Characterization of dengue virus NS1 stably expressed in 293T cell lines

Dengue virus NS1 is a viral nonstructural protein detected in sera of infected individuals and in infected cells. Multiple NS1 structural forms have been reported but the functional characteristics of these forms remain unknown. In this study, a set of 293T cell lines stably expressing recombinant dengue NS1 without additional C-terminal sequence (rNS1s), with a heterologous transmembrane segment (rNS1tm), or with the 26-residue N-terminal portion of NS2A (rNS1v1) was established to aid in the characterization of different NS1 forms. Each NS1 protein form had distinct phenotypes and the following properties were documented: (1) dissipated expression in the cytoplasm, dimerization, and N-glycosylation were observed, regardless of the forms of NS1 expressed; (2) the rNS1v1 and rNS1tm forms, but not the rNS1s, were observed prominently on the surface membrane; (3) only the rNS1v1 form incorporated ethanolamine, a precursor of the glycosylphosphatidylinositol moiety, and was partially sensitive to digestion with phosphatidylinositol-specific phospholipase C. The stable 239T transfectants expressing multiple forms of dengue NS1 may be a useful model to investigate the function of NS1 and the mechanism by which NS1 associates with membrane.