cell lines heparg 39 n a hepargntcp julie lucifora n a huh7 5 charles rice n a hek293t atcc crl 3216  (ATCC)

Journal: Journal of Virology

Article Title: Perturbation of de novo lipogenesis hinders MERS-CoV assembly and release, but not the biogenesis of viral replication organelles

doi: 10.1128/jvi.02282-24

Figure Lengend Snippet: Gene expression of host factors of the DNL pathway increases upon MERS-CoV infection. ( A ) Schematic representation of the main steps of the de novo lipogenesis pathway, the key enzymes involved (ACLY, ACC, and FASN), and the downstream biological functions of the resulting fatty acids. Double arrows indicate that multiple reactions are involved but not depicted here. Small-molecule inhibitors used in this study are denoted in red. PTMs: posttranslational modifications. ( B ) Gene expression analysis of key host factors participating in DNL and phospholipid synthesis (dark gray) or fatty acid oxidation (light gray) in mock-infected Huh7 cells in comparison with MERS-CoV-infected cells (MOI 5) at 12 h p.i. The mRNA levels of ACLY, ACC, FASN, SCD-1, DGAT1, ADRP, CPT1a , and ACOX were determined by RT-qPCR and normalized against the mock-infected control. ( C ) Western blot analysis of ACC, phospho-ACC, and FASN in MERS-CoV-infected Huh7 cells at 9 and 12 h p.i. Data are represented as mean ± SD of two biological replicates. Statistical significance was calculated using two-way ANOVA and applying Bonferroni multiple comparison correction, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Huh7 cells (kindly provided by Dr. Ralf Bartenschlager, Heidelberg University, Germany) and MRC5 cells (purchased from ATCC, CCL-171) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 8% (v/v) fetal calf serum (FCS), 100 IU/mL penicillin/streptomycin, 2 mM L-glutamine, and non-essential amino acids at 37°C and 5% CO 2 .

Techniques: Gene Expression, Infection, Comparison, Quantitative RT-PCR, Control, Western Blot

Journal: Journal of Virology

Article Title: Perturbation of de novo lipogenesis hinders MERS-CoV assembly and release, but not the biogenesis of viral replication organelles

doi: 10.1128/jvi.02282-24

Figure Lengend Snippet: DNL inhibition strongly reduces the release of infectious MERS-CoV progeny, without affecting intracellular viral RNA synthesis. ( A ) Huh7 cells or ( B ) MRC5 were infected with MERS-CoV (MOI 5) and treated with either DMSO (vehicle control) or 10 μM TOFA from 1 h p.i. onward. Cell lysates were harvested at 12 h p.i. (for Huh7 cells) or 16 h p.i. (for MRC5) and intracellular viral RNA copies were measured by RT-qPCR. ( C ) and ( D ) Infectious viral progeny was quantified by plaque assay on Huh7 cells using the harvested culture supernatant (see panel A and B, respectively). ( E ) Huh7 cells were infected with MERS-CoV (MOI 5) and treated with either DMSO (vehicle control) or 15 μM TVB-2640 from 1 h p.i. onward. At 16 h p.i., cells were harvested, and intracellular viral RNA copies were measured by RT-qPCR. ( F ) Infectious viral progeny from TVB-2640-treated and untreated Huh7 cells (see panel E) was quantified by plaque assay on Huh7 cells. ( G ) Huh7 cells were infected with MERS-CoV (MOI 5) and treated with either DMSO (vehicle control) or 10 μM TOFA from 1 h p.i. onward. Samples were collected at 12, 14, and 16 h p.i. Infectious viral progeny was quantified by plaque assay on Huh7 cells. While in the samples collected from TOFA-treated cells at 12 h p.i, the number of infectious viral particles was below the detection limit of the assay, viral titers increased over time at 14 and 16 h p.i. ( H ) Analysis of the secretory pathway’s functionality upon TOFA treatment. Huh7 cells were transfected with a plasmid expressing Gaussia luciferase, a naturally secreted reporter protein. From 8 h post-transfection., transfected cells were treated with either 10 μM TOFA or 5 μM Brefeldin A. At 24 h post-compound addition, the levels of extracellular Gaussia luciferase secreted in the supernatant were measured using an enzymatic assay (see Materials and Methods). ( I ) The cell monolayers from the same wells (panel H) were also lysed, and intracellular Gaussia luciferase expression was measured and compared with the amount of secreted reporter protein. Dotted lines indicate background luciferase levels measured in cell culture medium. Data are represented as mean ± SD of two ( D ), three ( B, E, G ) or four ( H, I ) biological replicates. Statistical significance was calculated using one-way ANOVA and applying Bonferroni multiple comparison correction, *** P < 0.001.

Article Snippet: Huh7 cells (kindly provided by Dr. Ralf Bartenschlager, Heidelberg University, Germany) and MRC5 cells (purchased from ATCC, CCL-171) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 8% (v/v) fetal calf serum (FCS), 100 IU/mL penicillin/streptomycin, 2 mM L-glutamine, and non-essential amino acids at 37°C and 5% CO 2 .

Techniques: Inhibition, Infection, Control, Quantitative RT-PCR, Plaque Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Enzymatic Assay, Cell Culture, Comparison

Journal: Journal of Virology

Article Title: Perturbation of de novo lipogenesis hinders MERS-CoV assembly and release, but not the biogenesis of viral replication organelles

doi: 10.1128/jvi.02282-24

Figure Lengend Snippet: TOFA induces lipolysis in uninfected and MERS-CoV-infected cells. ( A ) Uninfected Huh7 cells were treated with DMSO or 10 μM TOFA. ( B ) Huh7 cells were infected with MERS-CoV (MOI 5) and treated with either DMSO (vehicle control) or 10 μM TOFA from 1 h p.i. onward. For both uninfected and infected cells, samples were collected at 8 and 11 h p.t. The next day, the fixed cells were stained with BODIPY 493/503 (lipid droplets) and Hoechst 33342 (nuclei), and the presence of lipid droplets was analyzed by wide-field fluorescence microscopy.

Article Snippet: Huh7 cells (kindly provided by Dr. Ralf Bartenschlager, Heidelberg University, Germany) and MRC5 cells (purchased from ATCC, CCL-171) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 8% (v/v) fetal calf serum (FCS), 100 IU/mL penicillin/streptomycin, 2 mM L-glutamine, and non-essential amino acids at 37°C and 5% CO 2 .

Techniques: Infection, Control, Staining, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: Perturbation of de novo lipogenesis hinders MERS-CoV assembly and release, but not the biogenesis of viral replication organelles

doi: 10.1128/jvi.02282-24

Figure Lengend Snippet: Subcellular localization of MERS-CoV envelope proteins following TOFA treatment. Huh7 cells were infected with MERS-CoV, treated with TOFA as previously described, fixed at 12 h p.i and analyzed using immunofluorescence microscopy. Cells were triple-labeled for (A) S (green, panels i and ii) and or E proteins (green, panels iii and iv) in combination with labeling for giantin (red), as a marker for the Golgi complex or (B) S (green, panels i and ii) and M (green, panels iii and iv) proteins in combination with labeling for giantin (red). Confocal images are representative of at least two independent biological replicates. White arrows point to co-localization foci of two or three markers.

Article Snippet: Huh7 cells (kindly provided by Dr. Ralf Bartenschlager, Heidelberg University, Germany) and MRC5 cells (purchased from ATCC, CCL-171) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 8% (v/v) fetal calf serum (FCS), 100 IU/mL penicillin/streptomycin, 2 mM L-glutamine, and non-essential amino acids at 37°C and 5% CO 2 .

Techniques: Infection, Immunofluorescence, Microscopy, Labeling, Marker

Journal: Journal of Virology

Article Title: Perturbation of de novo lipogenesis hinders MERS-CoV assembly and release, but not the biogenesis of viral replication organelles

doi: 10.1128/jvi.02282-24

Figure Lengend Snippet: Effect of TOFA treatment on MERS-CoV structural protein expression and post-translational modifications. Expression levels of S (using separate antibodies against the S1 and S2 subunits), N, M, and E proteins were analyzed by Western blot, using β-actin as internal control. ( A ) Huh7 cells were infected with MERS-CoV and treated with DMSO or TOFA as previously described. Supernatants were collected at 16 h p.i., and progeny viral particles were pelleted through a sucrose cushion by ultracentrifugation, lysed, and used for Western blot analysis. ( B ) Huh7 cells were infected with MERS-CoV and DMSO- or TOFA-treated as previously described, and samples (WCL: whole cell lysate) were collected at 9 and 12 h p.i. for analysis by Western blotting. ( C ) Western blot for PNGase assay (described in Materials and Methods) using lysates collected from MERS-CoV-infected Huh7 cells at 12 h p.i. Blots are representative of at least two independent biological replicates.

Article Snippet: Huh7 cells (kindly provided by Dr. Ralf Bartenschlager, Heidelberg University, Germany) and MRC5 cells (purchased from ATCC, CCL-171) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 8% (v/v) fetal calf serum (FCS), 100 IU/mL penicillin/streptomycin, 2 mM L-glutamine, and non-essential amino acids at 37°C and 5% CO 2 .

Techniques: Expressing, Western Blot, Control, Infection

Journal: Journal of Virology

Article Title: Perturbation of de novo lipogenesis hinders MERS-CoV assembly and release, but not the biogenesis of viral replication organelles

doi: 10.1128/jvi.02282-24

Figure Lengend Snippet: TEM analysis of TOFA-treated Huh7 cells infected with MERS-CoV. Cells were infected with MOI 5, treated with TOFA or with DMSO and fixed and processed for electron microscopy at 12 h p.i. ( A ) Morphologically comparable virus-induced DMVs (asterisks) can be readily observed in both conditions. Close-ups of the boxed areas are provided in the insets. ( B ) Extracellular virions abundantly present in the extracellular space of DMSO-treated MERS-CoV-infected cells (inset: zoomed-in image of the boxed area), but not in the extracellular space of TOFA-treated and virus-infected cells. ( C ) Consecutive stages of MERS-CoV particle assembly in DMSO-treated and infected cells; assembly events were split in two categories for simplicity (see Materials and Methods), denoted as early-stage assembly events of budding nucleocapsid complexes in membranes of the secretory pathway (panels i and ii) and late-stage assembly events, including nearly fully formed viral particles and virions fully budded inside large vesicles (iii). ( D ) In TOFA-treated MERS-CoV-infected cells, early assembly stage events, as seen in panels (i) and (ii), were abnormally abundant, revealing a stalling of viral particle assembly in the presence of the compound. Mitochondria are annotated with M, convoluted membranes with CM, and cell nuclei with N.

Article Snippet: Huh7 cells (kindly provided by Dr. Ralf Bartenschlager, Heidelberg University, Germany) and MRC5 cells (purchased from ATCC, CCL-171) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 8% (v/v) fetal calf serum (FCS), 100 IU/mL penicillin/streptomycin, 2 mM L-glutamine, and non-essential amino acids at 37°C and 5% CO 2 .

Techniques: Infection, Electron Microscopy, Virus

Journal: Journal of Virology

Article Title: Perturbation of de novo lipogenesis hinders MERS-CoV assembly and release, but not the biogenesis of viral replication organelles

doi: 10.1128/jvi.02282-24

Figure Lengend Snippet: Exogenous palmitic acid supplementation partially rescues MERS-CoV assembly after TOFA treatment. ( A ) Alignment of S and E protein C-terminal sequences from selected coronaviruses using the following accession numbers: MERS-CoV ( NC_019843 ), SARS-CoV ( NC_004718 ), SARS-CoV-2 ( NC_045512 ), MHV ( AY700211 ), TGEV ( AJ271965 ), IBV ( NC_001451 ), and HCoV-229E ( NC_002645.1 ). Cysteines next to the transmembrane domain that can serve as palmitoylation substrates are highlighted in yellow and noted with an asterisk. ( B ) Huh7 cells were infected with MERS -CoV (MOI 5) and treated with either DMSO (vehicle control) or 30 μM 2-BP from 1 h p.i. onward. At 16 h p.i., cell lysates were harvested, and intracellular viral RNA copies were measured by RT-qPCR. ( C ) Infectious viral progeny was quantified by plaque assay on Huh7 cells. Data are represented as mean ± SD of three independent experiments. ( D ) Huh7 cells were infected with MERS-CoV (MOI 5) and treated from 1 h p.i. onward with DMSO supplemented with 100 μM BSA, TOFA, TOFA supplemented with 100 μM BSA-PA, or TOFA supplemented with 100 μM BSA-OA. At 16 h p.i., samples were collected, and infectious viral progeny was quantified by plaque assay on Huh7 cells. Data are represented as mean ± SD of two independent experiments. Statistical significance was calculated using one-way ANOVA and applying Dunnet multiple comparison correction, **** P < 0.0001 and ** P < 0.01, ns: not significant. ( E ) Huh7 cells were infected with MERS-CoV and treated with DMSO, TOFA, or TOFA with BSA-PA from 1 h p.i. onward as previously described, and samples were collected at 12 h p.i. for analysis by Western blotting. Blots are representative of at least two independent biological replicates. ( F-I ) Huh7 cells were infected with MERS-CoV and treated as described above. At 12 h p.i., samples were fixed and processed for TEM. ( F ) Post-addition of palmitic acid, the number of extracellular virus particles present in TOFA-treated virus-infected cells is increased. ( G ) Virions either in the late stage (advanced/fully assembled particles) or early stage were quantified (see Materials and Methods), and the ratio of both classes was determined. ( H ) Number of DMVs per µm 2 of cytoplasm observed (see Materials and Methods) in the DMSO and BSA-treated, TOFA-treated, or TOFA- and BSA-PA-treated virus-infected cells. ( I ) In the same samples, the area of CMs (in nm 2 ) was also determined per µm 2 of cytoplasm (see Materials and Methods). Data are represented as mean ± SD of three separate stiches. Statistical significance was calculated using one-way ANOVA and applying Tukey multiple comparison correction, *** P < 0.001, ** P < 0.01, and * P < 0.05.

Article Snippet: Huh7 cells (kindly provided by Dr. Ralf Bartenschlager, Heidelberg University, Germany) and MRC5 cells (purchased from ATCC, CCL-171) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 8% (v/v) fetal calf serum (FCS), 100 IU/mL penicillin/streptomycin, 2 mM L-glutamine, and non-essential amino acids at 37°C and 5% CO 2 .

Techniques: Infection, Control, Quantitative RT-PCR, Plaque Assay, Comparison, Western Blot, Virus