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

Figure 3.

Figure 3

ZIKV 5′ UTR Is Dispensable for Expression-Based Induction of VPs

(A and B) Schematic of the pTM/5′UTR/NS1-5/3′UTR ZIKV expression plasmid (abbreviated as 5′ WT) (A) and the 5′ UTR deletion introduced into this construct (B). The number in the bottom refers to position in the ZIKV H/PF/2013 genome (GenBank accession number KJ776791.2).

(C) Immunoblot showing abundance of ZIKV NS3, NS1, NS4A, and NS2B in transfected and infected (MOI = 10) cells (left and right panel, respectively). GAPDH served as sample loading control.

(D) Relative abundance of ZIKV proteins was determined by densitometry and levels of NS1, NS2B, and NS4A normalized to NS3 signal. Values represent mean and standard error of three independent experiments. n.s., not significant.

(E) Thin-section TEM images showing VPs induced upon transfection of constructs specified on the top. Cells were transfected or infected with ZIKV for 18 h and 24 h, respectively, before being processed for EM analysis. GAA 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, VPs contained in whole-cell sections from at least 20 cells were counted. Means ± SEM from three independent quantifications are given. n.s., not significant; ∗∗p < 0.01.

(G) Vesicle diameters were measured manually using Fiji software. Means ± SEM were calculated from three independent measurements. For each condition, 50 vesicles were counted per experiment. n.s., not significant.

(H) Detection of ZIKV NS4B by immunofluorescence microscopy to determine transfection efficiency for the constructs given on the top of each panel. Scale bar: 100 μm.

(I) Transfection efficiencies were quantified by counting NS4B-containing cells and normalization to the total number of cells. 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 a fraction of them had detectable amounts of ZIKV NS4B that were used to determine transfection efficiency (I). The higher percentage of cells with VPs attained with some constructs is most likely due to lower sensitivity of the immunofluorescence, allowing detection only of cells expressing high amounts of NS4B.