vr 1986hk 1 92×105  (ATCC)

Journal: Nature Communications

Article Title: Super-resolution atlas of SARS-CoV-2 infection reveals protease-dependent organelle maturation, dsRNA landscapes, and intracellular structural proteins

doi: 10.1038/s41467-026-75289-x

Figure Lengend Snippet: A Schematic of the SARS-CoV-2 genome. Targets explicitly labeled and investigated in this study are indicated in black text, while uninvestigated regions are shown in gray. Red markers indicate the binding sites for the vgRNA FISH probes. B Model of a SARS-CoV-2–infected A549-ACE2 cell highlighting the structural evolution of viral replication organelles (ROs) and their previously reported intracellular localizations. Key cellular targets investigated in this study are indicated. C Membrane topology of the uncleaved viral polyproteins pp1a and pp1ab. The schematic illustrates the relative positions of the individual non-structural protein (NSP) domains with respect to the endoplasmic reticulum (ER) membrane following translation. Arrows indicate known proteolytic cleavage sites targeted by nsp3 (orange) and nsp5 (blue).

Article Snippet: For nirmatrelvir washout experiments, cells were washed once with PBS and incubated with SARS-CoV-2 WA 1 (USA-WA1/2020) at an MOI of 10 in 50 μL of DMEM containing 2% FBS for 6 h. The viral inoculum was then removed, and cells were cultured in 100 μL of DMEM containing 2% FBS and 288 nM nirmatrelvir (MedChemExpress, HY-138687) for 18 h. A subset of cells was then washed with PBS and fixed by 4% PFA and 0.1% glutaraldehyde in PBS for 1 h. The remaining cells were washed and cultured in fresh DMEM containing 2% FBS for an additional 24 h before being washed and fixed as described above.

Techniques: Labeling, Binding Assay, Infection, Membrane

Journal: Nature Communications

Article Title: Super-resolution atlas of SARS-CoV-2 infection reveals protease-dependent organelle maturation, dsRNA landscapes, and intracellular structural proteins

doi: 10.1038/s41467-026-75289-x

Figure Lengend Snippet: A Representative three-color SR image of a SARS-CoV-2–infected A549-ACE2 cell at an early infection stage, labeled for nsp1 (yellow), nsp4 (magenta) and dsRNA (cyan). Nsp1 localizes diffusely throughout the cytoplasm, while nsp4 encapsulates round dsRNA clusters (double-membrane vesicles, DMVs). B Representative two-color SR image of nsp1 (yellow) and 18S ribosomal RNA (rRNA) (magenta) indicates diffused localization of both targets within the same regions of the cytoplasm. Inset: pair-correlation functions between nsp1 and 18S localizations peak at r = 0 nm, suggesting an association between nsp1 and 18S rRNA. The blue dashed line approximates the position of a nucleolus, characterized by an increased density of 18S rRNA. C Representative SR image of an infected cell at an early infection stage, labeled for nsp2 (magenta), nsp3 (yellow) and dsRNA (cyan). Nsp2 and nsp3 co-encapsulate dsRNA clusters (DMVs) and localize between accumulations of DMVs (putative convoluted membranes, CMs). D In late infection, a significant portion of cellular nsp2 (magenta) localizes to fragmented and dispersed Golgi bodies (labeled with an anti-Giantin antibody, green). Bottom panels (1–3) display magnified views of selected Golgi bodies; yellow arrowheads indicate additional examples of Golgi bodies within the large image. E SR image of a cell in early infection suggests localization of nsp5 (magenta) within DMVs, as indicated by nsp3 (yellow, DMV membrane) and dsRNA (cyan, DMV core). F SR image of a cell at a late infection stage indicates that nsp5 (magenta) localizes to vgRNA-labeled (green) DMVs. Inset: pair-correlation functions between nsp5 and vgRNA localizations in late infection peak at r = 0 nm, indicating a strong correlation between these two targets. In the pair-correlation function plots in ( B ) and ( F ), faint blue lines represent individual cells, faint red lines represent g 12 (r) calculated assuming the complete spatial randomness (CSR), and bold lines represent the mean values across all analyzed cells. Color-framed boxes indicate magnified regions shown in the corresponding panels below. White dashed curves denote the approximate nuclear edge. White arrowheads in ( A ), ( C ), ( E ) indicate selected individual DMVs, whereas green arrowheads point to examples of DMV aggregates associated with nsp3-labeled CMs. Scale bars: 2 µm (large images), 500 nm (magnified images in ( C ), ( D )), and 200 nm (magnified images in ( E ), ( F )).

Article Snippet: For nirmatrelvir washout experiments, cells were washed once with PBS and incubated with SARS-CoV-2 WA 1 (USA-WA1/2020) at an MOI of 10 in 50 μL of DMEM containing 2% FBS for 6 h. The viral inoculum was then removed, and cells were cultured in 100 μL of DMEM containing 2% FBS and 288 nM nirmatrelvir (MedChemExpress, HY-138687) for 18 h. A subset of cells was then washed with PBS and fixed by 4% PFA and 0.1% glutaraldehyde in PBS for 1 h. The remaining cells were washed and cultured in fresh DMEM containing 2% FBS for an additional 24 h before being washed and fixed as described above.

Techniques: Infection, Labeling, Membrane

Journal: Nature Communications

Article Title: Super-resolution atlas of SARS-CoV-2 infection reveals protease-dependent organelle maturation, dsRNA landscapes, and intracellular structural proteins

doi: 10.1038/s41467-026-75289-x

Figure Lengend Snippet: A Schematic of a SARS-CoV-2 DMV with dsRNA in the center and nsp4 with nsp3 at the membrane pores. The positions of all remaining components were determined with respect to the known positions of these reference targets. B Representative three-color SR image of the cytoplasm of a typical A549-ACE2 cell at an early infection stage. DMVs (dashed circles) appear as round dsRNA clusters (cyan) surrounded by nsp4 (yellow) and nsp3 (red). A magnified view of a single DMV (white box, top right) reveals the three-layer organization of dsRNA, nsp4, and nsp3. The corresponding radial density distribution functions, g ( r ) (top rightmost plot), and rotationally averaged images (bottom right) display the three-layer structure averaged across n DMVs from 16 different cells. Representative three-color SR images of individual DMVs, corresponding radial density distribution functions, g ( r ), and rotationally averaged images for vgRNA, 18S rRNA, nsp1, nsp2, nsp5 and nsp9 relative to nsp4 and dsRNA. Data were obtained from 10 ( C ), 15 ( D ), 26 ( E ), 13 ( F ), 25 ( G ), and 22 ( H ) individual early-stage infected cells. Where applicable in ( B – H ), the average peak positions, R , of g ( r ), are indicated above the plots as the mean ± 95% CI, obtained via Gaussian fitting of the peak portion of g ( r ) following resampling with replacement (bootstrapping). n represents the total number of analyzed DMVs for each labeling combination. I Mean positions ± 95% CI for all NSPs associated with DMVs, calculated after rescaling the nsp4 positions within individual target groups ( B – H ) to match the global mean position of nsp4. The global position of nsp4 ± 95% CI (yellow error bar) was calculated across 13 groups ( B , F–H ) comprising n = 36,662 DMVs. Detailed data for the remaining NSPs are provided in Supplementary Fig. . J Angular bivariate pair-correlation functions, p ( θ ), of nsp4 with the indicated targets, represented as mean ± 95% CI. All targets peak at θ = 0, except 18S rRNA and nsp1. K Model of a mature, early-stage DMV based on the SR data. Scale bars: 1 µm ( B , overview image), 100 nm ( C – H , K and magnified individual DMVs and rotational averages in B ).

Article Snippet: For nirmatrelvir washout experiments, cells were washed once with PBS and incubated with SARS-CoV-2 WA 1 (USA-WA1/2020) at an MOI of 10 in 50 μL of DMEM containing 2% FBS for 6 h. The viral inoculum was then removed, and cells were cultured in 100 μL of DMEM containing 2% FBS and 288 nM nirmatrelvir (MedChemExpress, HY-138687) for 18 h. A subset of cells was then washed with PBS and fixed by 4% PFA and 0.1% glutaraldehyde in PBS for 1 h. The remaining cells were washed and cultured in fresh DMEM containing 2% FBS for an additional 24 h before being washed and fixed as described above.

Techniques: Membrane, Infection, Labeling

Journal: Nature Communications

Article Title: Super-resolution atlas of SARS-CoV-2 infection reveals protease-dependent organelle maturation, dsRNA landscapes, and intracellular structural proteins

doi: 10.1038/s41467-026-75289-x

Figure Lengend Snippet: Representative 3D dSTORM images of DMVs acquired using a double-helix point spread function (DHPSF), colabeled for dsRNA and nsp5 ( A ), nsp8 ( B ), or nsp10 ( C ) in SARS-CoV-2–infected A549-ACE2 cells. Each blue-framed set displays the same DMVs from different viewing angles. Axes are indicated in the corner of each panel (red, X; green, Y; blue, Z). D , E 3D-DHPSF imaging of dsRNA in an infected cell. The magnified view in ( E ) corresponds to the pink box in ( D ). The axial (Z) position of the fluorophores is color-coded according to the provided scale, where 0 µm represents the coverslip. F 2D dSTORM images of dsRNA colabeled with nsp4 reveal thin dsRNA connectors between larger dsRNA structures. These images correspond to the yellow boxes in the whole-cell image shown in Supplementary Fig. . G Examples of a cell in an early infection stage that contains large, approximately round dsRNA granules decorated with nsp9 puncta but lacking an nsp4 shell. The magnified panels on the right correspond to the green box in the large image. Green arrowheads highlight additional examples of dsRNA connectors, and white arrowheads indicate selected dsRNA granules lacking nsp4. Examples of large dsRNA granules largely lacking nsp4 but decorated with nsp5 ( H ), nsp13 ( I ), and nsp8 ( J ). White dashed curves denote the approximate nuclear edge. Scale bars: 5 µm (whole-cell images in D , G ), 500 nm ( H – J , and magnified panels in G ), and 200 nm ( A – C , E , F ).

Article Snippet: For nirmatrelvir washout experiments, cells were washed once with PBS and incubated with SARS-CoV-2 WA 1 (USA-WA1/2020) at an MOI of 10 in 50 μL of DMEM containing 2% FBS for 6 h. The viral inoculum was then removed, and cells were cultured in 100 μL of DMEM containing 2% FBS and 288 nM nirmatrelvir (MedChemExpress, HY-138687) for 18 h. A subset of cells was then washed with PBS and fixed by 4% PFA and 0.1% glutaraldehyde in PBS for 1 h. The remaining cells were washed and cultured in fresh DMEM containing 2% FBS for an additional 24 h before being washed and fixed as described above.

Techniques: Infection, Imaging

Journal: Nature Communications

Article Title: Super-resolution atlas of SARS-CoV-2 infection reveals protease-dependent organelle maturation, dsRNA landscapes, and intracellular structural proteins

doi: 10.1038/s41467-026-75289-x

Figure Lengend Snippet: A Representative SR image of the M protein reveals distinct morphological structures. Magnified panels on the right correspond to the blue, green, and red boxes in the main image. B Representative SR image of the M protein shows virions arrayed on the cell membrane. Insets (yellow boxes) display magnified individual virions. C Representative two-color SR image of M protein (magenta) and the Golgi marker Giantin (green) indicates that M protein localizes within the Golgi apparatus. Bottom panels show magnified views of the region enclosed by the yellow box. D Representative two-color SR image of M protein (magenta) and the lysosomal marker LAMP1 (green) details the distribution of M protein inside and outside lysosomes. Panels on the right show magnified views of the regions enclosed by the corresponding colored boxes. E Representative two-color SR images demonstrate that M protein (magenta) codistributes with S1 protein (green) in both hollow (top) and solid (bottom) clusters. F Histogram of the equivalent radius of solid (blue, n = 276 clusters) and hollow (red, n = 180 clusters) M protein structures reveals two distinct size populations. Data were obtained from 22 cells and 8 independent experiments. G Representative two-color SR image of vgRNA (magenta) and N protein (green) at 24 hpi demonstrates colocalization exclusively in the cytoplasm outside ROs. Panels on the right show magnified views of the regions enclosed by the yellow and blue boxes. H Bivariate pair-correlation functions, g 12 ( r ), calculated between vgRNA and N protein localizations in the cytoplasm outside ROs indicate their close spatial association. I Representative two-color SR image of vgRNA (magenta) and S2 protein (green) shows a lack of colocalization both within and outside ROs. Panels on the right show magnified views of the regions enclosed by the corresponding colored boxes. J Bivariate pair-correlation functions, g 12 ( r ), calculated between vgRNA and S2 protein localizations in the cytoplasm outside ROs indicate a nanoscale spatial anti-correlation between them. White dashed lines in A , C , D , G , and I denote the approximate edge of the cell nucleus (large dark region). Color bars in A , B , D , E indicate the number of single-molecule localizations per SR pixel (20 × 20 nm 2 , except for B , 16 × 16 nm 2 ). Scale bars: 10 µm (large image in A ), 5 µm (large images in B – D , G , I ), 1 µm (magnified images in A, C), and 500 nm ( E and magnified images in D , G , I ). Images and data in A – F were obtained from SARS-CoV-2–infected A549-ACE2 cells, whereas those in G – J were obtained from infected Vero E6 cells. All cells were fixed at 24 hpi.

Article Snippet: For nirmatrelvir washout experiments, cells were washed once with PBS and incubated with SARS-CoV-2 WA 1 (USA-WA1/2020) at an MOI of 10 in 50 μL of DMEM containing 2% FBS for 6 h. The viral inoculum was then removed, and cells were cultured in 100 μL of DMEM containing 2% FBS and 288 nM nirmatrelvir (MedChemExpress, HY-138687) for 18 h. A subset of cells was then washed with PBS and fixed by 4% PFA and 0.1% glutaraldehyde in PBS for 1 h. The remaining cells were washed and cultured in fresh DMEM containing 2% FBS for an additional 24 h before being washed and fixed as described above.

Techniques: Membrane, Marker, Infection

Journal: Nature Communications

Article Title: Super-resolution atlas of SARS-CoV-2 infection reveals protease-dependent organelle maturation, dsRNA landscapes, and intracellular structural proteins

doi: 10.1038/s41467-026-75289-x

Figure Lengend Snippet: A Representative SR image of an infected cell (MOI = 2) treated with 144 nM nirmatrelvir from 0 to 24 hpi and fixed at 24 hpi. Two regions with large multilayered bodies (MLBs) are magnified in the central panels. Density profiles (right, panels 1 and 2) show the localization density of nsp4 and nsp5 calculated perpendicular to the corresponding numbered white curves, in the direction indicated by the arrows (localization density orthogonal to the arrows was averaged). Both nsp4 and nsp5 localize in layered structures with a periodicity of ∼75 nm. SR images of infected cells (MOI = 10) treated with 288 nM nirmatrelvir from 6 to 24 hpi and fixed at 24 hpi. Nsp10 ( B ), nsp16 ( C ) and nsp3 ( D ) localize to the same multilayered structures as nsp5. E Magnified view of the region in ( D ) demonstrates the localization of nsp3 on both sides of nsp5-labeled layers. The averaged density profiles of nsp3 and nsp5 (right), calculated perpendicular to the numbered white curves in the direction of the arrows, reveal a double structure of nsp3 layers centered on single nsp5 layers. F , G Additional examples of MLBs from different cells in the same sample group, exhibiting this double nsp3 layer structure. H Confocal images of infected cells (MOI = 10) treated with 288 nM nirmatrelvir from 6 to 24 hpi and fixed at either 24 hpi (left) or 48 hpi (right), utilizing lysosome-associated membrane protein 1 (LAMP1, green) as a general cellular marker. In the left panel, only 6 cells in the bottom right corner display a weak punctate nsp5 signal (magenta) typical for an early infection phenotype, whereas the remaining cells show only LAMP1 signal and no detectable nsp5. In the right panel, nearly all cells exhibit a strong perinuclear nsp5 signal consistent with late-stage infection. I , J SR images of infected cells (MOI = 10) treated with 288 nM nirmatrelvir from 6 to 24 hpi and fixed at 48 hpi. A typical cell under these conditions displays a late-infection phenotype with highly dense perinuclear NSP and dsRNA localizations ( I ), whereas another typical cell shows prominent cytoplasmic MLBs ( J ). Among 32 infected cells imaged in SR at 48 hpi, 16 (50%) contained MLBs. In contrast, among 41 similarly treated infected cells fixed at 24 hpi (examples shown in B–G), 29 (∼71%) contained MLBs. White dashed curves denote the approximate nuclear edge. Scale bars: 50 µm ( H ), 5 µm (large images in A ; D , I , J ), 1 µm (middle panels in A ; B , C ), and 500 nm ( E – G ). All images and data were obtained from SARS-CoV-2–infected A549-ACE2 cells.

Article Snippet: For nirmatrelvir washout experiments, cells were washed once with PBS and incubated with SARS-CoV-2 WA 1 (USA-WA1/2020) at an MOI of 10 in 50 μL of DMEM containing 2% FBS for 6 h. The viral inoculum was then removed, and cells were cultured in 100 μL of DMEM containing 2% FBS and 288 nM nirmatrelvir (MedChemExpress, HY-138687) for 18 h. A subset of cells was then washed with PBS and fixed by 4% PFA and 0.1% glutaraldehyde in PBS for 1 h. The remaining cells were washed and cultured in fresh DMEM containing 2% FBS for an additional 24 h before being washed and fixed as described above.

Techniques: Infection, Labeling, Membrane, Marker