication of the virus. Most NCp7 inhibitors identified so far are small molecules. A small number of short peptides also act as NCp7 inhibitors by competing with its nucleic acid (NA) binding and chaperone activities but exhibit antiviral activity only at relatively high concentrations. In this work, in order to obtain more potent NCp7 competitors, we designed a library of longer peptides (10-17 amino acids) whose sequences include most of the NCp7 structural determinants responsible for its specific NA binding and destabilizing activities. Using an in vitro assay, the most active peptide (pE) was found to inhibit the NCp7 destabilizing activity, with a 50% inhibitory concentration in the nanomolar range, by competing with NCp7 for binding to its NA substrates. Formulated with a cell-penetrating peptide (CPP), pE was found to accumulate into HeLa cells, with low cytotoxicity. However, either formulated with a CPP or overexpressed in cells, pE did not show any antiviral activity. In vitro competition experiments revealed that its poor antiviral activity may be partly due to its sequestration by cellular RNAs. The selected peptide pE therefore appears to be a useful tool for investigating NCp7 properties and functions in vitro, but further work will be needed to design pE-derived peptides with antiviral activity.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Desenho de Fármacos , HIV-1/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Sequência de Aminoácidos , Avaliação Pré-Clínica de Medicamentos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/química , HIV-1/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Ácidos Nucleicos/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
BACKGROUND: In HIV-1 infected cells, the integrated viral DNA is transcribed by the host cell machinery to generate the full length HIV-1 RNA (FL RNA) that serves as mRNA encoding for the Gag and GagPol precursors. Virion formation is orchestrated by Gag, and the current view is that a specific interaction between newly made Gag molecules and FL RNA initiates the process. This in turn would cause FL RNA dimerization by the NC domain of Gag (GagNC). However the RNA chaperoning activity of unprocessed Gag is low as compared to the mature NC protein. This prompted us to search for GagNC co-factors. RESULTS: Here we report that RPL7, a major ribosomal protein involved in translation regulation, is a partner of Gag via its interaction with the NC domain. This interaction is mediated by the NC zinc fingers and the N- and C-termini of RPL7, respectively, but seems independent of RNA binding, Gag oligomerization and its interaction with the plasma membrane. Interestingly, RPL7 is shown for the first time to exhibit a potent DNA/RNA chaperone activity higher than that of Gag. In addition, Gag and RPL7 can function in concert to drive rapid nucleic acid hybridization. CONCLUSIONS: Our results show that GagNC interacts with the ribosomal protein RPL7 endowed with nucleic acid chaperone activity, favoring the notion that RPL7 could be a Gag helper chaperoning factor possibly contributing to the start of Gag assembly.
Assuntos
HIV-1/fisiologia , Modelos Moleculares , RNA Viral/química , Proteínas Ribossômicas/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Dimerização , HIV-1/genética , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , RNA Viral/metabolismo , Proteínas Ribossômicas/genética , Montagem de Vírus , Dedos de Zinco , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and selecting the cell plasma membrane for budding. Both gRNA and membrane selections are thought to be mediated by the compact form of Gag. This compact form binds to gRNA through both its matrix (MA) and nucleocapsid (NC) domains in the cytoplasm. At the plasma membrane, the membrane competes with gRNA for Gag binding, resulting in a transition to the extended form of Gag found in immature particles with MA bound to membrane lipids and NC to gRNA. The Gag compact form was previously evidenced in vitro. Here, we demonstrated the compact form of Gag in cells by confocal microscopy, using a bimolecular fluorescence complementation approach with a split-GFP bipartite system. Using wild-type Gag and Gag mutants, we showed that the compact form is highly dependent on the binding of MA and NC domains to RNA, as well as on interactions between MA and CA domains. In contrast, Gag multimerization appears to be less critical for the accumulation of the compact form. Finally, mutations altering the formation of Gag compact form led to a strong reduction in viral particle production and infectivity, revealing its key role in the production of infectious viral particles.
Assuntos
HIV-1 , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana , HIV-1/metabolismo , HIV-1/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Montagem de Vírus/genética , Humanos , Vírion/metabolismo , Vírion/genética , Ligação Proteica , RNA Viral/metabolismo , RNA Viral/genética , Membrana Celular/metabolismo , Membrana Celular/virologiaRESUMO
The Human Immunodeficiency Virus-1 (HIV-1) nucleocapsid protein (NC) as a mature protein or as a domain of the Gag precursor plays important roles in the early and late phases of the infection. To better understand its roles, we searched for new cellular partners and identified the RNA-binding protein Unr/CSDE1, Upstream of N-ras, whose interaction with Gag and NCp7 was confirmed by co-immunoprecipitation and FRET-FLIM. Unr interaction with Gag was found to be RNA-dependent and mediated by its NC domain. Using a dual luciferase assay, Unr was shown to act as an ITAF (IRES trans-acting factor), increasing the HIV-1 IRES-dependent translation. Point mutations of the HIV-1 IRES in a consensus Unr binding motif were found to alter both the IRES activity and its activation by Unr, suggesting a strong dependence of the IRES on Unr. Interestingly, Unr stimulatory effect is counteracted by NCp7, while Gag increases the Unr-promoted IRES activity, suggesting a differential Unr effect on the early and late phases of viral infection. Finally, knockdown of Unr in HeLa cells leads to a decrease in infection by a non-replicative lentivector, proving its functional implication in the early phase of viral infection.
Assuntos
HIV-1 , Proteínas de Ligação a DNA/metabolismo , Genes ras , HIV-1/genética , HIV-1/metabolismo , Células HeLa , Humanos , Proteínas de Ligação a RNA/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
BACKGROUND: In vertebrates and invertebrates, sensory neurons adapt to variable ambient conditions, such as the duration or repetition of a stimulus, a physiological mechanism considered as a simple form of non-associative learning and neuronal plasticity. Although various signaling pathways, as cAMP, cGMP, and the inositol 1,4,5-triphosphate receptor (InsP3R) play a role in adaptation, their precise mechanisms of action at the cellular level remain incompletely understood. Recently, in Drosophila, we reported that odor-induced Ca2+-response in axon terminals of olfactory receptor neurons (ORNs) is related to odor duration. In particular, a relatively long odor stimulus (such as 5 s) triggers the induction of a second component involving intracellular Ca2+-stores. RESULTS: We used a recently developed in-vivo bioluminescence imaging approach to quantify the odor-induced Ca2+-activity in the axon terminals of ORNs. Using either a genetic approach to target specific RNAs, or a pharmacological approach, we show that the second component, relying on the intracellular Ca2+-stores, is responsible for the adaptation to repetitive stimuli. In the antennal lobes (a region analogous to the vertebrate olfactory bulb) ORNs make synaptic contacts with second-order neurons, the projection neurons (PNs). These synapses are modulated by GABA, through either GABAergic local interneurons (LNs) and/or some GABAergic PNs. Application of GABAergic receptor antagonists, both GABAA or GABAB, abolishes the adaptation, while RNAi targeting the GABABR (a metabotropic receptor) within the ORNs, blocks the Ca2+-store dependent component, and consequently disrupts the adaptation. These results indicate that GABA exerts a feedback control. Finally, at the behavioral level, using an olfactory test, genetically impairing the GABABR or its signaling pathway specifically in the ORNs disrupts olfactory adapted behavior. CONCLUSION: Taken together, our results indicate that a relatively long lasting form of adaptation occurs within the axon terminals of the ORNs in the antennal lobes, which depends on intracellular Ca2+-stores, attributable to a positive feedback through the GABAergic synapses.
Assuntos
Adaptação Fisiológica/fisiologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Drosophila melanogaster/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Cálcio/fisiologia , Drosophila melanogaster/genética , Feminino , Neurônios Receptores Olfatórios/citologia , Transmissão Sináptica/fisiologiaRESUMO
During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins. The protective capsid shell disassembles during a process called uncoating. The viral genome is transported into the cell nucleus and integrates into the host cell chromatin. Unlike biochemical approaches that provide global data about the whole population of viral particles, imaging techniques enable following individual viruses on a single particle level. In this context, quantitative microscopy has brought original data shedding light on the dynamics of the viral entry into the host cell, the cytoplasmic transport, the nuclear import, and the selection of the integration site. In parallel, multi-color imaging studies have elucidated the mechanism of action of host cell factors implicated in HIV-1 viral cycle progression. In this review, we describe the labeling strategies used for HIV-1 fluorescence imaging and report on the main advancements that imaging studies have brought in the understanding of the infection mechanisms from the viral entry into the host cell until the provirus integration step.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Internalização do Vírus , Animais , Núcleo Celular/virologia , HIV-1/química , HIV-1/genética , Humanos , Microscopia de Fluorescência , Integração ViralRESUMO
We wanted to develop a therapeutic approach against rabies disease by targeting the lyssavirus transcription/replication complex. Because this complex (nucleoprotein N-RNA template processed by the L polymerase and its cofactor, the phosphoprotein P) is similar to that of other negative-strand RNA viruses, we aimed to design broad-spectrum antiviral drugs that could be used as a complement to postexposure vaccination and immunotherapy. Recent progress in understanding the structure/function of the rabies virus P, N, and L proteins predicts that the amino-terminal end of P is an excellent target for destabilizing the replication complex because it interacts with both L (for positioning onto the N-RNA template) and N (for keeping N soluble, as needed for viral RNA encapsidation). Thus, peptides mimicking various lengths of the amino-terminal end of P have been evaluated, as follows: (i) for binding properties to the N-P-L partners by the two-hybrid method; (ii) for their capacity to inhibit the transcription/replication of a rabies virus minigenome encoding luciferase in BHK-21-T7 cells; and (iii) for their capacity to inhibit rabies virus infection of BHK-21-T7 cells and of two derivatives of the neuronal SK-N-SH cell line. Peptides P60 and P57 (the first 60 and first 57 NH2 residues of P, respectively) exhibited a rapid, strong, and long-lasting inhibitory potential on luciferase expression (>95% from 24 h to 55 h). P42 was less efficient in its inhibition level (75% for 18 to 30 h) and duration (40% after 48 h). The most promising peptides were synthesized in tandem with the Tat sequence, allowing cell penetration. Their inhibitory effects were observed on BHK-21-T7 cells infected with rabies virus and Lagos bat virus but not with vesicular stomatitis virus. In neuronal cells, a significant inhibition of both nucleocapsid inclusions and rabies virus release was observed.
Assuntos
Antivirais/farmacologia , Peptídeos/farmacologia , Fosfoproteínas/química , Vírus da Raiva/efeitos dos fármacos , Proteínas Estruturais Virais/química , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Humanos , Imunoprecipitação , Chaperonas Moleculares , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/virologia , Peptídeos/síntese química , Peptídeos/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Vírus da Raiva/patogenicidade , Técnicas do Sistema de Duplo-Híbrido , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismoRESUMO
The human immunodeficiency virus (HIV-1) polyprotein Gag (Group-specific antigen) plays a central role in controlling the late phase of the viral lifecycle. Considered to be only a scaffolding protein for a long time, the structural protein Gag plays determinate and specific roles in HIV-1 replication. Indeed, via its different domains, Gag orchestrates the specific encapsidation of the genomic RNA, drives the formation of the viral particle by its auto-assembly (multimerization), binds multiple viral proteins, and interacts with a large number of cellular proteins that are needed for its functions from its translation location to the plasma membrane, where newly formed virions are released. Here, we review the interactions between HIV-1 Gag and 66 cellular proteins. Notably, we describe the techniques used to evidence these interactions, the different domains of Gag involved, and the implications of these interactions in the HIV-1 replication cycle. In the final part, we focus on the interactions involving the highly conserved nucleocapsid (NC) domain of Gag and detail the functions of the NC interactants along the viral lifecycle.
Assuntos
HIV-1/genética , Interações entre Hospedeiro e Microrganismos/genética , Nucleocapsídeo/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Nucleocapsídeo/genética , Ligação Proteica , Transporte Proteico , Vírion , Montagem de Vírus , Replicação ViralRESUMO
The HIV-1 nucleocapsid protein 7 (NC) is a potential target for effective antiretroviral therapy due to its central role in virus replication, mainly linked to nucleic acid (NA) chaperone activity, and low susceptibility to drug resistance. By screening a compounds library, we identified the aminopyrrolic compound CN14_17, a known carbohydrate binding agent, that inhibits the NC chaperone activity in the low micromolar range. Different from most of available NC inhibitors, CN14_17 fully prevents the NC-induced annealing of complementary NA sequences. Using fluorescence assays and isothermal titration calorimetry, we found that CN14_17 competes with NC for the binding to NAs, preferentially targeting single-stranded sequences. Molecular dynamics simulations confirmed that binding to cTAR occurs preferably within the guanosine-rich single stranded sequence. Finally, CN14_17 exhibited antiretroviral activity in the low micromolar range, although with a moderate therapeutic index. Overall, CN14_17 might be the progenitor of a new promising class of NC inhibitors.
RESUMO
Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Mapeamento de Interação de Proteínas , Pseudomonas aeruginosa/metabolismo , Algoritmos , Sítios de Ligação , Cromossomos Bacterianos/genética , Corantes Fluorescentes/metabolismo , Genoma Bacteriano , Fótons , Plasmídeos/metabolismo , Pseudomonas aeruginosa/genética , SoftwareRESUMO
The nucleocapsid protein (NC) is a highly conserved protein that plays key roles in HIV-1 replication through its nucleic acid chaperone properties mediated by its two zinc fingers and basic residues. NC is a promising target for antiviral therapy, particularly to control viral strains resistant to currently available drugs. Since calixarenes with antiviral properties have been described, we explored the ability of calixarene hydroxymethylphosphonic or sulfonic acids to inhibit NC chaperone properties and exhibit antiviral activity. By using fluorescence-based assays, we selected four calixarenes inhibiting NC chaperone activity with submicromolar IC50 values. These compounds were further shown by mass spectrometry, isothermal titration calorimetry, and fluorescence anisotropy to bind NC with no zinc ejection and to compete with nucleic acids for the binding to NC. Molecular dynamic simulations further indicated that these compounds interact via their phosphonate or sulfonate groups with the basic surface of NC but not with the hydrophobic plateau at the top of the folded fingers. Cellular studies showed that the most soluble compound CIP201 inhibited the infectivity of wild-type and drug-resistant HIV-1 strains at low micromolar concentrations, primarily targeting the early steps of HIV-1 replication. Moreover, CIP201 was also found to inhibit the flipping and polymerization activity of reverse transcriptase. Calixarenes thus form a class of noncovalent NC inhibitors, endowed with a new binding mode and multitarget antiviral activity.
Assuntos
Fármacos Anti-HIV/farmacologia , Calixarenos/farmacologia , HIV-1/química , Chaperonas Moleculares/antagonistas & inibidores , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Organofosfonatos/farmacologia , Animais , Calixarenos/classificação , HIV-1/efeitos dos fármacos , Concentração Inibidora 50 , Camundongos , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Ligação ProteicaRESUMO
The HIV-1 nucleocapsid (NC) protein is a small basic DNA and RNA binding protein that is absolutely necessary for viral replication and thus represents a target of great interest to develop new anti-HIV agents. Moreover, the highly conserved sequence offers the opportunity to escape the drug resistance (DR) that emerged following the highly active antiretroviral therapy (HAART) treatment. On the basis of our previous research, nordihydroguaiaretic acid 1 acts as a NC inhibitor showing moderate antiviral activity and suboptimal drug-like properties due to the presence of the catechol moieties. A bioisosteric catechol replacement approach led us to identify the 5-dihydroxypyrimidine-6-carboxamide substructure as a privileged scaffold of a new class of HIV-1 NC inhibitors. Hit validation efforts led to the identification of optimized analogs, as represented by compound 28, showing improved NC inhibition and antiviral activity as well as good ADME and PK properties.
RESUMO
Lyssaviruses are highly neurotropic viruses associated with neuronal apoptosis. Previous observations have indicated that the matrix proteins (M) of some lyssaviruses induce strong neuronal apoptosis. However, the molecular mechanism(s) involved in this phenomenon is still unknown. We show that for Mokola virus (MOK), a lyssavirus of low pathogenicity, the M (M-MOK) targets mitochondria, disrupts the mitochondrial morphology, and induces apoptosis. Our analysis of truncated M-MOK mutants suggests that the information required for efficient mitochondrial targeting and dysfunction, as well as caspase-9 activation and apoptosis, is held between residues 46 and 110 of M-MOK. We used a yeast two-hybrid approach, a coimmunoprecipitation assay, and confocal microscopy to demonstrate that M-MOK physically associates with the subunit I of the cytochrome c (cyt-c) oxidase (CcO) of the mitochondrial respiratory chain; this is in contrast to the M of the highly pathogenic Thailand lyssavirus (M-THA). M-MOK expression induces a significant decrease in CcO activity, which is not the case with M-THA. M-MOK mutations (K77R and N81E) resulting in a similar sequence to M-THA at positions 77 and 81 annul cyt-c release and apoptosis and restore CcO activity. As expected, the reverse mutations, R77K and E81N, introduced in M-THA induce a phenotype similar to that due to M-MOK. These features indicate a novel mechanism for energy depletion during lyssavirus-induced apoptosis.
Assuntos
Apoptose , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Lyssavirus/patogenicidade , Mitocôndrias/fisiologia , Mitocôndrias/virologia , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Caspase 9/metabolismo , Linhagem Celular , Cricetinae , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Humanos , Imunoprecipitação , Lyssavirus/genética , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Viral proteins are frequently multifunctional to accommodate the high density of information encoded in viral genomes. Matrix (M) protein of negative-stranded RNA viruses such as Rhabdoviridae is one such example. Its primary function is virus assembly/budding but it is also involved in the switch from viral transcription to replication and the concomitant down regulation of host gene expression. In this study we undertook a search for potential rabies virus (RV) M protein's cellular partners. In a yeast two-hybrid screen the eIF3h subunit was identified as an M-interacting cellular factor, and the interaction was validated by co-immunoprecipitation and surface plasmon resonance assays. Upon expression in mammalian cell cultures, RV M protein was localized in early small ribosomal subunit fractions. Further, M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5'-UTRs. Interestingly, translation of hepatitis C virus IRES-containing mRNA, which recruits eIF3 via a different noncanonical mechanism, was unaffected. Together, the data suggest that, as a complement to its functions in virus assembly/budding and regulation of viral transcription, RV M protein plays a role in inhibiting translation in virus-infected cells through a protein-protein interaction with the cellular translation machinery.
Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Biossíntese de Proteínas , Vírus da Raiva/patogenicidade , Proteínas da Matriz Viral/metabolismo , Sequência de Aminoácidos , Fator de Iniciação 3 em Eucariotos/imunologia , Imunoprecipitação , Dados de Sequência Molecular , Mutação , Subunidades Proteicas/metabolismo , RNA Mensageiro/química , RNA Viral/química , Ribossomos/virologia , Ressonância de Plasmônio de Superfície , Técnicas do Sistema de Duplo-Híbrido , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
Fluorescence microscopy imaging of individual HIV-1 viruses necessitates a specific labeling of viral structures that minimally perturbs the infection process. Herein, we used HIV-1 pseudoviruses containing NCp7 fused to a tetracystein (TC) tag, labeled by a biarsenical fluorescein derivative (FlAsH) to quantitatively monitor the NCp7 protein concentration in the viral cores during the early stages of infection. Single particle imaging of individual pseudoviruses with defined ratios of TC-tagged to non tagged NCp7 proteins, together with theoretical modeling of energy transfer between FlAsH dyes, showed that the high packaging of TC-tagged proteins in the viral cores causes a strong fluorescence quenching of FlAsH and that the fluorescence intensity of individual viral complexes is an appropriate parameter to monitor changes in the amount of NCp7 molecules within the viral particles during infection. Interestingly, we observed a dramatic fluorescence increase of individual FlAsH-labeled pseudoviruses containing 100% TC-tagged NCp7 proteins in infected cells at 8 and 16 h post-infection. This effect was significantly lower for pseudoviruses expressing TC-tagged integrase. Therefore, this fluorescence increase is likely related to the cytoplasmic viral transformation and the release of NCp7 molecules from the viral complexes. This loss of quenching effect is largely reduced when reverse transcriptase is inhibited, showing that NCp7 release is connected to viral DNA synthesis. A spatial analysis further revealed that NCp7-TC release is more pronounced in the perinuclear space, where capsid disassembly is thought to be completed. Quantification of NCp7-TC content based on fluorescence quenching presented in this study evidences for the first time the cytoplasmic release of NCp7 during the remodeling of HIV-1 viral particles on their journey toward the nucleus. The developed approach can be applied to quantify dye concentrations in a wide range of nano-objects by fluorescence microscopy techniques.