Lunapark-dependent formation of a virus-induced ER exit site contains multi-tubular ER junctions that promote viral ER-to-cytosol escape

Abstract

Viruses rearrange host membranes to support different entry steps. Polyomavirus simian virus 40 (SV40) reorganizes the endoplasmic reticulum (ER) membrane to generate focus structures that enable virus ER-to-cytosol escape, a decisive infection step. The molecular architecture of the ER exit site that might illuminate why it is ideally suited for membrane penetration is unknown. Here 3D focused ion beam scanning electron microscopy (FIB-SEM) reconstruction reveals that the ER focus structure consists of multi-tubular ER junctions where SV40 preferentially localizes, suggesting that tubular branch points are virus ER-to-cytosol penetration sites. Functional analysis demonstrates that lunapark-an ER membrane protein that typically stabilizes three-way ER junctions-relocates to the ER foci, where it supports focus formation, leading to SV40 ER escape and infection. Our results reveal how a virus repurposes the activity of an ER membrane protein to form a virus-induced ER substructure required for membrane escape and suggest that ER tubular junctions are vulnerable sites exploited by viruses for membrane penetration.

Keywords: Endoplasmid reticulum; FIB-SEM; Lunapark; Membrane trafficking; Non-enveloped virus; Polyomavirus; SV40.

Figure 1.. Identification of the SV40-induced focus structure by confocal and electron microscopy

(A) Mock-infected CV-1 cells or CV-1 cells infected with SV40 for 7 h were fixed and stained with the indicated antibodies and analyzed by confocal microscopy. (B) CV-1 cells infected with SV40 for 7 h were fixed and analyzed by scanning electron microscopy. (C) Immunogold-stained transmission electron microscopy (TEM) images of SV40-induced foci in CV-1 cells at 7 and 24 hpi. 18-nm gold particles label SV40 VP2/VP3 at the focus structure. N, nucleus.

Figure 2.. Virus-induced foci consist of multi-tubular ER junctions with SV40 localized at the junctions

(A) FIB-SEM image of an ER focus (obtained at slice 134) induced by SV40 in CV-1 cells at 7 hpi. (B–D) 3D reconstruction of the zoomed-in focus ultrastructure shown in (A) from different angles, where slice 134 serves as the mid-point. (E) FIB-SEM image of an ER focus (obtained at slices 46 and 68) induced by SV40 in CV-1 cells at 7 hpi. (F–H) 3D reconstruction of the zoomed-in focus ultrastructure shown in (E) from different angles, starting at slice 46. (I) FIB-SEM image of an ER focus (obtained at slice 144) induced by SV40 in CV-1 cells at 7 hpi. (J–L) 3D reconstruction of the zoomed-in focus ultrastructure shown in (I) from different angles, with slice 144 serving as the mid-point. The green structures mark the ER, and the red structures represent SV40. The blue arrows identify the ER tubular junctions. L, ER lumen.

Figure 3.. LNP facilitates SV40 infection

(A) CV-1 cells were transfected with the indicated siRNA for 48 h and lysed with 1% Triton X-100, and the resulting extract was subjected to SDS-PAGE and immunoblotting with the indicated antibodies. The relative LNP band intensity is quantified in the graph below by using FIJI/ImageJ (NIH) software. The data were normalized to scrambled siRNA. The values represent means and standard deviations (SDs) of the results of three independent experiments. A two-tailed t test was used. (B) CV-1 cells transfected with the indicated siRNA for 72 h were treated with trypan blue after transfection to stain dead cells. The live cells were quantified relative to the total cells to check the cell viability. Data represent the mean ± SD of three independent experiments. (C) Large T antigen-positive nuclei were scored in SV40-infected CV-1 cells transfected with the indicated siRNAs. The data were normalized to the scrambled siRNA, with the values representing means and SDs (n ≥ 3). (D) CV-1 cells were transfected with the indicated siRNA for 48 h and lysed with 1% Triton X-100, and the resulting extract was subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (E) Large T antigen-positive nuclei were scored in SV40-infected CV-1 cells transfected with the indicated siRNAs. The data were normalized to the scrambled siRNA, with the values representing means and SDs (n ≥ 3). (F) (Diagram) Structure of LNP transmembrane protein in the ER membrane. The C-terminal domain of LNP in the cytosol contains the zinc-finger (ZnF) domain and LNP motif. The indicated LNP constructs were expressed in CV-1 cells at 24 h post-transfection. LNP 235–428-mCherry is a cytosolic soluble protein. (G) CV-1 cells were transfected with the indicated siRNA for 24 h before transfection with the indicated plasmids for 30 h. The cells were then infected with SV40, fixed, and immunostained with anti-large T antigen antibodies. The percentage of large T antigen-positive cells was determined only in cells (≥600) expressing the indicated GFP- or mCherry-tagged protein by immunofluorescence microscopy. The data were normalized to the scrambled siRNA, with the values representing means and SDs (n ≥ 3).

Figure 4.. LNP is required for ER-to-cytosol escape of SV40

(A) CV-1 cells infected with SV40 (multiplicity of infection [MOI] 5) and transfected with the indicated siRNA were subjected to the ER-to-cytosol transport assay (see STAR Methods). The ER luminal protein BiP served as the membrane marker, whereas cytosolic Hsp90 served as the cytosol marker. (B) Relative VP1 band intensities in the cytosol fraction from (A) were determined by using FIJI/ImageJ software. The data were normalized to scrambled siRNA. The values represent means and SD of the results of three independent experiments. A two-tailed t test was used. (C) Relative VP1 band intensities in ER-localized fraction from (A) were determined by using FIJI/ImageJ software. A two-tailed t test was used.

Figure 5.. SV40 triggers LNP reorganization into the ER focus structure to stabilize this ER exit site

(A) CV-1 cells were immunostained with LNP (green), ER marker BAP31 (red), and DAPI (blue) with or without SV40 infection (MOI 5) for 16 h. The BAP31 focus structure represents the SV40 ER exit site. (B) Quantification of the percentage of LNP foci co-localizing with BAP31 foci in (A). More than 400 cells were counted in each experiment, and three independent experiments were performed. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 for 16 h. Cells were fixed; immunostained for BAP31 (red), SV40 VP2/3 (green), and DAPI (blue); and analyzed by immunofluorescence microscopy. Scale bar is 10 μm. The far-right (zoom) panels show 3.3× enlargements of the boxed focus regions.

Figure 6.. Depletion of LNP blunts formation of the large and intact ER focus

(A) CV-1 cells transfected with scrambled siRNA or LNP (#1) siRNA were infected with SV40 and, at the indicated time point, fixed, stained with BAP31 (red) and DAPI (blue), and analyzed by epifluorescence microscopy. Cells with small and dispersed foci are marked as thin arrows, and cells with large and intact focus are marked as thick arrows. (B) Quantification of the percentage of cells with small and dispersed foci, as well as the percentage of cells with the large and intact focus, under the scrambled or LNP KD condition from (A). At least 200 cells were analyzed for each condition, and three independent biological replicates were performed. The error bar represents the standard deviation of the results of three independent experiments.