Journal: ACS Nano

Article Title: Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography

doi: 10.1021/acsnano.3c07265

Figure Lengend Snippet: Different distributions of SARS-CoV-2 N protein and dsRNA in infected human lung adenocarcinoma cells at different stages of the infection. A549-ACE2 cells were inoculated with SARS-CoV-2 (MOI 0.01), incubated at 37 °C for 24 h, chemically fixed and processed for immunofluorescence microscopy using antibodies against dsRNA (red) and SARS-CoV-2 N protein (green). Nuclei were counterstained with DAPI (blue). Scale bars represent 10 μm. (A–D) Examples of cells with increasing amounts of dsRNA signal (red) indicating different stages of the infection: (A) early, (B, C) intermediate, and (D) late stages of infection.

Article Snippet: A549 expressing hACE2 (A549-ACE2) was generated by transduction of parental A549 cells (ATCC CRM-CCL-185) with a retroviral vector expressing human ACE2 and a selection marker that confers resistance to blasticidin. pCMV3-hACE2 was obtained from Sino Biologicals S.L. (Beijing, China).

Techniques: Infection, Incubation, Immunofluorescence, Microscopy

Journal: ACS Nano

Article Title: Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography

doi: 10.1021/acsnano.3c07265

Figure Lengend Snippet: dsRNA-positive structures near centrosomes surrounded by vimentin during SARS-CoV-2 infection. A549-ACE2 cells were inoculated with SARS-CoV-2 (MOI 0.01), incubated at 37 °C for 24 h, chemically fixed, and processed for immunofluorescence microscopy using antibodies against dsRNA (red) and vimentin (A, B; green) or pericentrin (C; green). Nuclei and actin filaments were counterstained with DAPI (blue) and Alexa 660-conjugated phalloidin (A, B; cyan), respectively. (B) corresponds to a zoomed-in image of the indicated area in (A). Scale bars represent 10 μm.

Article Snippet: A549 expressing hACE2 (A549-ACE2) was generated by transduction of parental A549 cells (ATCC CRM-CCL-185) with a retroviral vector expressing human ACE2 and a selection marker that confers resistance to blasticidin. pCMV3-hACE2 was obtained from Sino Biologicals S.L. (Beijing, China).

Techniques: Infection, Incubation, Immunofluorescence, Microscopy

Journal: ACS Nano

Article Title: Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography

doi: 10.1021/acsnano.3c07265

Figure Lengend Snippet: Golgi apparatus dispersion in SARS-CoV-2-infected cells. A549-ACE2 cells were inoculated with SARS-CoV-2 (MOI 0.01), incubated at 37 °C for 24 h, chemically fixed, and processed for immunofluorescence microscopy using antibodies against dsRNA (red) and Golgi marker giantin (green). (A) and (B) show two different representative fields of infected cells. Arrowheads show late stages of infection and (n) dsRNA-negative, noninfected cells. Nuclei were counterstained with DAPI (blue). Scale bars represent 10 μm.

Article Snippet: A549 expressing hACE2 (A549-ACE2) was generated by transduction of parental A549 cells (ATCC CRM-CCL-185) with a retroviral vector expressing human ACE2 and a selection marker that confers resistance to blasticidin. pCMV3-hACE2 was obtained from Sino Biologicals S.L. (Beijing, China).

Techniques: Dispersion, Infection, Incubation, Immunofluorescence, Microscopy, Marker

Journal: ACS Nano

Article Title: Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography

doi: 10.1021/acsnano.3c07265

Figure Lengend Snippet: Transmission electron microscopy of SARS-CoV2-infected human cells. A549-ACE2 cells were inoculated at MOI 3 and incubated for 20 h before chemical fixation and processing for TEM. Representative images of (A) mock infected cells, where the inset shows the ultrastructure of a multilamellar body (MLB), and (B–F) SARS-CoV-2-infected cells. Definitions: N, nucleus; VRO, viral replication organelle; m, mitochondrion; VIR, putative virions; DMV, double-membrane vesicle; black arrowheads, enlarged ER cisternae; white arrowheads, centriole. Scale bars: (A, B) 5 μm; (C–E) 1 μm; (F) 0.5 μm. The scale bar in the inset to (A) is 1 μm. (H) corresponds to the area delimited by a dashed line in (G).

Article Snippet: A549 expressing hACE2 (A549-ACE2) was generated by transduction of parental A549 cells (ATCC CRM-CCL-185) with a retroviral vector expressing human ACE2 and a selection marker that confers resistance to blasticidin. pCMV3-hACE2 was obtained from Sino Biologicals S.L. (Beijing, China).

Techniques: Transmission Assay, Electron Microscopy, Infection, Incubation, Membrane

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 In Vitro

doi: 10.1128/AAC.02680-20

Figure Lengend Snippet: Potency of a panel of HCV PIs and an HCV NS4A inhibitor against SARS-CoV-2 in vitro

Article Snippet: Our extensive results in Vero E6 and A549-hACE2 cells using short-term drug interaction assays demonstrated that the mode of interaction between PI and remdesivir depended on the PI structure.

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 In Vitro

doi: 10.1128/AAC.02680-20

Figure Lengend Snippet: Potency of selected HCV PIs against SARS-CoV-2 was confirmed in A549-hACE2 cells. A549-hACE2 cells were seeded in 96-well plates and, the following day, infected with SARS-CoV-2 at an MOI of 0.003 followed by treatment with specified concentrations of the PIs boceprevir, simeprevir, grazoprevir, glecaprevir, and voxilaprevir, as described in Materials and Methods. After 46 to 50 h of incubation, SARS-CoV-2-infected cells were visualized by immunostaining for the SARS-CoV-2 spike protein and quantified by automated counting, as described in Materials and Methods. Data points (red dots) are means from 7 replicates ± SEMs and represent percent residual infectivity, determined as percent SARS-CoV-2-positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration-response curves (red lines) were fitted and EC 50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means from 3 replicate cultures ± SEMs and represent percent cell viability relative to mean absorbance from 12 nontreated controls. Sigmoidal concentration-response curves were fitted and CC 50 values were determined, as shown in Fig. S6. The red dotted lines represent the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to <70%, according to Fig. S2. The blue dotted lines represent the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to <90%, according to Fig. S2.

Article Snippet: Our extensive results in Vero E6 and A549-hACE2 cells using short-term drug interaction assays demonstrated that the mode of interaction between PI and remdesivir depended on the PI structure.

Techniques: Infection, Incubation, Immunostaining, Concentration Assay, Colorimetric Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 In Vitro

doi: 10.1128/AAC.02680-20

Figure Lengend Snippet: Analysis of interactions of selected HCV PIs with remdesivir in A549-hACE2 cells. A549-hACE2 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 at an MOI of 0.003 followed by treatment with serial dilutions of the linear PI boceprevir (BOC), the macrocyclic PI simeprevir (SIM) or grazoprevir (GRA), polymerase inhibitor remdesivir (REM), or a combination of these PIs and remdesivir, as described in Materials and Methods. After 46 to 50 h of incubation, SARS-CoV-2-infected cells were visualized by immunostaining for the SARS-CoV-2 spike protein and quantified by automated counting, as described in Materials and Methods. For each inhibitor pair to be evaluated, 8 to 10 treatment conditions were used (indicated on x axes). Each treatment condition was defined by a given concentration of PI applied singly, a given concentration of remdesivir applied singly, and a combination of these same concentrations of PIs and remdesivir, as specified in Table S2, resulting in 3 data points per treatment condition. Data points are means from 7 replicates ± SEMs and represent percent residual infectivity, determined as percent SARS-CoV-2-positive cells relative to means of counts from infected nontreated control cultures. Sigmoidal concentration-response curves were fitted as described in Materials and Methods. DMSO was kept constant in all cultures. The tested inhibitor concentrations did not impair cell viability (Fig. S8). DMSO did not induce antiviral effects in the tested concentration ranges (Fig. S2).

Article Snippet: Our extensive results in Vero E6 and A549-hACE2 cells using short-term drug interaction assays demonstrated that the mode of interaction between PI and remdesivir depended on the PI structure.

Techniques: Infection, Incubation, Immunostaining, Concentration Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 In Vitro

doi: 10.1128/AAC.02680-20

Figure Lengend Snippet: Comparison of barriers to escape for HCV PIs in A549-hACE2 cells. A549-hACE2 cells seeded the previous day in T25 flasks were infected with SARS-CoV-2 at an MOI of 0.0005, followed by treatment with indicated concentrations of specified inhibitors administered immediately after infection and subsequently at the listed time points when cells were split, as described in Materials and Methods. BOC, boceprevir; SIM, simeprevir; GRA, grazoprevir; REM, remdesivir. Upon splitting of cells, cell culture supernatant was harvested and subjected to RT-qPCR for determination of SARS-CoV-2 RNA titers determined as genome copies per milliliter. The black lines indicate the LLOQs. To facilitate comparisons, bars are color coded according to the day postinfection, and blue and red dotted lines were inserted to highlight day 1 and 3 values of the nontreated culture, respectively. Cultures summarized in this figure are derived from different experimental setups, each including an infected nontreated control culture, which showed viral spread comparable to that in the depicted representative culture. (Left) Treatment with 1-fold EC 50 boceprevir, simeprevir, or grazoprevir. (Middle) Treatment with 1-, 2-, 3-, 4-, 5-, and 8-fold EC 50 boceprevir. *, culture was terminated due to virus- or drug-induced cytotoxicity; # , culture was maintained for a total of 13 days without indication of infection (RNA titers were around the LLOQ and no observation of single SARS-CoV-2 spike protein-positive cells). (Right) Treatment with 0.8-fold EC 50 remdesivir, 1-fold EC 50 boceprevir, 1-fold EC 50 simeprevir, or 1-fold EC 50 grazoprevir singly or with a combination of remdesivir with either of the PIs.

Article Snippet: Our extensive results in Vero E6 and A549-hACE2 cells using short-term drug interaction assays demonstrated that the mode of interaction between PI and remdesivir depended on the PI structure.

Techniques: Infection, Cell Culture, Quantitative RT-PCR, Derivative Assay