Figure 1.

Subcellular targeting strategies tested for increasing accumulation of NS1 in N. benthamiana leaves. (A) Schematic representation of the DENV-NS1 expression constructs evaluated in this study: p35S, double enhanced 35S promoter from Cauliflower Mosaic Virus 35S gene; tCUP, translation enhancer from the tobacco cryptic upstream promoter; nos, nopaline synthase transcription terminator; Pr1b, tobacco pathogenesis-related 1b protein secretory signal peptide; C-Myc, detection/purification tag; KDEL, endoplasmic reticulum retrieval tetrapeptide; CTPP, vacuole sorting peptide; RuBisCo T.P., rubisco small subunit transit peptide; HFBI, hydrophobin I; ELP, elastin-like polypeptide. The schematic is not to scale. (B) Western blot analysis of NS1 targeting the protein to five different subcellular compartments in N. benthamiana leaves 4 days post-infiltration. Protein was extracted with PEB buffer, and an equal volume of TSP (20μl/lane) was loaded on the gel and detected with an anti-c-Myc antibody. Expression levels were measured by densitometry of western blot using known amounts of c-Myc-tagged CBD protein (25–200 ng) as reference; p19-infiltrated tissue was used as negative control. (C) Accumulation of NS1 and NS1 fusions in different subcellular compartments, results are the average of three experiments, and error bars represent the standard error of the mean.