RESUMEN
The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif neutralizes the human DNA-editing enzyme APOBEC3 protein, an antiretroviral cellular factor from the innate immune system, allowing the virus to escape the host defence system. It was shown that Vif is packaged into viral particles through specific interactions with the viral genomic RNA. Conserved and structured sequences from the 5'-noncoding region, such as the Tat-responsive element (TAR) or the genomic RNA dimerization initiation site (DIS), are primary binding sites for Vif. In the present study we used isothermal titration calorimetry to investigate sequence and structure determinants important for Vif binding to short viral RNA corresponding to TAR and DIS stem-loops. We showed that Vif specifically binds TAR and DIS in the low nanomolar range. In addition, Vif primarily binds the TAR UCU bulge, but not the apical loop. Determinants for Vif binding to the DIS loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-TAR inhibitors or DIS small molecule binders might be also effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions.
Asunto(s)
Duplicado del Terminal Largo de VIH , VIH-1/genética , ARN Viral/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Secuencia de Bases , Sitios de Unión , Calorimetría , Escherichia coli/genética , Secuencias Invertidas Repetidas , Tamaño de la Partícula , Unión Proteica , Estructura Secundaria de Proteína , ARN Ribosómico 23S/química , ARN Ribosómico 23S/genética , ARN Viral/genética , TermodinámicaAsunto(s)
Fármacos Anti-VIH/química , Inhibidores de la Proteasa del VIH/química , Transcriptasa Inversa del VIH/química , Compuestos Heterocíclicos con 3 Anillos/química , Indoles/química , Modelos Biológicos , Modelos Moleculares , Nitrilos/química , Piridonas/química , Pirimidinas/química , Inhibidores de la Transcriptasa Inversa/química , Compuestos de Vinilo/química , Fármacos Anti-VIH/uso terapéutico , Cristalografía por Rayos X , Inhibidores de la Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , Transcriptasa Inversa del VIH/metabolismo , Humanos , Estructura Molecular , Inhibidores de la Transcriptasa Inversa/uso terapéuticoAsunto(s)
Aminoglicósidos/química , Antibacterianos/química , VIH-1/efectos de los fármacos , Conformación de Ácido Nucleico , ARN Viral/química , Replicación Viral/efectos de los fármacos , Aminoglicósidos/farmacología , Antibacterianos/farmacología , Secuencia de Bases , Dimerización , Genoma Viral , HumanosRESUMEN
Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.
