Fig 3.

Viral and PolyA mRNA concentrate with eIF4G, PABP, and RSV M2-1 in the differentially phase-separated IB microdomains/IBAGs. (A and B) Vero cells were left uninfected or infected with bRSV at an MOI of 1 for 24 h. Cells were then fixed and immunostained for eIF4G (magenta) and RSV N (green) or RSV M2-1 (cyan) in A, and for PABP (magenta) and RSV M2-1 (cyan) in B, and nuclei stained with DAPI (blue) for confocal analysis. Graph shows signal intensities along the white line drawn in the “Merge + DAPI” panel. (C and D) Vero cells infected with bRSV at an MOI of 1 were fixed at 24 hpi. FISH analyses were performed (as described in the Materials and Methods) with specific biotinylated probes to detect the indicated viral mRNAs (C; yellow) or total polyadenylated mRNA (D; yellow). Negative controls included uninfected cells stained with the same probes or bRSV-infected cells stained with probes against VSV G mRNA. Co-immunostaining was performed with RSV N (green in C) and M2-1 (cyan in D). Presented images are single-plane confocal images taken in the median section of the cells. Arrowheads indicate sites of mRNA and M2-1 concentration. Bottom panel of D, Imaris reconstruction of confocal image Z-stacks of the same cell from the top panel (left) and another example (right). (E) Enlarged images of the IBs boxed in D (i and ii) are shown with other examples (iii to v). Scale bars, 1 µm. (F) Graph shows volume of the IBs in the panel above (represented as cyan bars) and volumes of the individual IBAGs identified within them (presented as yellow dots), also quantified using the Imaris software, as described in the Materials and Methods. IBs i–v were stained for PolyA and M2-1, and vi–x with PolyA and P. (G) Graph shows longest diameter of IBs containing identifiable IBAGs (+ IBAGs) and those without (− IBAGs), also quantified using the Imaris software. An unpaired t-test was used for statistical analysis; ****, P < 0.0001. (H and I) CLEM analysis of (H) Vero cells infected with bRSV and (I) A549 cells infected with hRSV. TEM was done of the same cells following confocal imaging for PolyA mRNA (yellow) and RSV P (green) and images merged in the CLEM overlay panels. Zoom panels are higher magnification images of the IBs boxed in the panels above.