Inhibiting rotavirus infection by membrane-impermeant thiol/disulfide exchange blockers and antibodies against protein disulfide isomerase.

OBJECTIVES: Determining the effect of membrane-impermeant thiol/disulfide exchange inhibitors on rhesus rotavirus infectivity in MA104 cells and investigating protein disulfide isomerase (PDI) as a potential target for these inhibitors. METHODS: Cells were treated with DTNB [5,5-dithio-bis-(2-nitrobenzoic acid)], bacitracin or anti-PDI antibodies and then infected with virus. Triple-layered particles (TLPs) were also pretreated with inhibitors before inoculation. The effects of these inhibitors on α-sarcin co-entry, virus binding to cells and PDI-TLP interaction were also examined. FACS analysis, cell-surface protein biotin-labeling, lipid-raft isolation and ELISA were performed to determine cell-surface PDI expression. RESULTS: Infectivity became reduced by 50% when cells or TLPs were treated with 1 or 6 mM DTNB, respectively; infectivity became reduced by 50% by 20 mM bacitracin treatment of cells whereas TLPs were insensitive to bacitracin treatment; anti-PDI antibodies decreased viral infectivity by about 45%. The presence of DTNB (2.5 mM) or bacitracin (20 mM) was unable to prevent virus binding to cells and rotavirus-induced α-sarcin co-entry. CONCLUSIONS: It was concluded that thiol/disulfide exchange was involved in rotavirus entry process and that cell-surface PDI was at least a potential target for DTNB and bacitracin-induced infectivity inhibition.

[Interaction of rotavirus with protein disulfide isomerase in vitro and cell system]. / Interacción de rotavirus con la proteína disulfuro-isomerasa in vitro y en sistemas celulares.

INTRODUCTION: Rotavirus entry process involves a multi-step mechanism, the first of which is when the outermost viral proteins interact with four different integrins and Hsc70. Recently, rotavirus infection reportedly has been decreased after blocking cell surface protein disulfide isomerase (PDI). This suggested that this protein interacts with rotavirus during the entry process. OBJECTIVES: The aim was to establish the rotavirus-PDI interaction in an in vitro system using PDI isolated from bovine liver, and in a cell system consisting of MA104 cells and mouse small intestinal villi. MATERIALS AND METHODS: Protein disulfide isomerase was isolated from a bovine liver homogenate using anti-PDI antibodies coupled to agarose through hydrazone bonds. Purity of purified protein was assessed by SDS-PAGE and Western blot. The purified PDI was used to study its in vitro interaction with the rotavirus particles. This interaction was compared with that taking place in MA104 cells and small intestinal villi isolated from sucking mice ICR. RESULTS: The purified PDI showed an electrophoretic homogeneity and was able to bind rotavirus particles in vitro. Rotavirus-PDI interaction was detected by capture ELISA using purified protein and rotavirus strains RRV and wild-type ECwt. Interaction between rotavirus particles and cellular PDI was detected by ELISA using cell lysates after virus inoculation. CONCLUSIONS: Rotavirus-PDI interaction was demonstrated in vitro as well as inMA104 cells and intestinal villi from suckling mice.

Interacción de rotavirus con la proteína disulfuro-isomerasa in vitro y en sistemas celulares / Interaction of rotavirus with protein disulfide isomerase in vitro and cell system

Introducción. La entrada del rotavirus a la célula implica un mecanismo de múltiples pasos; las proteínas virales externas interaccionan con cuatro diferentes integrinas y Hsc70. Recientemente reportamos que la infección por rotavirus disminuye cuando se bloquea la proteína disulfuro-isomerasa de la superficie celular, lo que sugiere su interacción con el rotavirus en el proceso de entrada. Objetivo. Establecer la interacción del rotavirus con la proteína disulfuro-isomerasa en un sistema in vitro utilizando la proteína aislada de hígado bovino y, en un sistema celular, utilizando vellosidades intestinales de ratón y células MA104. Materiales y métodos. Se aisló la proteína disulfuro-isomerasa a partir de un homogenizado de hígado bovino utilizando anticuerpos anti-proteína disulfuro-isomerasa acoplados a agarosa mediante enlace hidrazona. La proteína disulfuro-isomerasa purificada se examinó por SDS-PAGE y Western blot y se utilizó para estudiar su interacción in vitro con rotavirus. Esta interacción se comparó con aquella observada en células MA104 y en las vellosidades intestinales de ratón. Resultados. La proteína disulfuro-isomerasa purificada mostró homogeneidad electroforética y fue capaz de unirse a rotavirus en un sistema in vitro. La interacción proteína-rotavirus fue detectada por ELISA de captura usando la proteína disulfuro-isomerasa bovina purificada y rotavirus de las cepas RRV y silvestre ECwt. La interacción de partículas de rotavirus purificadas con la proteína disulfuro-isomerasa celular se evidenció con ELISA, usando lisado celular después de la inoculación viral. Conclusión. La interacción rotavirus-proteína disulfuro-isomerasa fue demostrada in vitro, en células MA104 y en vellosidades intestinales de ratón lactante.

Introduction. Rotavirus entry process involves a multi-step mechanism, the first of which is when the outermost viral proteins interact with four different integrins and Hsc70. Recently, rotavirus infection reportedly has been decreased after blocking cell surface protein disulfide isomerase (PDI). This suggested that this protein interacts with rotavirus during the entry process. Objectives. The aim was to establish the rotavirus-PDI interaction in an in vitro system using PDI isolated from bovine liver, and in a cell system consisting of MA104 cells and mouse small intestinal villi. Materials and methods. Protein disulfide isomerase was isolated from a bovine liver homogenate using anti-PDI antibodies coupled to agarose through hydrazone bonds. Purity of purified protein was assessed by SDS-PAGE and Western blot. The purified PDI was used to study its in vitro interaction with the rotavirus particles. This interaction was compared with that taking place in MA104 cells and small intestinal villi isolated from sucking mice ICR. Results. The purified PDI showed an electrophoretic homogeneity and was able to bind rotavirus particles in vitro. Rotavirus-PDI interaction was detected by capture ELISA using purified protein and rotavirus strains RRV and wild-type ECwt. Interaction between rotavirus particles and cellular PDI was detected by ELISA using cell lysates after virus inoculation. Conclusions. Rotavirus-PDI interaction was demonstrated in vitro as well as in MA104 cells and intestinal villi from suckling mice.

Mycobacterium tuberculosis ESAT-6 antigen immunogenicity in Owl Monkeys

La única vacuna disponible contra la tuberculosis es la cepa Mycobacterium bovis BCG, que ofrece una eficacia protectiva variable (0/100-80/100), siendo urgente un nuevo agente profiláctico. Se han evaluado diversos candidatos a vacuna contra este patógeno, en los modelos animales de experimentación convencionales (murino, cobayo, conejo), obteniéndose información básica sobre el efecto de la vacuna en la carga bacterial frente a un reto infeccioso, así como también la reducción o prevención de la patología en los pulmones u otros órganos blanco; además de los aspectos relacionados con la respuesta inmune hacia el Mycobacterium tuberculosis. Los primates no humanos tienen ventajas sobre los modelos convencionales en la evaluación de vacunas, de hecho se ha verificado el comportamiento de agentes terapéuticos en humanos después de haber sido medida la capacidad protectiva de éstos en monos con tuberculosis inducida. Los primates mas estudiados en la infección por micobacterias son el cynomulgus, y el rhesus, observándose que estos animales mantienen la infección en un estado subclínico, muy similar a la tuberculosis humana donde el 90/100 de la población infectada mantiene la infección en un estado latente. Dado que el modelo animal debe semejar el comportamiento de las proteínas estudiadas en el ser humano, el mono Aotus puede representar ventajas en la investigación de tuberculosis por ser un primate con aproximadamente un 90/100 de similitud al humano en cuanto a las moléculas del sistema inmune estudiadas hasta hoy. La proteína ESAT-6 de (early secretory antigenic target 6 kD) de Mycobacterium tuberculosis es un componente minoritario del filtrado de cultivo de corto tiempo (CFP), ha sido genética y químicamente caracterizada e induce una potente respuestainmunogénica del tipo TH1. Este antígeno es secretado durante la fase inicial de crecimiento siendo fuertementereconocido por animales y humanos infectados por Mycobacterium tuberculosis...

Characterising Mycobacterium tuberculosis Rv1510c protein and determining its sequences that specifically bind to two target cell lines.

The process of Mycobacterium tuberculosis infection of the macrophage implies a very little-known initial recognition and adherence step, important for mycobacterial survival; many proteins even remain like hypothetical. The Rv1510c gene, encoding a putatively conserved membrane protein, was investigated by analysing the M. tuberculosis genome sequence data reported by Cole et al. and a previous report that used PCR assays to show that the Rv1510 gene was only present in M. tuberculosis. This article confirmed all the above and identified the transcribed gene in M. tuberculosis, Mycobacterium africanum, and in M. tuberculosis clinical isolates. Antibodies raised against peptides from this protein recognised a 44 kDa band, corresponding to Rv1510c theoretical mass (44,294 Da). Assays involving synthetic peptides covering the whole protein binding to U937 and A549 cell lines led to recognising five high activity binding peptides in the Rv1510 protein: 11094, 11095, 11105, 11108, and 11111. Their affinity constants and Hill coefficients were determined by using U937 cells. Cross-linking assays performed with some of these HABPs showed that they specifically bound to a U937 cell line 51 kDa protein, but not to Hep G2 or red blood cell proteins, showing this interaction's specificity.