RESUMEN
The 2'-5' oligoadenylate synthetases and the protein kinase PKR are both interferon-induced, double-stranded RNA-dependent proteins that play important roles in the antiviral effects of the interferons and in cellular growth control. Both enzymes are activated by natural or synthetic dsRNAs and by single-stranded RNAs that possess extensive secondary structure. This report describes the effects of the small Epstein-Barr virus-encoded RNA EBER-1 on the regulation of 2-5(A) synthetase activity. We demonstrate that EBER-1 RNA binds to and activates the human 40-kDa 2-5(A) synthetase in a dose-dependent manner. The efficiency of EBER-1 as an activator of 2-5(A) synthetase is approximately 25% of that of the synthetic double-stranded RNA poly(I)/poly(C), and poly(I)/poly(C) further stimulates enzyme activity even in the presence of a high concentration of EBER-1. Conversely, EBER-1 neither stimulates nor inhibits 2-5(A) synthetase that has been activated by a high concentration of poly(I)/poly(C). Competitive binding assays suggest that the relative affinity of the enzyme for poly(I)/poly(C) is considerably higher than that for EBER-1. Our data indicate that EBER-1, like VAI RNA of adenovirus, TAR RNA of HIV-1, and Rex-RE RNA of HTLV-1, is able to activate the 2-5(A) synthetases. The significance of why several viruses may activate the 2-5(A) synthetase/RNase L-mediated RNA degradation pathway is discussed.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Herpesvirus Humano 4/genética , ARN Viral/metabolismo , 2',5'-Oligoadenilato Sintetasa/genética , Animales , Línea Celular , Activación Enzimática , Humanos , Inductores de Interferón/metabolismo , Interferones , Poli I-C/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/citologíaRESUMEN
Previous evidence has shown that the majority of the interferon-inducible, double-stranded RNA-dependent protein kinase PKR is associated with ribosomes in vivo. Here we show that ribosomes are inhibitory for PKR activity since they compete with dsRNA for binding to PKR, inhibit the activation of the protein kinase by dsRNA, and prevent the phosphorylation of the PKR substrate eIF2alpha. We suggest that ribosomes constitute a reservoir of inactive PKR and that the protein kinase must be displaced from the ribosome by dsRNA in order to become activated.
Asunto(s)
Ribosomas/metabolismo , eIF-2 Quinasa/antagonistas & inhibidores , Animales , Inducción Enzimática , Factor 2 Eucariótico de Iniciación/metabolismo , Genes myc , Células HeLa , Humanos , Cinética , Mamíferos , Ratones , Fosforilación , Cloruro de Potasio/farmacología , ARN Bicatenario/metabolismo , eIF-2 Quinasa/biosíntesisRESUMEN
Forty-five patients with moderately severe perennial bronchial asthma were challenged by ingestion of: acetylsalicyclic acid (ASA); 4 azo dyes (tartrazine, sunset yellow, amaranth, and ponceau); 3 non-azo dyes (erythrosine, brilliant blue, and indigotin); sodium benzoate (NaB); parahydroxybenzoic acid (OHBA); butylated hydroxyanisole (BHA); and butylated hydroxytoluene (BHT). A fall in forced expiratory volume is one second (FEV1) greater than 25% from baseline was considered positive. Seven patients who gave an unequivocal history of aspirin intolerance were not challenged with ASA; an additional 13 had positive open challenges to ASA, giving an apparent incidence of aspirin sensitivity of 20/45. The presence of nasal polyps, simusitis, or the regular use of corticosteroids, either singly or in combination, was not associated with an increased incidence of reactions to ASA. Significant bronchoconstriction to open challenges with agents other than ASA was less frequent. Positive open challenges to all substances except aspirin were followed by double-blind challenges which were positive in only 3 instances: 1 each with erythrosine, ponceau, and NaB/OHBA. Our findings confirm that ASA intolerance is relatively common but suggest on the other hand that reactions to dyes and preservatives are uncommon cause of clinically significant bronchoconstriction in moderately severe perennial asthmatics.