Fig 2: The expression of the SARS-CoV-2 E protein is restricted to intracellular compartments of the cell.COS-7 cells were transfected with the vector expressing the SARS-CoV-2 E-HA protein. At 24 h, cells were processed for immunostaining. The cells on coverslips were reacted with a mouse monoclonal antibody against HA-tag and with rabbit antibodies against ERGIC53, Golgin-97, or LAMP-1. The cells were washed and reacted with a secondary goat anti-rabbit antibody conjugated to AlexaFluor488 and with a chicken anti-mouse antibody conjugated to AlexaFluor594 for 1 h. Cells on coverslips were counterstained with DAPI and mounted on glass slides with a glycerol-containing mounting medium. To examine the intracellular localization of the E protein with the RER, cis-medial Golgi, or trans Golgi network (TGN), COS-7 cells were co-transfected with the plasmid expressing the E-HA protein, and vectors expressing ERMoxGFP, mNeonGreen-Giantin, or TGN38-GFP. Cells were prepared as above and immunostained with an anti-HA antibody and mounted as above. Coverslips were viewed with a Leica TCS SP8 Confocal Microscope with a 100X objective and a 2X digital zoom using the Leica Application Suite X (LASX) as previously described. A 405nm filter was used to visualize the DAPI, a 594nm filter to visualize HA staining, and a 488 nm filter to visualize the cellular markers. Panels A. Cells co-transfected with vectors expressing SARS-CoV-2 E-HA and ERMoxGFP and immunostained with an anti-HA antibody. Scale bar= 20µm. Panel B. Cells transfected with a vector expressing SARS-CoV-2 E-HA and immunostained with anti-HA and anti-ERGIC53. Panel C. Cells co-transfected with vectors expressing SARS-CoV-2 E-HA and mNeonGreen-Giantin and immunostained with an anti-HA antibody. Panel D. Cells transfected with a vector expressing SARS-CoV-2 E-HA and immunostained with anti-HA and anti-Golgin97. Panel E. Cells co-transfected with vectors expressing SARS-CoV-2 E-HA and TGN38-GFP and immunostained with an anti-HA antibody. Panel F. Cells transfected with the vector expressing SARS-CoV-2 E-HA and immunostained with an anti-HA and anti-LAMP1.

Fig 3: Hypoxia-induced Fc?RIIA expression increases uptake of antibody-opsonized DENV A, BViral RNA copy numbers at 2 hpi with DENV-2 opsonized with h3H5 (ADE) (A) or DENV-2 only (B). Blue and red bars represent cells cultured in normoxia and hypoxia, respectively (n = 4 biological replicates for each condition).CViral RNA copy numbers after pronase treatment at 2 hpi under ADE or DENV-2 only conditions (n = 4 biological replicates for each condition).DImmunofluorescence microscopy of surface Fc?RIIA (blue, AF532), total Fc?RIIA (red, AF647) with DENV (green, AF488). Co-localization of internalized Fc?RIIA with DENV in the cytoplasm of THP-1 is indicated by the white arrows. Scale bars represent 4 µm.EImaris spot detection of total Fc?RIIA (red, AF647), surface Fc?RIIA (blue, AF532), and DENV-2 (green, AF488) interaction. Colocalization of red and blue spots represents surface Fc?RIIA, whereas red-only spots represent internalized Fc?RIIA. Images were acquired at 100× magnification using a Leica TCS SP8 confocal DMI6000 Microscope with hybrid detectors (HyD) and processed using Leica Application Suite X 2.0.1.14392 (LAS X). Deconvolution analysis was performed using Huygens Professional v15.05 and Imaris x64 8.1.2 software. White arrow in the merged image indicates co-localization of DENV (green) with internalized Fc?RIIA (red only). This co-localization was identified with an additional 6× magnification and tilting of the image for better visualization, as shown in the accompanying video within the Source Data. Scale bars represent 4 µm.FPlaque titers following ADE infection in shControl and shFc?RIIA THP-1 cells at 72 hpi. Fc?RIIA silencing efficiency was assessed by Western blot with LAMP1 as a loading control. Numbers under the Western blot indicate levels of Fc?RIIA normalized to LAMP1 (n = 4 biological replicates for each condition).Data information: In (A–C, F), data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired t-test). Source data are available online for this figure.

Fig 4: Intracellular and surface expression of HIV-1 Env in the presence of gB. We examined the intracellular and surface expression of gB, HIV-1 Env, or both using COS-7 cells. For intracellular expression, cells grown on cover slips were transfected with vectors expressing gB and HIV-1. At 24 h, cells were fixed, permeabilized, reacted with a primary antibody against HIV-1 Env and gB for 16 h at 4C. The cells washed and reacted with appropriate secondary antibodies. Cells were counterstained with DAPI. For surface staining, cells on cover slips were transfected with vectors expressing gB, HIV-1 Env or both. At 24 h, cells were washed reacted with primary antibodies against gB and/or HIV-1 Env for 1 h at 4C, cells washed and fixed. The cells were then reacted with appropriate secondary antibodies and counterstained with DAPI. The cells were washed and the cover slips were mounted. The cells on coverslips were examined using a Leica TCS SPE confocal microscope using a 63 × objective with a 2 × digital zoom using the Leica Application Suite X (LAS X, LASX) software package. A 405 nm filter used to visualize the DAPI, 488 nm filter was used to visualize the gB, and a 594 nm filter to visualize HIV-1 Env staining. a–d Cells co-transfected with vectors expressing gB and HIV-1 Env that were fixed prior to staining. e–h Cells transfected with a vector expressing gB and reacted with the primary antibody against gB at 4C prior to fixation. i–l Cells transfected with vectors expressing HIV-1 Env and reacted with the primary antibody against HIV-1 Env at 4C prior to fixation. m–p Cells transfected with vectors expressing HIV-1 Env and gB, reacted with the primary antibodies against HIV-1 Env and gB at 4C prior to fixation. The results shown are representative of examining 50 transfected cells. a, e, i, m Visualization of gB staining. b, f, j, n Visualization of HIV-1 Env staining. c, g, k, o Visualization of DAPI staining. d, h, l, p Merge of the three panels to the left

Fig 5: The HSV-1 gD co-localizes with Gag-EGFP. 293 cells were transfected with either the vector expressing gD, Gag-EGFP, or co-transfected with gD and Gag-EGFP. At 24 h, cells on cover slips were washed, fixed, permeabilized and blocked as described in the Materials and Methods section. The cover slips were reacted with a mouse monoclonal antibody against gD and an appropriate secondary antibody and counterstained with DAPI (1 µg/ml) for 5 min. The cover slips were mounted and examined using a Leica TCS SPE confocal microscope using a 63X objective with a 2X digital zoom using the Leica Application Suite X (LAS X, LASX) software package. A 405 nm filter used to visualize the DAPI, 488 nm filter was used to visualize the Gag-EGFP, and a 594 nm filter to visualize gD staining. a–d Cells transfected with vector expressing gD. e–h Cells transfected with vector expressing Gag-EGFP. i–l Cells co-transfected with vectors expressing gD and Gag-GFP. a, e, i Visualization of DAPI staining. b, f, j Visualization of gD staining. c, g, k Visualization of Gag-EGFP. d, h, l Merge of the three panels to the left