RESUMO
Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51.
Assuntos
Regulação para Baixo , Infecções por HIV/enzimologia , HIV-1/fisiologia , Rad51 Recombinase/metabolismo , Integração Viral , Linhagem Celular , Reparo do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Infecções por HIV/genética , Infecções por HIV/virologia , Integrase de HIV/genética , Integrase de HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , Humanos , Ligação Proteica , Rad51 Recombinase/química , Rad51 Recombinase/genética , Recombinação GenéticaRESUMO
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.
Assuntos
Nucleossomos , Spumavirus , Humanos , Histonas/genética , Integração Viral , Cromatina , Retroviridae/genética , Integrases/genética , DNA Viral/química , Spumavirus/genéticaAssuntos
Fármacos Anti-HIV/administração & dosagem , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Integrase de HIV/genética , HIV/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Mutação de Sentido Incorreto , HIV/genética , HIV/isolamento & purificação , Infecções por HIV/virologia , Humanos , Masculino , Oxazinas , Piperazinas , PiridonasRESUMO
We described chemical inhibitors of Mos1 transposition. Some were already known to affect a related prokaryotic transposase (Tn5) or HIV-1 integrase, whereas the other were new compounds in this field. The new compounds were all organized around a bis-(heteroaryl)maleimides scaffold. Their mechanism of action depended on the chemical substitutions on the scaffold. The cross-activity, between HIV-1 integrase and Mos1 transposase, of the new group of inhibitors showed that Mos1 transposase could constitute an excellent surrogate HIV-1 inhibitor screen.