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. 2020 Jul 7;32(1):107859. doi: 10.1016/j.celrep.2020.107859

Figure 2.

Figure 2

Induction of VPs upon Expression of the Minimal DENV Replicase Requires Sequences in the 3′ UTR but Not the 5′ UTR

(A) Schematic of the pTM/5′UTR/NS1-5/3′UTR DENV expression plasmid (abbreviated 5′ WT).

(B) Schematic representation of 5′ UTR truncations introduced into the construct shown in (A). Numbers below each construct refer to the position in the DENV-2 genome (GenBank accession number NC_001474.2).

(C) Immunoblot showing the expression of NS3, NS1, NS5, and NS4B in cells 20 h after transfection with constructs specified on the top (left panel) or 48 h after DENV infection (right panel). β-actin served as sample loading control.

(D) Relative abundance of viral proteins was determined by densitometry and levels of NS1, NS4B, and NS5 normalized to NS3 signal. Values represent mean and standard error of three independent experiments. n.s., not significant.

(E) Thin-section TEM images of VPs induced upon transfection of indicated constructs. Cells were transfected or infected with DENV for 20 h and 48 h, respectively, before being processed for EM analysis. GND indicates the NS5 polymerase-inactivating mutation. Lower panels are magnified views of areas indicated with yellow squares in the upper panel images. Yellow arrowheads indicate individual VPs. Scale bars: 500 nm and 200 nm (upper and lower panels, respectively).

(F) For each condition, whole-cell sections of 20 cells were counted. Means ± SEM from three independent quantifications are given. n.s., not significant (∗p < 0.05).

(G) Vesicle diameters were measured manually using Fiji software. Means ± SEM were based on three independent measurements. For each condition, 50 vesicles were counted per experiment. n.s., not significant; ∗∗∗p < 0.001.

(H) Detection of NS3 by immunofluorescence microscopy to determine transfection efficiency for the indicated constructs. Scale bar: 100 μm.

(I) Transfection efficiencies were quantified according to NS3 staining. Error bars represent the standard error of three independent experiments. n.s., not significant.

Note that percentages given in (F) refer to all cells analyzed by TEM, but only ~70% of them had been transfected (I); therefore, not more than 70% of cells can contain VPs.