RESUMEN
Betulinic acid (1) has been modified to ionic derivatives (2-5) to improve its water solubility and biological activities. The binding properties of these derivatives with respect to human serum albumin (HSA) was examined and found to be similar to current anti-HIV drugs. These compounds did not inhibit HIV reverse transcriptase, however, 1, 2 and 5 inhibited herpes simplex type 2 (HSV-2) replication at concentrations similar to those reported for acyclovir (IC50 â¼ 0.1-10 µM) and with minimal cellular cytotoxicity. IC50 values for antiviral activity against HSV-2 186 were 1.6, 0.6, 0.9, 7.2, and 0.9 µM for compounds 1-5, respectively.
Asunto(s)
Herpesvirus Humano 2/fisiología , Triterpenos/química , Aciclovir/farmacología , Animales , Antivirales/química , Antivirales/farmacología , Chlorocebus aethiops , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Humanos , Triterpenos Pentacíclicos , Triterpenos/farmacología , Células Vero , Replicación Viral/efectos de los fármacos , Ácido BetulínicoRESUMEN
Sam68 functionally complements for, as well as synergizes with, HIV-1 Rev in Rev response element (RRE)-mediated gene expression and virus production. Furthermore, C-terminal deletion/point mutants of Sam68 (Sam68DeltaC/Sam68-P21) exert a transdominant negative phenotype for Rev function and HIV-1 production. However, the relevance of Sam68 in Rev/RRE function is not well defined. To gain more insight into the mechanism of Sam68 in Rev function, we used an RNAi (RNA interference) strategy to create stable Sam68 knockdown HeLa (SSKH) cells. In SSKH cells, Rev failed to activate both RRE-mediated reporter gene [chloramphenicol acetyltransferase (CAT) and/or gag] expressions. Importantly, reduction of Sam68 expression led to a dramatic inhibition of HIV-1 production. Inhibition of the reporter gene expression and HIV production correlated with the failure to export RRE-containing CAT mRNA and unspliced viral mRNAs to the cytoplasm, confirming that SSKH cells are defective for Rev-mediated RNA export. Taken together, these results suggest that Sam68 is involved in Rev-mediated RNA export and is absolutely required for HIV production.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Productos del Gen rev/metabolismo , VIH-1/genética , Fosfoproteínas/fisiología , Proteínas de Unión al ARN/fisiología , Proteínas Adaptadoras Transductoras de Señales , Línea Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Regulación Viral de la Expresión Génica , Genes Reporteros , VIH-1/fisiología , Células HeLa , Humanos , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Interferencia de ARN , Transporte de ARN , ARN Mensajero/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Elementos de Respuesta , Activación Transcripcional , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
Sam68 associates with c-Src kinase during mitosis. We previously demonstrated that Sam68 functionally replaces and/or synergizes with HIV-1 Rev in rev response element (RRE)-mediated gene expression and virus production. Furthermore, we reported that knockdown of Sam68 inhibited Rev-mediated RNA export and it is absolutely required for HIV-1 production. In the present study, we identified small heat shock protein, hsp22, as a novel interacting partner of Sam68. Hsp22 binds to Sam68 in vitro and in vivo. Overexpression of hsp22 significantly inhibits Sam68-mediated RRE- as well as CTE (constitutive transport element)-dependent reporter gene expression. Furthermore, exposing 293T cells to heat shock inhibits Sam68/RRE function by virtue of elevating hsp22. The critical domain of hsp22 that interacts with Sam68 resides between amino acids 62 and 133. Our studies provide evidence for the first time that hsp22 specifically binds to Sam68 and modulates its activity, thus playing a role in the post-transcriptional regulation of gene expression.