Simultaneous Packaging of Two Different RNA Segments into an Influenza C Virus-like Particle Occurs Inefficiently

Figures

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  Figure 1. A flow chart of the experimental design. Eleven plasmids expressing reporter gene RNA genomes (GFP-vRNA and DsRed2-vRNA) and virus proteins (PB2, PB1, P3, HEF, NP, M1, CM2, NS1, and NS2) were transfected into 293T cells. In these cells, GFP-vRNA and DsRed2-vRNA s (each flanked by the non-coding regions of Segments 5 and 6, respectively) were replicated and transcribed by the RNA polymerase complex comprising the PB2, PB1, P3, and NP proteins, resulting in the expression of GFP and DsRed2 proteins. At 48 h post transfection, the cells were subjected to flow cytometry, and cells expressing both GFP and DsRed2 were sorted. The sorted cells were then incubated for 48 h, and the influenza C virus-like particles (C-VLPs) generated from the cells were collected. HMV-II cells were infected with these C-VLPs, followed by superinfection with a helper virus (C/Ann Arbor/1/50), and the fluorescence-positive cells were examined.

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  Figure 2. Gene expressions of plasmid-transfected 293T cells. (A) The transfected 293T cells were subjected to flow cytometry 48 h post transfection. The x- and y-axes in the graph indicate the intensities of GFP and DsRed2, respectively. Each 293T cell analyzed is expressed as a dot. The graph area is divided into four regions according to the proteins expressed: DsRed2, cells expressing DsRed2 alone; EGFP, cells expressing GFP alone; Q2, cells expressing both GFP and DsRed2; and Q3, cells expressing neither GFP nor DsRed2. (B) The sorted 293T cells were observed under a fluorescence microscope to detect GFP and DsRed2 expression. Two independent fields are shown (fields 1 and 2). Scale bar, 20 µm. (C) Total RNA extracted from the sorted 293T cells was treated with DNase I, reverse-transcribed, and PCR-amplified with a primer set specific to GFP or DsRed2. The cDNA preparation setup without reverse transcription was PCR-amplified using primers specific to GFP and electrophoresed in the lane RT(−). The main products in the corresponding lanes are shown (DsRed2, 537 bp; GFP, 504 bp). M: DNA size marker (bp).

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  Figure 3. HMV-II cells infected with C-VLPs. The culture supernatant of the sorted 293T cells was added to the HMV-II monolayers, followed by superinfection with the helper virus. At 48 h post infection, the cells were observed under a fluorescence microscope (A). Cells expressing GFP alone, DsRed2 alone, and both GFP and DsRed2 are shown in the upper, middle, and lower panels, respectively. Merged images are shown on the right side. Scale bar, 20 µm. (B) The proportion of fluorescence-positive HMV-II cells expressing GFP or DsRed2 (left bar), or GFP and DsRed2 (right bar). Data obtained from three independent experiments are shown. Comparisons between groups were statistically evaluated using paired t-tests.