Assuntos
COVID-19/complicações , COVID-19/terapia , Linfoma não Hodgkin/complicações , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , COVID-19/imunologia , Técnicas de Cultura de Células , Tomada de Decisão Clínica , Feminino , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Hospedeiro Imunocomprometido , Imunoterapia , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/terapia , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Prevenção SecundáriaRESUMO
Epstein-Barr virus (EBV) is a potent mitogenic and antiapoptotic agent for B lymphocytes and is associated with several different types of human tumour. The abundantly expressed small viral RNA, EBER-1, binds to the growth inhibitory and pro-apoptotic protein kinase R (PKR) and blocks activation of the latter by double-stranded RNA. Recent evidence has suggested that expression of EBER-1 alone in EBV-negative B cells promotes a tumorigenic phenotype and that this may be related to inhibition of the pro-apoptotic effects of PKR. The ribosomal protein L22 binds to EBER-1 in virus-infected cells, but the significance of this has not previously been established. We report here that L22 and PKR compete for a common binding site on EBER-1. As a result of this competition, L22 interferes with the ability of the small RNA to inhibit the activation of PKR by dsRNA. Transient expression of EBER-1 in murine embryonic fibroblasts stimulates reporter gene expression and partially reverses the inhibitory effect of PKR. However, EBER-1 is also stimulatory when transfected into PKR knockout cells, suggesting an additional, PKR-independent, mode of action of the small RNA. Expression of L22 prevents both the PKR-dependent and -independent effects of EBER-1 in vivo. These results suggest that the association of L22 with EBER-1 in EBV-infected cells can attenuate the biological effects of the viral RNA. Such effects include both the inhibition of PKR and additional mechanism(s) by which EBER-1 stimulates gene expression.
Assuntos
RNA Viral/antagonistas & inibidores , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Animais , Ligação Competitiva , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica , Genes Reporter , Células HeLa , Herpesvirus Humano 4/genética , Humanos , Camundongos , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/farmacologia , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Transfecção , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Recent studies have suggested a role for the Epstein-Barr virus-encoded RNA EBER-1 in malignant transformation. EBER-1 inhibits the activity of the protein kinase PKR, an inhibitor of protein synthesis with tumour suppressor properties. In human 293 cells and murine embryonic fibroblasts, transient expression of EBER-1 promoted total protein synthesis and enhanced the expression of cotransfected reporter genes. However reporter gene expression was stimulated equally well in cells from control and PKR knockout mice. NIH 3T3 cells stably expressing EBER-1 exhibited a greatly increased frequency of colony formation in soft agar, and protein synthesis in these cells was relatively resistant to inhibition by the calcium ionophore A23187. Nevertheless clones containing a high concentration of EBER-1 were not invariably tumourigenic. We conclude that EBER-1 can enhance protein synthesis by a PKR-independent mechanism and that, although this RNA may contribute to the oncogenic potential of Epstein-Barr virus, its expression is not always sufficient for malignant transformation.
Assuntos
Fibroblastos/citologia , Fibroblastos/virologia , Herpesvirus Humano 4/patogenicidade , Biossíntese de Proteínas , RNA Viral/fisiologia , Animais , Divisão Celular , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Viral , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Neoplasias/fisiopatologia , Transfecção , eIF-2 Quinase/metabolismoRESUMO
The dsRNA-activated protein kinase PKR is involved in signal transduction pathways that mediate cellular processes as diverse as cell growth and differentiation, the stress response, and apoptosis. PKR was originally described as an interferon-inducible elF2alpha kinase involved in the antiviral defense mechanism of the cell. The interaction of the kinase with specific viral RNAs has been studied in much detail, but information about cellular mRNAs, which are able to bind and activate PKR, is scarce. In search for such cellular mRNAs, we developed a cloning strategy to identify individual mRNA species from the dsRNA-rich fraction of Daudi cell poly(A)+ RNA. Two out of five cDNA clones we obtained contained sequences derived from the mRNA of the translationally controlled tumor protein P23/TCTP, indicating that this mRNA is present in the dsRNA-rich fraction. Secondary structure predictions and gel electrophoretic mobility investigations on P23/TCTP transcripts confirmed the potential of this mRNA to form extensive secondary structure. A full-length P23 transcript, but not a truncated version thereof, was able to bind to PKR in vitro and in vivo. Transient transfection experiments in human 293 cells showed that coexpression of full-length P23 mRNA leads to partial inhibition of the expression of a beta-galactosidase reporter gene in trans. Additional coexpression of a dominant negative mutant of PKR or of adenovirus VA1 RNA suppressed this inhibition, indicating that it is mediated by PKR. Studies on P23/TCTP expression in cells from PKR-knockout mice suggest that P23/TCTP mRNA translation is regulated by PKR. Hence, our results demonstrate that the mRNA of P23/TCTP may both activate PKR and be subject to translational regulation by this kinase.