FIG 3.

In situ fluorescence protease protection assay. (A) Topology model of DENV NS4B on the ER membrane (17). Two predicted transmembrane domains (pTMD1 and pTMD2), three validated transmembrane domains (TMD3 to -5), and the amino acid positions in NS4B are indicated. (B) Schematic diagram of 2K-NS4B truncate-EGFP fusion constructs. Each NS4B construct was fused with the N-terminal 2K signal peptide (black boxes) and a C-terminal EGFP tag. The transmembrane domains (pTMDs and TMDs) are shown as white boxes. MTase derived from the N-terminal 300 amino acids of NS5 is indicated with an oval. (C) FPP assay. BHK-21 cells were transfected with the indicated NS4B truncate-EGFP constructs. At 24 h posttransfection, the sensitivities of EGFP were examined by FPP assay (see Materials and Methods for details). Curves of relative intensities (%) versus time (s) were plotted. The relative intensities were calculated according to previously described formulas (15). The NS4B constructs are indicated in the corresponding fluorescence panels. (D) Summary of relative fluorescence intensities and initial rates of fluorescence degradation. Average values for at least two independent experiments are presented. (E) Schematic diagram of the membrane topology of NS4B before (left) and after (right) cleavage of the MTase of NS5 by viral NS2B/NS3 protease. The cleavage of MTase from NS4B regulates the translocation of TMD5 from the cytosol to the ER lumen.