Determinación de la interacción del rotavirus WTEW con las proteínas de choque térmico en la membrana citoplasmática de las células KATO III

Abstract

In the Molecular Biology of Virus located at Universidad Nacional de Colombia, the research to determine whether rotavirus WWM, Wt1-5, O-ECwt and WTEW (generated in the laboratory) can be used as oncolytic virus. Preliminary research suggests that rotaviral isolates infected tumor cell lines Sp2/0-Ag14 (murine myeloma), U937 (histiocytic lymphoma), REH (Acute Lymphocytic Leukemia), PC3 (prostate adenocarcinoma grade IV). MDA and MCF-7 (breast adenocarcinoma). This study was aimed to determine whether there was or not WTEW rotavirus interaction with heat shock protein Hsp90, Hsp70, Hsp60, Hsp40 during the entry to the tumor cell line KATO III (gastric carcinoma). For this, fractions enriched with cytoplasmic membranes were isolated, incubated with rotavirus or blocked with specific anti-Hsp (complete or portion F(ab')2) and was added rotavirus MOI 8. The interaction was observed by capture ELISA, co-immunoprecipitation and western blotting. To determine the functional role of HSPs, the cells were incubated with antibody F(ab')2 and then with rotavirus for 12 h at 37 °C. Rotavirus was incubated with soluble proteins HSPs complete for 1 h at 37 °C and then the HSPs rotavirus complex was added to the cells for 12 h at 37 °C. In both cases the infection was assessed by immunocytochemistry. Finally cells rotavirus WTEW incubated for 1 h at 4 °C and then were added the synthetic peptides each Hsp for 1 h at 4 °C leaving 12 h at 37 °C in CO2 chamber. Infection was evaluated capture ELISA. It was determined that there is interaction between the rotavirus WTEW and HSPs was direct and functional manner as to block or alter binding said infection is decreased, suggesting that interacts rotavirus WTEW and HSPs of facilitating the entry of rotavirus to the cell KATO III line.