Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane

Figures

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  Figure 1. Three approaches to visualize genome dimerization in Rous Sarcoma virus (RSV). (A) Schematic of pRC.V8, the wild-type RSV proviral construct that forms the backbone plasmid that was modified to generate pRC.V8.GagCFP, which contains CFP fused to NC, disrupting the gagpol reading frame, resulting in a non-functional PR and pol (designated as pr* and pol*). pRC.V8.Gag-CFP contains insertion of either RNA binding stem-loops (MS2SL or BglSL) or a non-coding mCherry (ATG-mCherry) sequence. Below are schematics of the fluorophore-fused MS2 and Bgl binding proteins, which contain an NLS; (B) In the first approach, proviral constructs containing a noncoding mCherry sequence (pRC.GagCFP.ATG-mCherry) or PP7-derived sequence encoding RNA stem-loops (pRC.GagCFP.PP7) were co-transfected, each serving as a target for specific smFISH probes labeled with Alexa 647 (against mCherry; red) or Alexa555 (against PP7, green). WGA-Alexa 594 (gray) staining was used to outline the plasma membrane (image shown is a single z-plane); (C) In the second approach, proviral constructs containing the noncoding mCherry sequence (pRC.GagCFP.ATG-mCherry) were detected by smFISH probes labeled with Alexa 647 (red)…

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  Figure 2. Examples of genome heterodimers located in the nucleus and at the plasma membrane. Shown are representative images of genome heterodimers within the nucleus (A–C), as defined by DAPI staining (blue), and at the plasma membrane, as defined by WGA-Alexa 594 staining (D,E) or Alexa 647 staining (F) in gray, for each of the described imaging methods. See Materials and Methods for further detail on the selection of the appropriate WGA conjugate. For the images showing genome heterodimers within the nucleus, the crosshairs expand into x,z and y,z planes to show the genome heterodimer is located within the DAPI staining in three dimensions (as outlined in white dashed line). For the images showing genome heterodimers at the plasma membrane, the two images on the left are a max projection image and inset showing the genome heterodimer at the plasma membrane (outline in white dashed line), while the two images on the right show the WGA-Alexa 594 or WGA-Alexa 647 staining (in gray) with Imaris Spots representing genome heterodimers. White arrowheads mark representative genome heterodimers at the plasma membrane (D,E) and at the plasma membrane and in the nucleus (F). Image histograms were…

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  Figure 3. Genome heterodimer formation dependent on presence of dimerization initiation signal (DIS) (A) Schematic of pRC.V8 and pRC.V8.∆DIS proviral constructs, which contains a 77-nucleotide deletion (bp 219-296) removing the L3 stem-loop DIS. Each construct contains either 24x-MS2 RNA stem-loops or a non-coding mCherry RNA sequence after env; (B) Cells on the left were co-transfected with pRC.V8.ATG-mCherry, pRC.V8.MS2, and MS2-YFP (green) and subjected to mCherry RNA smFISH (red). On the right, cells were transfected with pRC.∆DIS.ATG-mCherry, pRC.∆DIS.MS2, and MS2-YFP (green) and mCherry RNA smFISH (red) was performed. Cells were imaged through the width of the nucleus, as assessed by DAPI staining, using a 63× objective at 3.5× optical zoom. The top rows of each panel show maximum projection images and the bottom row images indicate the location of foci identified using the Imaris Spots function; (C) Colocalization of dual-colored foci was analyzed using the Imaris Spots colocalization function (colocalization threshold 0.5 µm). Statistical significance was assessed by using negative binomial regression model, and a total of 30 cells were analyzed in two biological replicates, (*** p…

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  Figure 4. Single virion analysis in RSV shows preferential formation of genome homodimers: (A) Confocal microscopy of virions harvested from cell supernatant. QT6 fibroblasts were transiently transfected with proviruses containing MS2 and Bgl RNA stem-loop-containing and their respective fluorophore-labeled coat proteins. Visualized RNA genome foci are considered to be genome dimers, as they were present in virions harvested from the supernatant of transiently transfected cells. The top row shows the confocal microscopy images and the bottom row shows Spots labeled using the Imaris software. Images of virions were obtained using a 63× objective at 6× optical zoom; (B) Detection of Gag-CFP from virions harvested from cell supernatant expressing loop-containing provirus and their coat proteins by immunoblotting. The Gag-CFP is uncleaved, as expected, since CFP is located between NC and protease. pRC.V8, the wild-type parental proviral construct, which has normal protease activity, was used as a positive control for virus budding; (C) Quantitation of the number of genome homodimers and genome heterodimers present within harvested virions. Only RNA genomes that colocalized with Gag-CFP were…

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  Figure 5. Contribution of spliced vRNA to intracellular genome heterodimers in RSV: (A) Schematic of the provirus constructs used to visualize only unspliced vRNAs (top) or spliced and unspliced vRNAs (bottom). Each proviral construct contains either a MS2-24x loop cassette or a non-coding mCherry RNA sequence used for RNA smFISH. The location of the 5′ splice donor (5′ SD) and 3′ splice acceptor (3′ SA) are indicated; (B) Representative confocal microscopy images of genome heterodimers in cells co-transfected with the proviral constructs containing ATG-mCherry (red) and MS2-24x loop cassette and MS2-YFP (green) are shown here. Cells were imaged by a z-stack through the width of the cell, as determined by WGA membrane staining, using a 63× objective at 3.5× optical zoom. A colocalization threshold of 0.5 µm was used to determine whether two Spots were colocalized; (C) Comparison of the average number of heterodimers per cell between only unspliced vRNAs and spliced and unspliced vRNAs (n = 19 cells and n = 22 cells, respectively). The average number of heterodimers per cell is 3.79 ± 0.49 in unspliced only and 2.79 ± 1.10 in spliced and unspliced vRNAs. Significant outliers were identified…