Fig 2.

An intact N-terminal NS4A AH is required for DENV replication. (A) A helical wheel plot of NS4A amino acids 3 to 20 showing the location of the inserted mutations (black, L6E and M10E). (B) Comparable expression levels of full-length wild-type and L6E;M10E mutant NS4A as determined by Western blot analysis of FLAG-tagged wild-type or mutant NS4A expression levels using an anti-FLAG antibody. Monoclonal mouse anti-actin antibody was used as a loading control. (C) Spectra of the purified mutant peptide was recorded in 10 mM sodium phosphate buffer (pH 7.0) (dashed dotted line) or in the presence of 100 mM sodium dodecyl sulfate (SDS; solid line), n-dodecyl phosphocholine (DPC; long dashed line), n-dodecyl-β-d-maltoside (DDM; short dashed line), or 1 mM cetyl trimethylammonium bromide (CTAB; dotted line). (D) Schematic diagram of the DENV2 luciferase reporter replicon (pDRrep) used (11). Amino acids 1 to 30 of NS4A containing the AH region are shown for the wild-type and mutant replicons. Amino acid changes are in bold. (E) Replication assay for NS4A-AH mutated dengue replicons. Wild-type and mutated replicons were transfected into BHK21 cells. Luciferase activity was measured at 6, 24, 48, and 72 h posttransfection. Luciferase activity (relative light units [RLU]) is plotted for each time point. The data were normalized to the 6-h values that reflect transfection efficiency and to luciferase levels in replicon with a mutated polymerase. The mean values of at least two independent experiments performed in triplicates are shown. Error bars indicate standard errors of the means.