Purification and amino-terminal sequence of the dengue virus-induced cytotoxic factor

Abstract

The present study was undertaken to purify the dengue type 2 virus (DV)-induced cytotoxic factor (CF) and analyse its amino-terminal sequence. Spleens collected from DVinfected moribund mice were made into a single cell suspension and cultured for 24 h. The culture supernatant was purified using fast protein liquid chromatography (FPLC) and polyacrylamide gel electrophoresis (PAGE). CF could be purified in a single step by FPLC and native PAGE. The isoelectric point of CF was pH 6.5. Purified CF had a molecular weight of 45 kDa on native-PAGE while on SDS-PAGE it dissociated into two bands of 20 and 2 5 kDa. Anti- CF-antisera reacted specifically with CF bands separated on native and SDS-PAGE in a Western blot assay. A sequence of I9 amino-acids of the N-terminus of CF was analysed which on comparison with that of other known cytotoxic proteins indicated that CF differs from them. Thus, CF appears to be a unique cytotoxic protein induced by a virus.