RESUMEN
The genomic RNA of hepatitis C virus (HCV) encodes the viral polyprotein precursor that undergoes proteolytic cleavage into structural and nonstructural proteins by cellular and the viral NS3 and NS2-3 proteases. Nonstructural protein 4A (NS4A) is a cofactor of the NS3 serine protease and has been demonstrated to inhibit protein synthesis. In this study, GST pull-down assay was performed to examine potential cellular factors that interact with the NS4A protein and are involved in the pathogenesis of HCV. A trypsin digestion followed by LC-MS/MS analysis revealed that one of the GST-NS4A-interacting proteins to be eukaryotic elongation factor 1A (eEF1A). Both the N-terminal domain of NS4A from amino acid residues 1-20, and the central domain from residues 21-34 interacted with eEF1A, but the central domain was the key player involved in the NS4A-mediated translation inhibition. NS4A(21-34) diminished both cap-dependent and HCV IRES-mediated translation in a dose-dependent manner. The translation inhibitory effect of NS4A(21-34) was relieved by the addition of purified recombinant eEF1A in an in vitro translation system. Taken together, NS4A inhibits host and viral translation through interacting with eEF1A, implying a possible mechanism by which NS4A is involved in the pathogenesis and chronic infection of HCV.
Asunto(s)
Proteínas Portadoras/metabolismo , Factor 1 de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas , Caperuzas de ARN , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/química , Cartilla de ADN , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas no Estructurales Virales , Proteínas Virales/químicaRESUMEN
CCAAT/enhancer-binding proteins, C/EBPalpha and C/EBPbeta, are required for fat cell differentiation and maturation. Previous studies showed that replacement of C/EBPalpha with C/EBPbeta, generating the beta/beta alleles in the mouse genome, prevents lipid accumulation in white adipose tissue (WAT). In this study, beta/beta mice lived longer and had higher energy expenditure than their control littermates due to increased WAT energy oxidation. The WAT of beta/beta mice was enriched with metabolically active, thermogenic mitochondria known for energy burning. The beta/beta allele exerted its effect through the elevated expression of the G protein alpha stimulatory subunit (Galphas) in WAT. Galphas, when overexpressed in fat-laden 3T3-L1 cells, stimulated mitochondrial biogenesis similar to that seen in the WAT of beta/beta mice, and effectively diminished the stored lipid pool.