Estudio de la localizaciĆ³n subcelular de las isoformas de la proteĆna X (HBx) del virus de la hepatitis B (HBV)
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Fecha
2017
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Idioma
es
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Universidad AndrƩs Bello
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Licencia CC
Licencia CC
Resumen
El Virus de la Hepatitis B (HBV) codifica para la proteĆna X (IiBx), una pequeƱa proteĆna no
estructural a la cual se le atribuyen mĆŗltiples funciones dentro de la cĆ©lula infectada, tales
como controlar el ciclo replicativo viral, regular el ciclo celular y participar en el desarrollo
del Carcinoma Hepatocelular (HCC). El transcrito de HBx posee codones internos de inicio
de la traducciĆ³n, lo cual sugiere la traducciĆ³n de isoformas de HBx. La localizaciĆ³n subcelular
de HBx varĆa segĆŗn su nivel de expresiĆ³n, localizĆ”ndose en el nĆŗcleo a bajos niveles de
expresiĆ³n y en el citoplasma a altos niveles de expresiĆ³n. Nuestra hipĆ³tesis considera que las
isoformas de HBx tienen una localizaciĆ³n subcelular distinta cuando son expresadas de
manera individual y que varĆa segĆŗn su nivel de expresiĆ³n. Para investigar esto se generaron
mutantes que expresan de manera individual cada isoforma, y luego, mediante tƩcnicas de
fluorescencia, se determinĆ³ la localizaciĆ³n subcelular de cada isoforma, variando ademĆ”s su
nivel de expresiĆ³n. Los resultados de microscopĆa indican que cada isoforma de manera
individual presenta una localizaciĆ³n subcelular diferente. AdemĆ”s, al expresarlas en
combinaciones, la localizaciĆ³n final tambiĆ©n varĆa. Finalmente serĆa importante analizar a
futuro cĆ³mo estas isoformas interactĆŗan entre sĆ, siendo de manera directa o mediante otras
proteĆnas, asĆ como tambiĆ©n analizar el comportamiento de las isoformas en el contexto viral.
Hepatitis B virus (HBV) codes for protein X (HBx), a small non-structural protein with multiple functions within infected cell, such as control the viral replicative cycle, regulate the cell cycle and participate in the development of Hepatocellular Carcinoma (HCC). The HBx transcript has three in-frame translational initiation codons, which suggests the translation of three HBx isoforms. The subcellular HBx localization varies according to its expression level, localizing in the nucleus at low expression levels and in the cytoplasm at high expression levels. Our hypothesis considers that the isoforms of HBx have a different subcellular location when they are expressed individually and varies according to their expression level. To investigate this, different DNA constructs were generated by mutagenesis to individually express each isoform, and then, by indirect immunofluorescence or fluorescence, the subcellular localization of each isoform was determined at different expression levels. In addition, the co-expression of these isoforms was analysed. Microscopy results indicated that each isoform individually exhibits a different subcellular localization. In addition, when expressed in combinations, the fmal location also varied. Finally it would be important to analyse, in the future, how these isoforms interact with one another, directly or through other proteins, as well as to analyse the behaviour of the isoforms in the viral context.
Hepatitis B virus (HBV) codes for protein X (HBx), a small non-structural protein with multiple functions within infected cell, such as control the viral replicative cycle, regulate the cell cycle and participate in the development of Hepatocellular Carcinoma (HCC). The HBx transcript has three in-frame translational initiation codons, which suggests the translation of three HBx isoforms. The subcellular HBx localization varies according to its expression level, localizing in the nucleus at low expression levels and in the cytoplasm at high expression levels. Our hypothesis considers that the isoforms of HBx have a different subcellular location when they are expressed individually and varies according to their expression level. To investigate this, different DNA constructs were generated by mutagenesis to individually express each isoform, and then, by indirect immunofluorescence or fluorescence, the subcellular localization of each isoform was determined at different expression levels. In addition, the co-expression of these isoforms was analysed. Microscopy results indicated that each isoform individually exhibits a different subcellular localization. In addition, when expressed in combinations, the fmal location also varied. Finally it would be important to analyse, in the future, how these isoforms interact with one another, directly or through other proteins, as well as to analyse the behaviour of the isoforms in the viral context.
Notas
Tesis (BioquĆmico, MagĆster en BioquĆmica)
Esta tesis se realizĆ³ en el Laboratorio de Cromatina y EpigenĆ©tica de la FundaciĆ³n Ciencia y Vida y fue fmanciada por el proyecto ANILLOS ACT 1119 titulado "Mecanismos moleculares del cĆ”ncer. Examinando la funciĆ³n de la cromatina en la replicaciĆ³n de HBV y leucemia" proyecto BASAL PFB-16, FONDECYT 1160480 y beca CONICYT 21140956 (Sergio HernĆ”ndez, 2014).
Esta tesis se realizĆ³ en el Laboratorio de Cromatina y EpigenĆ©tica de la FundaciĆ³n Ciencia y Vida y fue fmanciada por el proyecto ANILLOS ACT 1119 titulado "Mecanismos moleculares del cĆ”ncer. Examinando la funciĆ³n de la cromatina en la replicaciĆ³n de HBV y leucemia" proyecto BASAL PFB-16, FONDECYT 1160480 y beca CONICYT 21140956 (Sergio HernĆ”ndez, 2014).
Palabras clave
Isoformas de ProteĆnas, Hepatitis B