Fig 1.
Cell type independent astrovirus ER remodeling.
(A, C, E) Representative immunofluorescence staining of mock or HAstV1 infected (MOI = 3; 24 hpi) cells of the indicated type. Scale bars represent 20 µm. (B, D, F) Quantification of the total area of ER signal (top) and mean area of ER particles (bottom) between groups by particle analysis using FIJI ImageJ software and an unpaired t-test (*** p < 0.0001). Thresholding was applied uniformly between groups for n = 50 cells across 3 independent experiments. Calnexin was used as an ER marker for analysis.
Fig 2.
Temporal HAstV1-driven restructuring of the ER by long-term, time-lapse imaging.
(A) Representative immunofluorescence staining of mock or HAstV1 infected (MOI = 3; 24 hpi) Huh7 cells expressing the mCherry-KDEL ER marker (magenta). Scale bars represent 20 µm. Quantification of the total area of ER signal (left) and mean area of ER particles (right) between groups was performed by particle analysis using FIJI ImageJ software and an unpaired t-test (*** p < 0.0001). Thresholding was applied uniformly between groups for n = 15 cells across 3 independent experiments. mCherry-KDEL was used as an ER marker for analysis. (B) Still frames from live-cell imaging of mock or HAstV1 infected (MOI = 3), mCherry-KDEL (gray) expressing Huh7 cells at the indicated times post infection. Infected cells are indicated by an asterisk (*). Scale bars represent 20 µm. (C) A higher magnification of the cell marked with a cyan asterisk from panel B, representing images taken every 20 minutes between 14:20 and 18:00 hpi. Remodeled ER is segmented in cyan. The region indicated by the white box highlights recruitment of remodeled ER to the existing aggregate.
Fig 3.
Temporal HAstV1-driven membrane alterations by transmission electron microscopy.
Transmission electron microscopy (TEM) of sectioned HAstV1 infected (MOI = 3) Huh7 cells fixed at the indicated times post infection. Black arrowheads indicate ER membrane connected to DMVs. Scale bars represent the indicated distances. Insets show a higher magnification of the indicated regions.
Fig 4.
Astrovirus nonstructural proteins colocalize with dsRNA and remodeled ER.
(A) Representative immunofluorescence staining of rHAstV1-V5 infected (MOI = 3; 24 hpi) Cos7 cells expressing the mCherry-KDEL ER marker. Percent maximum intensity profiles of dsRNA (yellow), mCherry-KDEL (magenta), and V5 (cyan) staining were obtained across a 40 µm linescan shown in merged images (white line) using FIJI ImageJ software. (B) Quantification of the colocalization of V5-tagged nsps with remodeled ER and (C) dsRNA. Analysis was performed by calculating the Pearson correlation coefficient (PCC) for n = 15 cells from each rHAstV1-V5 group and (D) pooling of all virus-infected groups (total n = 45) to quantify colocalization of dsRNA with remodeled ER across 3 independent experiments.
Fig 5.
Astrovirus replication occurs within ER-derived DMVs.
(A) Representative immunofluorescence staining of rHAstV1-V5-1a/2 infected (MOI = 3; 24 hpi) Huh7 cells permeabilized with digitonin alone or digitonin followed by Triton X-100. Scale bars represent 20 µm. (B) Quantification of the mean fluorescence intensity of V5 signal (cyan), (C) dsRNA signal (yellow), and (D) HAstV1 capsid signal (magenta) for n = 50 cells based on permeabilization treatment. Mean fluorescence intensity was obtained using FIJI ImageJ software from cells imaged using the same acquisition settings. Data are analyzed by an unpaired t-test and shown as a percentage of the maximum mean intensity observed across three independent experiments (* p < 0.05, *** p < 0.0001).
Fig 6.
Nsp1a/1 and nsp1a/2 are the minimal constituents for HAstV1 DMV biogenesis.
(A) Schematic of nsp1a/1 and nsp1a/2 membrane topology and construction of dual-tagged HAstV1 expression plasmids. Nsp1a/1 (light gray), nsp1a/2 (dark gray). Black arrowheads represent sites cleaved by host signal peptidase (SPase). Numbers indicate HAstV1 amino acid positions. (B) Representative immunofluorescence staining of Cos7 cells transfected with the indicated constructs or infected with rHAstV1-V5-1a/1 (MOI = 3) at 24 hours post treatment. Scale bars represent 20 µm. (C) Quantification of the total area of ER signal and (D) mean area of ER particles between groups by particle analysis using FIJI ImageJ software and one-way ANOVA with multiple comparisons versus mock treated cells (*** p < 0.0001). Thresholding was applied uniformly between groups for n = 20 cells across 3 independent experiments. Calnexin was used as an ER marker. (E) Transmission electron microscopy (TEM) of sectioned Cos7 cells transfected with the indicated constructs and fixed at 24 hours post transfection. Yellow arrowheads highlight typical ER membrane morphology. ER whorl (W), convoluted membranes (CM), high curvature ER (HCER). Yellow asterisks (*) indicate representative vesicles with a double membrane component. Scale bars represent 500 µm.
Fig 7.
Conservation of nsp1a/1- and nsp1a/2-mediated ER remodeling for HAstV-VA1.
(A) Construction of dual-tagged HAstV-VA1 expression plasmids. Black arrowheads represent sites cleaved by host signal peptidase (SPase). Numbers indicate VA1 amino acid positions. (B) Representative immunofluorescence staining of Cos7 cells transfected with the indicated constructs at 24 hours post transfection. Scale bars represent 20 µm. Arrowheads indicate regions of over- (yellow) or undersaturation (white) during imaging of nsp1a/1. (C) Quantification of the total area of ER signal and (D) mean area of ER particles between groups by particle analysis using FIJI ImageJ software and one-way ANOVA with multiple comparisons versus GFP-transfected cells (*** p < 0.0001). Thresholding was applied uniformly between groups for n = 20 cells across 3 independent experiments. Calnexin was used as an ER marker.
Fig 8.
Remodeled ER architecture by super resolution dSTORM.
(A) Representative dSTORM images of immunolabeled calnexin in mock or HAstV1 infected (MOI = 3; 24 hpi) Huh7 cells. Segmented particles greater than 1 µm2 within a single cell region of interest (ROI) are highlighted in cyan. Insets (1-4) show a higher magnification of the indicated regions. Scale bars represent the indicated distances. Quantification of the total area of dense ER particles in mock and HAstV1 infected cells was performed by thresholding and particle analysis using FIJI ImageJ software and an unpaired t-test (*** p < 0.0001). (B) Representative dSTORM images of immunolabeled V5 in rHAstV1-V5-1a/1 and -1a/2 infected (MOI = 3; 24 hpi) Huh7 cells. Insets (1-2) show a higher magnification of the indicated regions. Scale bars represent the indicated distances. Representative particles used for V5 puncta diameter analysis are segmented in cyan. Quantification of the average V5 puncta diameter from dSTORM images and DMV diameter from TEM micrographs shown in Fig 3 (24 hpi) was performed using the measure tool in FIJI ImageJ software and a one-way ANOVA with multiple comparisons between all groups (ns, not significant).