Journal: bioRxiv

Article Title: β-Coronavirus Nsp6 hijacks host ER translocation machineries into viral replication centers

doi: 10.64898/2025.12.16.694738

Figure Lengend Snippet: (A) (D) Cartoon of domain organization and topology of MHV nonstructural proteins Nsp3-6 derived from literature models and alphafold 3 predictions . (B-C) Representative images of FL83B cells co-transfected with (B) mNe-Nsp3 (green), mCh-Nsp4 (yellow), HALO-Nsp6 (JFX650 conjugated, magenta) and with either (B) BFP-KDEL (blue) or (C) BFP-Sec61β (blue). (D) Images of FL83B cells co-transfected with BFP-KDEL (magenta) and mNe-Nsp3 (top, green), mCh-Nsp4 (middle, green), or HALO-Nsp6 (conjugated to JFX650, bottom, green). (E) Images of FL83B cells co-transfected with BFP-Sec61β (magenta) and mNe-Nsp3 (top, green), mCh-Nsp4 (middle, green), or HALO-Nsp6 (conjugated to JFX650, bottom, green). (F-G) Images of FL83B cells co-transfected with BFP-KDEL (F, blue) or BFP-Sec61β (G, blue) and with mNe-Nsp3 (green) and mCh-Nsp4 (magenta). (H-J) Images of FL83B cells co-transfected with BFP-KDEL (H and J, blue) or BFP-Sec61β (I, blue) and Halo-Nsp6 (magenta) with either (H) mNe-Rtn3L, (I) mNe-SigmaR1, or (J) mCh-Climp63 (green). Inset white boxes shown in left panels are magnified in panels on the right. Scale bars = 5 µm.

Article Snippet: 293T cells (ATCC CRL-3216) were used to produce lentiviral particles for generating acGFP-Sec61β FL83B stable cells.

Techniques: Derivative Assay, Transfection

Journal: bioRxiv

Article Title: β-Coronavirus Nsp6 hijacks host ER translocation machineries into viral replication centers

doi: 10.64898/2025.12.16.694738

Figure Lengend Snippet: Representative images of FL83B cells co-transfected with (A) mCh-KDEL (magenta), mNe-Sec61β (green), and in the absence (top panels) or presence (bottom panels) of HALO-Nsp6 (JFX650 conjugated, blue). (B-D) A 10 x 10 µm ROIs were used to score protein co-localization (between channels indicated) within ER domains by Mander’s Correlation Coefficient (MCC) analyses. Control 90° and +Nsp6 90° are the MCC measured after rotating one channel 90° relative to the other channel to account for overlap due to chance and pixel density. (E) Images of FL83B cells co-transfected with mCh-KDEL (magenta), Sec61α-mNe, and in the absence (top panels) or presence (bottom panels) of HALO-Nsp6 (blue). (F-H) MCC were measured and graphed as in (B-D) for (E). (I) Images of FL83B cells co-transfected with mCh-KDEL (magenta), SRPRβ-mNe, and in the absence (top panels) or presence (bottom panels) of HALO-Nsp6 (blue). (J-L) MCC were performed as in (B-D) for (I). (M) Images of FL83B cells co-transfected with mCh-KDEL (magenta), WRB-mNe, and in the absence (top panels) or presence (bottom panels) of HALO-Nsp6 (blue). (N-P) MCC were measured and graphed as in (B-D) for (M). For MCC graphs, the large blue square, green circle, and magenta triangle represent the mean for independent biological replicates and corresponding small symbols are each individual data point within the respective replicate (3 biological replicates, n = 15 cells per replicate, error bars = ± standard error of the mean). Scale bars = 5 µm.

Article Snippet: 293T cells (ATCC CRL-3216) were used to produce lentiviral particles for generating acGFP-Sec61β FL83B stable cells.

Techniques: Transfection, Control

Journal: bioRxiv

Article Title: β-Coronavirus Nsp6 hijacks host ER translocation machineries into viral replication centers

doi: 10.64898/2025.12.16.694738

Figure Lengend Snippet: (A) Representative images of FL83B cells (top) mock transfected, (middle) transfected with TurboID-mNe empty vector or (bottom) TurboID-mNe-Nsp6 (magenta) and treated with biotin for 1 hour prior to immunostaining with streptavidin-405 antibody (green). (B) TurboID biotinylation experimental approach. (C) Log 2 iBAQ values plotted for the top 30 ER-specific biotinylation hits identified by mass-spectrometry. Nsp6 self-biotinylation (blue dot), translocation machineries (magent dots), post-translocon-associated factors (green dots), and all other ER proteins (black dots). Representative 10 x 10 µm images of (D) EMC5-mNe (H) mNe-OPTI, (L) TMCO1-mNe, or (P) DDOST-mNe (green) and mCh-KDEL (magenta) co-transfected in the absence (top) or presence of HALO-Nsp6 (bottom, blue). MCC analysis of EMC5-mNe, (I) mNe-OPTI, (M) TMCO1-mNe, or (Q) DDOST-mNe with mCh-KDEL without (Control) and with (+Nsp6) HALO-Nsp6 including an mCh-KDEL channel rotated control (Control 90° and +Nsp6 90°). MCC analysis of (E) EMC5-mNe, (I) mNe-OPTI, (M) TMCO1-mNe, or (Q) DDOST-mNe with HALO-Nsp6 (+Nsp6) compared to HALO-Nsp6 channel rotated control (+Nsp6 90°). MCC analysis of (G, K, O, S) HALO-Nsp6 with mCh-KDEL (+Nsp6) and mCh-KDEL rotated control (+Nsp6 90°). For MCC graphs, the large blue square, green circle, and magenta triangle represent the mean for independent biological replicates and corresponding small symbols are each individual data point within the respective replicate (3 biological replicates, n = 15 cells per replicate, error bars = ± standard error of the mean). Scale bars = 5 µm.

Article Snippet: 293T cells (ATCC CRL-3216) were used to produce lentiviral particles for generating acGFP-Sec61β FL83B stable cells.

Techniques: Transfection, Plasmid Preparation, Immunostaining, Mass Spectrometry, Translocation Assay, Control

Journal: bioRxiv

Article Title: β-Coronavirus Nsp6 hijacks host ER translocation machineries into viral replication centers

doi: 10.64898/2025.12.16.694738

Figure Lengend Snippet: Representative time-lapse images of FL83B cells transfected with (A) SRPRβ-mNe (white) or (A) SRPRβ-mNe (white) and HALO-Nsp6 (magenta) imaged before (prebleach) and after photobleaching a region of interest (ROI) in circle shown. Fluorescence recovery was measured within the ROI at times indicated following the photobleaching event and were plotted (on the right as indicated). Statistics measured during fluorescence recovery after photobleaching (FRAP) as indicated. (C-D) Experiments were performed and recorded as in (A-B) for FL83B cells transfected with (C) Sec61α-mNe (white) or (D) Sec61α-mNe (white) and HALO-Nsp6 within the general ER network (Control) or within an Nsp6 domain (Nsp6, magenta), respectively. (E-F) FL83B cells were transfected with (E) SRPRβ-mNe (white) or (F) SRPRβ-mNe (white) and HALO-Nsp6 (magenta) and were bleached in the white circle ROIs beginning at time = 0 and again every 50 seconds. Fluorescence loss in photobleaching (FLIP) was measured in another region of the cell at ROIs (yellow outline) that corresponded to control or Nsp6 domains. FRAP and FLIP curves were fit using a one phase association (FRAP) or one phase decay (FLIP) to extrapolate the mobile fraction, half-time recovery or loss (dashed line, t 1/2 ), recovery or decay rate (k), normalized intensity at t = 0 (I 0 ), and plateau (I ∞ ). Means for each time-point are shown with green (control) or magenta (+Nsp6) circles (3 replicates, n = 5 cells per replicate, error bars = ± standard deviation). Scale bars = 5 µm (A-D). or 20 µm (E-H).

Article Snippet: 293T cells (ATCC CRL-3216) were used to produce lentiviral particles for generating acGFP-Sec61β FL83B stable cells.

Techniques: Transfection, Fluorescence, Control, Standard Deviation

Journal: bioRxiv

Article Title: β-Coronavirus Nsp6 hijacks host ER translocation machineries into viral replication centers

doi: 10.64898/2025.12.16.694738

Figure Lengend Snippet: (A) Cartoon of experimental workflow performed for B-D. (B) FL83B cells or (C and D) FL83B cells stably expressing acGFP-Sec61β were transfected with (B and C) HALO-Nsp6 or (B and D, E) infected with 0.1 MOI MHV JHM IA and collected at time points indicated for (B) qPCR or (C and D) immunostaining analysis. (B) qPCR plot of ΔCt (Ct, cycle threshold) values calculated from the difference between Nsp6 and actin (actb – 5, see Methods) in samples extracted from infected cell culture pellets (green), HALO-Nsp6 transfected (magenta), or mock transfected cells (black). (C and D) Representative 63X objective images of fixed FL83B cells stably expressing acGFP-Sec61β (green) and (C) HALO-Nsp6 transfected (JFX650 conjugated, 500 ng, magenta) or (D) 0.1 MOI MHV JHM IA infected (mock control, 8, 12, and 18 hpi). Infected cells were immuno-stained with dsRNA to identify replication centers (magenta) relative to Sec61β signal (green). Scale bars = 5 µm (C, D).

Article Snippet: 293T cells (ATCC CRL-3216) were used to produce lentiviral particles for generating acGFP-Sec61β FL83B stable cells.

Techniques: Stable Transfection, Expressing, Transfection, Infection, Immunostaining, Cell Culture, Control, Staining

Journal: Nature Methods

Article Title: A highly photostable monomeric red fluorescent protein for dual-color 3D STED and time-lapse 3D SIM imaging

doi: 10.1038/s41592-025-02962-2

Figure Lengend Snippet: 3D reconstruction of the ER labeled by mScarlet3-S2 around the nucleus

Article Snippet: Scale bar, 5 μm. e , Stacked bar chart of the accumulated fluorescence intensity of whole-stack scanning. f , A 3D reconstruction of the entire ER in U-2 OS cells labeled with sec61β-mScarlet3-S2. g , Structures of sec61β-mScarlet3-S2-labeled invagination reconstructed by Imaris software.

Techniques:

Journal: Nature Methods

Article Title: A highly photostable monomeric red fluorescent protein for dual-color 3D STED and time-lapse 3D SIM imaging

doi: 10.1038/s41592-025-02962-2

Figure Lengend Snippet: 3D reconstruction of the ER labeled by mScarlet3-M163H around the nucleus

Article Snippet: Scale bar, 5 μm. e , Stacked bar chart of the accumulated fluorescence intensity of whole-stack scanning. f , A 3D reconstruction of the entire ER in U-2 OS cells labeled with sec61β-mScarlet3-S2. g , Structures of sec61β-mScarlet3-S2-labeled invagination reconstructed by Imaris software.

Techniques:

Journal: Nature Methods

Article Title: A highly photostable monomeric red fluorescent protein for dual-color 3D STED and time-lapse 3D SIM imaging

doi: 10.1038/s41592-025-02962-2

Figure Lengend Snippet: Actin structures in U-2 OS cells were labeled with Lifeact (an actin-binding peptide) fused to FPs. Cells were fixed 24 h after transfection. a , Confocal (left and right) and STED (middle) images of actin structure imaged with mScarlet3-S2, mScarlet3-M163H or mScarlet-H, respectively. The STED images (middle column) are magnified views of the boxed regions in the confocal images. The right column shows confocal images after STED imaging. Scale bar, 5 μm. b , Ratio of averaged fluorescence intensity in boxed areas after STED imaging (right column of a ) versus before STED (left column of a ). For each FP, five images were analyzed. Individual dots represent independent measurements derived from two independent experiments. Data are presented as mean ± s.e.m. Significance was calculated by two-tailed Student’s t -test; ** P < 0.01 ( P = 0.00185), **** P < 0.0001 ( P = 4.19 × 10 −9 ). c , FWHM measurements of actin filaments of confocal (Conf.) and STED images (arrows, a , top row). d , Color-coded 3D distributions ( XY and YZ views) of ER labeled by mScarlet3-S2, mScarlet3-M163H or mScarlet-H in U-2 OS cells. Scale bar, 5 μm. e , Stacked bar chart of the accumulated fluorescence intensity of whole-stack scanning. f , A 3D reconstruction of the entire ER in U-2 OS cells labeled with sec61β-mScarlet3-S2. g , Structures of sec61β-mScarlet3-S2-labeled invagination reconstructed by Imaris software. The arrowhead and arrows indicate structures resembling NR and NR-like channels. h , XZ view (11.4 µm y -slice) showing NR-like intranuclear channels in confocal (top) and STED (bottom) modes. The hollow arrowhead indicates the NF, while the solid arrowhead and solid arrow denote the NR and NR channels, respectively.

Article Snippet: Scale bar, 5 μm. e , Stacked bar chart of the accumulated fluorescence intensity of whole-stack scanning. f , A 3D reconstruction of the entire ER in U-2 OS cells labeled with sec61β-mScarlet3-S2. g , Structures of sec61β-mScarlet3-S2-labeled invagination reconstructed by Imaris software.

Techniques: Labeling, Binding Assay, Transfection, Imaging, Fluorescence, Derivative Assay, Two Tailed Test, Software

Journal: bioRxiv

Article Title: Proteolytic processing of the Marburg virus glycoprotein depends on Sec61β and is required for cell entry

doi: 10.1101/2025.06.26.660697

Figure Lengend Snippet: ( A ) SEC61B -knockout (KO) single cell clones were generated from HEK293T cells using CRISPR/Cas9. Sec61β expression in lysates of the indicated SEC61B -KO single cell clones was analyzed by immunoblot using polyclonal anti-Sec61β antibody. Expression of β-actin was analyzed as loading control. Results were confirmed in two additional experiments. ( B ) The indicated SEC61B -KO cell lines were stained with CFSE and analyzed via flow cytometry at day 0, 1, 2 and 3. The results of a single experiment are shown and were confirmed in two separate experiments. ( C ) Phase contrast microscopy of Sec61β single cell clones. Similar results were obtained in one additional experiment.

Article Snippet: Here, we report that the Sec61 subunit Sec61β, although dispensable for GP expression, is required for proteolytic cleavage of MARV- but not EBOV-GP and that an intact furin motif is essential for robust cell entry of Marburg- but not Ebolaviruses.

Techniques: Knock-Out, Clone Assay, Generated, CRISPR, Expressing, Western Blot, Control, Staining, Flow Cytometry, Microscopy

Journal: bioRxiv

Article Title: Proteolytic processing of the Marburg virus glycoprotein depends on Sec61β and is required for cell entry

doi: 10.1101/2025.06.26.660697

Figure Lengend Snippet: ( A ) WT and SEC61B -KO cells were transfected with plasmids expressing C-terminal V5-tagged GPs of VSV, EBOV (Makona) and MARV (Musoke) or empty vector (EV) as control. Glycoprotein expression was detected by immunoblot using a mouse monoclonal anti-V5 antibody. Detection of β-actin served as a loading control. Results were confirmed by one to three additional experiments. ( B ) The experiment was conducted as for panel A but the effect of coexpression of Sec61β on MARV-GP expression and cleavage was examined. The results of a representative immunoblot are shown and were confirmed in two separate experiments. ( C-D ) The expression of MARV-GP and EBOV-GP with the indicated mutations in the furin cleavage sites was analyzed in cell lysates (C) and pseudotyped VSV particles (D). The expression of β-actin (cell lysates) and VSV-M (particles) served as loading control. Results were confirmed in two (EBOV-GP) or three (MARV-GP) additional experiments. ( E ) HEK293T cells were inoculated with VSV particles pseudotyped with the indicated glycoproteins. Luciferase activity in cell lysates were quantified at 18-20 h post inoculation. Luminescence signals were normalized to background (empty vector control). Shown is the mean ± SEM of four separate experiments each conducted with four technical replicates. Statistical significances were determined using unpaired, two-tailed t-test with Welch‘s correction (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **)

Article Snippet: Here, we report that the Sec61 subunit Sec61β, although dispensable for GP expression, is required for proteolytic cleavage of MARV- but not EBOV-GP and that an intact furin motif is essential for robust cell entry of Marburg- but not Ebolaviruses.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Luciferase, Activity Assay, Two Tailed Test