RESUMEN
The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.
Asunto(s)
Endonucleasas , Elementos de Nucleótido Esparcido Largo , ADN Polimerasa Dirigida por ARN , Transcripción Reversa , Humanos , Microscopía por Crioelectrón , Endonucleasas/química , Endonucleasas/genética , Endonucleasas/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , ARN/genética , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Cristalografía por Rayos X , ADN/biosíntesis , ADN/genética , Inmunidad Innata , Interferones/biosíntesisRESUMEN
The mechanisms of congenital transmission of Chagas disease remain largely unknown. To better understand the role of maternal immunology during pregnancy in congenital Chagas transmission, we studied the cytokine production and the parasitic load in three groups of mothers: infected mothers who transmitted the disease to their babies (M+B+-), infected mothers who did not transmit the disease to their babies (M+B-) and not infected mothers as a control group (M-B-). M+B+ mothers produced less IFNgamma and more IL-10 than the M+B- mothers, and they are not able to produce IL-2. M+B+ mothers showed a higher parasitic load. These results, indicated that the congenital Chagas transmission is associated with an immunological imbalance and a high parasitic load in the M+B+ mothers.
Asunto(s)
Animales , Femenino , Humanos , Embarazo , Citocinas/biosíntesis , Complicaciones Infecciosas del Embarazo/inmunología , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/transmisión , Transmisión Vertical de Enfermedad Infecciosa , Trypanosoma cruzi/fisiología , Citocinas/inmunología , Enfermedad de Chagas/parasitología , Inmunidad Celular , Interferón gamma/biosíntesis , Interferones/biosíntesis , Portador Sano/inmunologíaRESUMEN
Genetically homogeneous and heterogeneous mouse populations were tested for resistance to experimental street rabies virus infection and their ability to synthesize interferon (IFN) during the infection. The genetically heterogenous HI mouse population was highly resistant (12 per cent mortality), and the genetically homogeneous BALB/c and C3H mice as well as the genetically heterogeneous Sw and LI mouse populations were susceptible (60 to 71 per cent mortality). The genetically homogeneous A/J mice were highly susceptible (85 per cent mortality) to experimental street rabies infection. The ability of these mice to synthesize IFN as measured in serum 4 days after the infection was directly related to the degree of resistance, with the highly resistant HI mice showing large amounts of IFN (850 U/ml), and the susceptible mice showing low amounts of IFN (50 to 280 U/ml). IFN induced within the central nervous system and measured in brain homogenates during infection was not correlated with resistance. The present data suggest that high levels of IFN occurring in serum early during infection with street rabies virus contribute to the resistance of these mice
Asunto(s)
Animales , Masculino , Ratones , Interferones/biosíntesis , Rabia/inmunología , Virus de la Rabia/inmunología , Sistema Nervioso Central/inmunología , Susceptibilidad a Enfermedades , Inmunidad Innata , Ratones Endogámicos BALB C , Factores de TiempoRESUMEN
Human amniotic interferon was investigated to define the species specificity of its antiviral action and compare its anti-cellular and NK cell stimulating activities with those of other human interferons. The antiviral effect was titrated in bovine (RV-IAL) and monkey (VERO) cells. Amniotic interferon exhibited, in bovine cells, 5% of the activity seen in monkey cells, while alpha interferon displayed 200%. No effect was detected with either beta or gamma interferon in bovine cells. Daudi cells were exposed to different concentrations of various interferons and the cell numbers were determined. The anticellular effect of the amniotic interferon reached its peak on the third day of incubation. Results suggested a higher activity for alpha and gamma interferons and a lower activity for beta when compared to amniotic interferon. Using total mononuclear cells as effector cells and K 562 as target cell in a 51Cr release assay, it was demonstrated that low concentrations of amniotic interferon consistently stimulated NK cell activity in cells derived from several donors, the results indicating a higher level of activity with this interferon than with alpha and beta interferons
Asunto(s)
Interferones/biosíntesis , Antivirales/análisisRESUMEN
Foi desenvolvido um sistema de produçäo de interferon humano de membranas amnióticas (IFN-MA) em microtécnica. Fragmentos de âmnio de 1,4 ou 2,2cm de diâmetro, em placas de microtécnica (24 câmaras), foram infectados por vírus Sendai e o IFN-MA resultante foi titulado. Maiores títulos (12.800 e 13.000 unidades por ml) foram obtidos quando utilizadas quantidades de meio de 0,5ml e 1,0ml); 6,4 unidades hemaglutinantes do vírus Sendai e um fragmento por câmara. Níveis de IFN-MA mais consistentes e altos foram alcançados na regiäo coriônica do âmnio (6.000 a 9.600 unidades/ml) quando comparados com aqueles da regiäo umbilical (860 a 2.500 unidades/ml) e reflexa (200 a 6.500 unidades/ml). O sistema com fragmentos de 2,2cm de diâmetro produziu, em média, quantidades de interferon/ml superiores (9.300 unidades/ml) quando comparado ao da produçäo do âmnio total (2.200 unidades/ml). Apesar da variabilidade nos títulos produzidos individualmente pelos fragmentos de 2,2cm de diâmetro, devido a econômia de tempo e de materiais e aos altos níveis de IFN resultantes, a microtécnica poderá se empregada quando um grande número de variáveis for investigada
Asunto(s)
Humanos , Interferones/biosíntesis , Membranas Extraembrionarias/análisisRESUMEN
No presente trabalho, foi investigada a produçäo de interferon nos tecidos da placenta humana induzidos por diferentes vírus. A membrana amniótica mostrou ser mais eficiente produtora de interferon quando infectada pelo vírus da doença de Newcastle (NDV) que a membrana ou as vilosidades coriônicas. A irradiaçäo do NDV com a luz ultravioleta (UV) diminuiu sua capacidade indutora nos três tecidos placentários. Os níveis de interferon encontrados em culturas da membrana amniótica ou coriônica, induzidos por vírus para-influenza I (Sendai), foram muito diferentes, dependendo da placenta utilizada, tanto com o vírus vivo como o tratado com a UV. Quando vilosidades coriônicas foram infectadas com este vírus, näo foi encontrada atividade de IFN. Testes comparativos mostraram que o NDV é melhor indutor de interferon do que o vírus Sendai em membrana amniótica. Os vírus Sindbis e Oriboca (vivos ou inativados pela UV) näo induziram títulos mensuráveis de interferon em qualquer dos tecidos mencionados. Concluiu-se que o sistema NDV - membrana amniótica é o mais eficiente na induçäo de IFN e merece investigaçöes mais abrangentes
Asunto(s)
Humanos , Femenino , Placenta/metabolismo , Interferones/biosíntesis , Virus de la Parainfluenza 1 Humana/fisiología , Amnios/metabolismo , Virus de la Enfermedad de Newcastle/fisiologíaRESUMEN
En el presente trabajo se describe la clonación del cDNA correspondiente al interferón leucocitario humano tipo A, preparado a partir de el RNAm purificado de un mieloblastoma. Asimismo, se detalla la estrategia seguida para la producción de esta proteína en E. coli. Para estos efectos se fusionó un adaptador de DNA sintético al cDNA de interferón, uniéndole luego el promotor y sitio de unión a ribosomas del operón de triptofano. Se determinó utilizando un sistema de minicélulas, la expresión de interferón