plvx ef1alpha sars cov 2 nsp5 2xstrep ires puro  (Addgene inc)

Journal: Journal of Virology

Article Title: The ORF6 accessory protein contributes to SARS-CoV-2 virulence and pathogenicity in the naturally susceptible feline model of infection

doi: 10.1128/jvi.00644-25

Figure Lengend Snippet: Role of SARS-CoV-2 accessory protein ORF6 in virus replication and spread in vitro . ( A ) Schematic representation of parental rSARS-CoV-2 WA1 (rWA1) and ORF6-deleted (rWA1ΔORF6) viruses used in this study. ( B ) Multi-step growth curves. Vero E6 TMPRSS2, Calu-3, and feline lung cACE2 cells were infected (MOI 0.1) with rWA1 and rWA1ΔORF6 viruses and incubated at 4°C for 1 h (virus adsorption) and then transferred and incubated at 37°C. Cells were harvested at the indicated time points, and the virus titers were determined by limiting dilutions and expressed as TCID 50 .mL −1 . The limit of detection (LOD) for infectious virus titration was 10 1.05 TCID 50 .mL −1 , which is marked with the dashed line. Data are the mean ± SEM of three replicates ( n = 3) from three independent experiments and two-way ANOVA with Tukey’s multiple comparison test, ns = not significant. ( C ) Plaque phenotype. Vero E6 cells were infected with rWA1 and rWA1ΔORF6 and overlaid with media containing 0.5% agarose. Plates were incubated at 37°C for 72 h, the agarose overlay was removed, cells were fixed, and the monolayer was stained with 0.5% crystal violet. Representative images of plaque phenotypes from three independent experiments are shown. ( D ) Viral plaque sizes. The diameters of viral plaques were measured using a scale in millimeters. Data indicate means ± SEM, n = 45, and two independent experiments. Mann-Whitney U test, **** P ≤ 0.0001.

Article Snippet: Initially, a lentiviral plasmid encoding the SARS-CoV-2 ORF6 protein (pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro, Addgene # #141387) was used in the luciferase reporter assays.

Techniques: Virus, In Vitro, Infection, Incubation, Adsorption, Titration, Comparison, Staining, MANN-WHITNEY

Journal: Journal of Virology

Article Title: The ORF6 accessory protein contributes to SARS-CoV-2 virulence and pathogenicity in the naturally susceptible feline model of infection

doi: 10.1128/jvi.00644-25

Figure Lengend Snippet: Innate immune inhibition by SARS-CoV-2 accessory protein ORF6. HEK293T cells were transfected with plasmids encoding ORF6 or an empty vector and with IFN-β ( A ), IRF3 ( B ), NF-κB ( C ), PRDII ( D ), and PRDIII ( E ) Firefly reporter plasmids. At 24 h post-transfection, the cells were stimulated with poly(I:C), SeV, or TNF-α for 12 h. Cell lysates were harvested, and Firefly luciferase activity was measured using a luminometer. The ratio of luminescence obtained from target reporters (IFN-β-Luc, IRF3-Luc, NF-κB-Luc, PRDII-Luc, and PRDIII-Luc) to luminescence from the control Renilla reporter (pRN-Luc) was determined to normalize for transfection efficiencies. The relative IFN-β-, IRF3-, NF-κB-, PRDII-, or PRDIII-driven luciferase activity was calculated as fold change over the unstimulated cells. HEK293T cells were transfected with plasmids encoding ORF6 or an empty vector and stimulated with poly(I:C) ( F ), SeV ( G ), or TNF-α ( H ) for 4 h. Cell lysates were harvested and analyzed by qPCR. ( A–H ) Data indicate mean ± SEM of 3–5 independent experiments. Mann-Whitney U test, * P ≤ 0.05, ns = not significant. One-way ANOVA with Tukey’s multiple comparison tests, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Initially, a lentiviral plasmid encoding the SARS-CoV-2 ORF6 protein (pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro, Addgene # #141387) was used in the luciferase reporter assays.

Techniques: Inhibition, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Control, MANN-WHITNEY, Comparison

Journal: Journal of Virology

Article Title: The ORF6 accessory protein contributes to SARS-CoV-2 virulence and pathogenicity in the naturally susceptible feline model of infection

doi: 10.1128/jvi.00644-25

Figure Lengend Snippet: Role of ORF6 on innate immune inhibition during SARS-CoV-2 infection in vitro . HEK293T-hACE2 cells were transfected with IFN-β ( A ) or NF-κB ( B, C ) Firefly reporter plasmids for 6 h. Cells were then infected with rWA1 or rWA1ΔORF6 (MOI = 3) for 12 h. After that, the cells were stimulated with SeV or TNF-α for 8 h, cell lysates were harvested, and the luciferase activity was measured using a luminometer. Untreated control cells were harvested after 20 h of infection. The ratio of luminescence obtained from the target reporters (IFN-β-Luc or NF-κB-Luc) to luminescence obtained from the control Renilla reporter (pRN-Luc) was determined to normalize for transfection efficiencies. The relative IFN-β or NF-κB promoter-driven luciferase activity was expressed as fold change over the unstimulated cells. Data indicate mean ± SEM of four independent experiments. One-way ANOVA with multiple comparison test, * P ≤ 0.05, ns = not significant.

Article Snippet: Initially, a lentiviral plasmid encoding the SARS-CoV-2 ORF6 protein (pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro, Addgene # #141387) was used in the luciferase reporter assays.

Techniques: Inhibition, Infection, In Vitro, Transfection, Luciferase, Activity Assay, Control, Comparison

Journal: Journal of Virology

Article Title: The ORF6 accessory protein contributes to SARS-CoV-2 virulence and pathogenicity in the naturally susceptible feline model of infection

doi: 10.1128/jvi.00644-25

Figure Lengend Snippet: ORF6 contributes to SARS-CoV-2 virulence and pathogenesis in cats. ( A ) Experimental study design. ( B ) Changes (°F) in body temperature and ( C ) body weight (%) in cats intranasally inoculated with rWA1 and rWA1ΔORF6. SARS-CoV-2 RNA loads quantified by rRT-PCR in nasal ( D ) and oral ( E ) secretions collected on days 0, 1, 3, and 5 pi and in bronchoalveolar lavage fluid ( F ) collected on days 3 and 5 pi. Infectious SARS-CoV-2 loads in nasal ( G ) and oral ( H ) secretions and BALF ( I ) were determined by virus titration in rRT-PCR-positive samples. Virus titers were determined using endpoint dilutions and expressed as TCID 50 .mL −1 . The limit of detection (LOD) for infectious virus titration was 10 1.05 TCID 50 .mL −1 , which is depicted with the dashed line. Data indicate the mean ± standard deviation of 3–6 animals per group per time point. Two-way ANOVA with Tukey’s multiple comparison test, ns = not significant, * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Initially, a lentiviral plasmid encoding the SARS-CoV-2 ORF6 protein (pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro, Addgene # #141387) was used in the luciferase reporter assays.

Techniques: Quantitative RT-PCR, Virus, Titration, Standard Deviation, Comparison

Journal: Journal of Virology

Article Title: The ORF6 accessory protein contributes to SARS-CoV-2 virulence and pathogenicity in the naturally susceptible feline model of infection

doi: 10.1128/jvi.00644-25

Figure Lengend Snippet: The rWA1ΔORF6 virus presents reduced replication and pathology in the respiratory tract of cats. Infectious SARS-CoV-2 in tissues was assessed by virus titration in rRT-PCR-positive tissue samples collected on days 3 ( A ) and 5 ( B ) pi. Virus titers were determined using endpoint dilutions and expressed as TCID 50 .mL −1 . The limit of detection (LOD) for infectious virus titration was 10 1.05 TCID 50 .mL −1 , which is depicted with the dashed line. Data indicate mean ± standard deviation of 3 animals per group per time point. ( C ) In situ hybridization (ISH) scoring of tissues. ( A–C ) Two-way ANOVA with Tukey’s multiple comparison test, * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.005. Hematoxylin and eosin (H&E) staining and ISH in the nasal turbinate ( D–F ), trachea ( G–I ), and lung ( J–L ) of cats inoculated with rWA1 and rWA1ΔORF6 and control cats. Nasal turbinates, trachea, and lungs were collected from rWA1 ( D, G, and J ) and rWA1ΔORF6 ( E, H, and K ) or mock ( F, I, and L ) inoculated cats on days 3 and 5 pi. In ISH, the viral RNA (red labeling) was performed using a probe targeting the SARS-CoV-2 S RNA. Lesion scores (established as described in Materials and Methods) for the tissues evaluated on days 3 and 5 pi are presented in ( M ) and ( N ), respectively.

Article Snippet: Initially, a lentiviral plasmid encoding the SARS-CoV-2 ORF6 protein (pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro, Addgene # #141387) was used in the luciferase reporter assays.

Techniques: Virus, Titration, Quantitative RT-PCR, Standard Deviation, In Situ Hybridization, Comparison, Staining, Control, Labeling

Journal: Journal of Virology

Article Title: The ORF6 accessory protein contributes to SARS-CoV-2 virulence and pathogenicity in the naturally susceptible feline model of infection

doi: 10.1128/jvi.00644-25

Figure Lengend Snippet: Heatmaps of differential gene expression in the nasal turbinate of cats infected with SARS-CoV-2. Nasal turbinate was collected from rWA1 and rWA1ΔORF6 or mock-inoculated cats on day 3 pi and subjected to RNA-seq analysis. ( A ) Heatmap of hierarchically clustered DEGs in nasal turbinate tissues of mock- (control), rWA1ΔORF6- and rWA1-infected cats. Each column represents the gene expression profiles from individual samples across three groups. A total of 5,000 genes represented by colored bars represent z-score values in the color key, where red represents upregulation, blue represents downregulation, and white represents unchanged gene expression. A group comparison of DEGs in rWA1 vs. control showed 394 genes up- and 104 down-regulated genes ( B ), 134 up- and 8 down-regulated genes in rWA1ΔORF6 vs. control ( C ), and 19 up- and 17 down-regulated genes in rWA1ΔORF6 vs. rWA1 groups ( D ). The up- and down-regulated DEGs are clustered across three samples from each group.

Article Snippet: Initially, a lentiviral plasmid encoding the SARS-CoV-2 ORF6 protein (pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro, Addgene # #141387) was used in the luciferase reporter assays.

Techniques: Gene Expression, Infection, RNA Sequencing, Control, Comparison