Subcellular localizations and time-course expression of dengue envelope and non-structural 1 proteins in human endothelial cells

We performed subcellular fractionation of human endothelial cells (EA.hy926) after a challenge with dengue virus serotype-2 (DEN-2) at multiplicity of infection (MOI) of 10 for 2 h. The cells were fractionated into four fractions including cytosol, membrane/organelle, nucleus and cytoskeleton, and their purity was nicely confirmed by immunofluorescence staining of markers for individual subcellular compartments. Western blot analysis of dengue envelope (E) and non-structural 1 (NS1) proteins showed that E was present only in membrane/organelle fraction, whereas NS1 was present in membrane/organelle, nucleus, and cytoskeleton fractions. Time-course study showed that E was present immediately after the challenge, whereas NS1 was present after 12 h of the challenge. Our data may provide a new roadmap for studying mechanisms of replication of dengue virus and its interactions with host cells.