ABSTRACT Infection by retroviruses as HIV-1 requires the stable integration of their genome into ... more ABSTRACT Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro . Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. IMPORTANCE In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.
Antimicrobial Agents and Chemotherapy, Jul 18, 2023
HIV-1 integrase-LEDGF allosteric inhibitors (INLAIs) share the binding site on the viral protein ... more HIV-1 integrase-LEDGF allosteric inhibitors (INLAIs) share the binding site on the viral protein with the host factor LEDGF/p75. These small molecules act as molecular glues promoting hyper-multimerization of HIV-1 IN protein to severely perturb maturation of viral particles.
LEDGF/p75 (LEDGF) main cellular cofactor of HIV-1 integrase (IN), acts as tethering factor to tar... more LEDGF/p75 (LEDGF) main cellular cofactor of HIV-1 integrase (IN), acts as tethering factor to target integration of HIV in actively transcribed genes. Recently a class of IN inhibitors based on inhibition of LEDGF-IN interaction has been developed. We describe here a new series of IN-LEDGF allosteric inhibitors (INLAIs), with potent anti-HIV-1 activity in the one-digit nanomolar range. These compounds inhibited IN-LEDGF interaction while enhancing IN-IN aberrant multimerization by allosteric mechanism. Compounds of this series were fully active on HIV-1 mutants resistant to IN strand transfer inhibitors (INSTIs) and other class of anti-HIV drugs, confirming that they belong to a new class of antiretrovirals. These compounds displayed most potent antiretroviral activity at post-integration due to aberrant IN polymerization responsible of infectivity defect of viral particles produced. INLAI-resistant mutants were selected by dose escalation method and the most detrimental mutation fo...
Completion of neuronal migration is critical for brain development. Kif21b is a plus-end directed... more Completion of neuronal migration is critical for brain development. Kif21b is a plus-end directed kinesin motor protein that promotes intracellular transport and controls microtubule dynamics in neurons. Here we report a physiological function of Kif21b during radial migration of projection neurons in the mouse developing cortex.In vivoanalysis in mouse and live imaging on cultured slices demonstrate that Kif21b regulates the radial glia-guided locomotion of new-born neurons independently of its motility on microtubules. Unexpectedly we show that Kif21b directly binds and regulates the actin cytoskeleton bothin vitroandin vivoin migratory neurons. We establish that Kif21b-mediated regulation of actin cytoskeleton dynamics influences branching and nucleokinesis during neuronal locomotion. Altogether, our results reveal atypical roles of Kif21b on the actin cytoskeleton during migration of cortical projection neurons.
Summary illustration. Model for HDAC6-mediated A3G protection and control of HIV-1 infectiveness.... more Summary illustration. Model for HDAC6-mediated A3G protection and control of HIV-1 infectiveness. HDAC6 directly interacts and forms a constitutive complex with A3G. This HDAC6/A3G complex is formed either in the absence (1) or in the presence of the HIV-1 Vif protein (2), and appears to be independent on the BUZ domain of HDAC6 (the different domains of HDAC6 are represented). Moreover, HDAC6 could concomitantly interact with Vif (3). The balance between the level of HDAC6 and Vif expression conditions the efficiency of HDAC6 to interact with A3G (1 and 2) and/or Vif (3 or 4; in the absence or presence of A3G, respectively), to avoid A3G-Vif interaction and subsequent Vif-mediated A3G ubiquitination and proteasome degradation (promoting HIV-1 infection) (pathway 5; the Vif-recruited E3 ligase complex (Vif-[CBF-β-EloB-EloC-Cul5-Rbx2/E2]) that targets A3G is shown). In fact, HDAC6 induces Vif autophagic clearance in a BUZ-dependent manner (pathway 3), thereby inhibiting HIV-1 infecti...
Background: The production of full length, biologically active proteins in mammalian cells is cri... more Background: The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10-50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures. Results: Here we report further improvements which allow an increase in the ease and speed of recombinant virus isolation, the scale-up of protein production and the simultaneous synthesis of several polypeptides belonging to a protein complex using a single virus vector. Isolation of MVA-T7 viruses encoding foreign proteins was simplified by combining positive selection for virus recombinants and...
Recent evidence indicated that HIV-1 Integrase (IN) binds genomic viral RNA (gRNA) playing a crit... more Recent evidence indicated that HIV-1 Integrase (IN) binds genomic viral RNA (gRNA) playing a critical role in viral particle morphogenesis and gRNA stability in host cells. Combining biophysical and biochemical approaches we show that the C-terminal flexible 18-residues tail of IN acts as a sensor of the peculiar apical structure of trans-activation response element RNA (TAR), directly interacting with its hexaloop. We highlighted how the whole IN C-terminal domain, once bound to TAR, can change its structure assisting the binding of Tat, the HIV trans-activator protein, which finally displaces IN from TAR. Our results are consistent with the emerging role of IN in early stage of proviral transcription and suggest new steps of HIV-1 life cycle that can be considered as therapeutic targets.
The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression ... more The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation and protein degradation) that together reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′untranslated region (5′UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky-scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G ...
Methods in molecular biology (Clifton, N.J.), 2018
Purification of proteins containing disordered regions and participating in transient complexes i... more Purification of proteins containing disordered regions and participating in transient complexes is often challenging because of the small amounts available after purification, their heterogeneity, instability, and/or poor solubility. To circumvent these difficulties, we set up a methodology that enables the production of stable complexes in large amounts for structural and functional studies. In this chapter, we describe the methodology used to establish the best cell culture conditions and buffer compositions to optimize soluble protein production and their stabilization through protein complex formation. Two examples of challenging protein families are described, namely, the human steroid nuclear receptors and the HIV-1 pre-integration complexes.
ABSTRACT Infection by retroviruses as HIV-1 requires the stable integration of their genome into ... more ABSTRACT Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro . Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. IMPORTANCE In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.
Antimicrobial Agents and Chemotherapy, Jul 18, 2023
HIV-1 integrase-LEDGF allosteric inhibitors (INLAIs) share the binding site on the viral protein ... more HIV-1 integrase-LEDGF allosteric inhibitors (INLAIs) share the binding site on the viral protein with the host factor LEDGF/p75. These small molecules act as molecular glues promoting hyper-multimerization of HIV-1 IN protein to severely perturb maturation of viral particles.
LEDGF/p75 (LEDGF) main cellular cofactor of HIV-1 integrase (IN), acts as tethering factor to tar... more LEDGF/p75 (LEDGF) main cellular cofactor of HIV-1 integrase (IN), acts as tethering factor to target integration of HIV in actively transcribed genes. Recently a class of IN inhibitors based on inhibition of LEDGF-IN interaction has been developed. We describe here a new series of IN-LEDGF allosteric inhibitors (INLAIs), with potent anti-HIV-1 activity in the one-digit nanomolar range. These compounds inhibited IN-LEDGF interaction while enhancing IN-IN aberrant multimerization by allosteric mechanism. Compounds of this series were fully active on HIV-1 mutants resistant to IN strand transfer inhibitors (INSTIs) and other class of anti-HIV drugs, confirming that they belong to a new class of antiretrovirals. These compounds displayed most potent antiretroviral activity at post-integration due to aberrant IN polymerization responsible of infectivity defect of viral particles produced. INLAI-resistant mutants were selected by dose escalation method and the most detrimental mutation fo...
Completion of neuronal migration is critical for brain development. Kif21b is a plus-end directed... more Completion of neuronal migration is critical for brain development. Kif21b is a plus-end directed kinesin motor protein that promotes intracellular transport and controls microtubule dynamics in neurons. Here we report a physiological function of Kif21b during radial migration of projection neurons in the mouse developing cortex.In vivoanalysis in mouse and live imaging on cultured slices demonstrate that Kif21b regulates the radial glia-guided locomotion of new-born neurons independently of its motility on microtubules. Unexpectedly we show that Kif21b directly binds and regulates the actin cytoskeleton bothin vitroandin vivoin migratory neurons. We establish that Kif21b-mediated regulation of actin cytoskeleton dynamics influences branching and nucleokinesis during neuronal locomotion. Altogether, our results reveal atypical roles of Kif21b on the actin cytoskeleton during migration of cortical projection neurons.
Summary illustration. Model for HDAC6-mediated A3G protection and control of HIV-1 infectiveness.... more Summary illustration. Model for HDAC6-mediated A3G protection and control of HIV-1 infectiveness. HDAC6 directly interacts and forms a constitutive complex with A3G. This HDAC6/A3G complex is formed either in the absence (1) or in the presence of the HIV-1 Vif protein (2), and appears to be independent on the BUZ domain of HDAC6 (the different domains of HDAC6 are represented). Moreover, HDAC6 could concomitantly interact with Vif (3). The balance between the level of HDAC6 and Vif expression conditions the efficiency of HDAC6 to interact with A3G (1 and 2) and/or Vif (3 or 4; in the absence or presence of A3G, respectively), to avoid A3G-Vif interaction and subsequent Vif-mediated A3G ubiquitination and proteasome degradation (promoting HIV-1 infection) (pathway 5; the Vif-recruited E3 ligase complex (Vif-[CBF-β-EloB-EloC-Cul5-Rbx2/E2]) that targets A3G is shown). In fact, HDAC6 induces Vif autophagic clearance in a BUZ-dependent manner (pathway 3), thereby inhibiting HIV-1 infecti...
Background: The production of full length, biologically active proteins in mammalian cells is cri... more Background: The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10-50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures. Results: Here we report further improvements which allow an increase in the ease and speed of recombinant virus isolation, the scale-up of protein production and the simultaneous synthesis of several polypeptides belonging to a protein complex using a single virus vector. Isolation of MVA-T7 viruses encoding foreign proteins was simplified by combining positive selection for virus recombinants and...
Recent evidence indicated that HIV-1 Integrase (IN) binds genomic viral RNA (gRNA) playing a crit... more Recent evidence indicated that HIV-1 Integrase (IN) binds genomic viral RNA (gRNA) playing a critical role in viral particle morphogenesis and gRNA stability in host cells. Combining biophysical and biochemical approaches we show that the C-terminal flexible 18-residues tail of IN acts as a sensor of the peculiar apical structure of trans-activation response element RNA (TAR), directly interacting with its hexaloop. We highlighted how the whole IN C-terminal domain, once bound to TAR, can change its structure assisting the binding of Tat, the HIV trans-activator protein, which finally displaces IN from TAR. Our results are consistent with the emerging role of IN in early stage of proviral transcription and suggest new steps of HIV-1 life cycle that can be considered as therapeutic targets.
The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression ... more The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation and protein degradation) that together reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′untranslated region (5′UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky-scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G ...
Methods in molecular biology (Clifton, N.J.), 2018
Purification of proteins containing disordered regions and participating in transient complexes i... more Purification of proteins containing disordered regions and participating in transient complexes is often challenging because of the small amounts available after purification, their heterogeneity, instability, and/or poor solubility. To circumvent these difficulties, we set up a methodology that enables the production of stable complexes in large amounts for structural and functional studies. In this chapter, we describe the methodology used to establish the best cell culture conditions and buffer compositions to optimize soluble protein production and their stabilization through protein complex formation. Two examples of challenging protein families are described, namely, the human steroid nuclear receptors and the HIV-1 pre-integration complexes.
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