Journal: Cellular & Molecular Biology Letters

Article Title: The SARS-CoV-2 envelope PDZ binding motif acts as a virulence factor disrupting host’s epithelial cell–cell junctions

doi: 10.1186/s11658-025-00758-y

Figure Lengend Snippet: Production and characterization of recombinant SARS-CoV-2 viruses lacking E protein PBM. A Sequences of the Envelope C-terminus end of the recombinant viruses generated by reverse genetics. The box highlights the PBM. rSARS-CoV-2-E-WT, rSARS-CoV-2-E-ΔPBM, and rSARS-CoV-2-E-MutPBM correspond to the wild-type recombinant virus, and respectively to the viruses that lack the PBM by deletion of the four last residues or by mutations in a quadruplex of glycine. B Plaque morphology of the recombinant viruses. Plaques were developed in Vero-E6 cells on day 4 (left) or 6 (right) after infection. Images are representative of at least 3 independent experiments. Scale bar = 1 cm. C In vitro viral growth curves of the different recombinant SARS-CoV-2 viruses along with the original clinical isolate Wuhan SARS-CoV-2 virus (Wuhan CoV-2) used as reference. Subconfluent monolayers of Vero-E6 cells were infected at different MOIs, primarily 0.01 (left) and 0.001 (right), to assess the effect of initial viral load on replication kinetics. Culture supernatants were collected at several time points postinfection and titrated by TCID 50 assay. Dots correspond to means with standard deviations and dashed lines indicate the limit of detection ( n = 3 independent replicates/time-point, nd: not detected). D Resistance dose response in Vero-E6 cells at 72 hpi. Impedance was measured every minute over the course of 96 h in wells that were mock infected or infected with recombinant SARS-CoV-2 viruses in tenfold dilutions ranging from MOIs of 1 to 0.0001. Sigmoid curves were generated using AxIS Z software. Dots indicate the median and vertical lines indicate the interquartile range ( n = 3). E Median time-to-death calculations based on raw resistance data for each MOI. It was noted that the median time to cell death did not show significant variation across different MOIs, particularly between 0.01 and 0.1, suggesting a potential saturation effect at higher MOIs. Horizontal lines indicate median with the interquartile range ( n = 3). Kruskal–Wallis test followed by the Dunn’s multiple comparisons test (the adjusted p value is indicated when significant)

Article Snippet: Lung sections immunohistochemistry was performed on a Bond RX ( Leica TM ) using a rabbit polyclonal antibody against SARS Nucleocapsid Protein antibody (1:500, NB100-56576, Novus Biologicals ) and biotinylated goat anti-rabbit Ig secondary antibody (1:600, E0432, Dako ® , Agilent ).

Techniques: Recombinant, Generated, Virus, Infection, In Vitro, Software

Journal: Cellular & Molecular Biology Letters

Article Title: The SARS-CoV-2 envelope PDZ binding motif acts as a virulence factor disrupting host’s epithelial cell–cell junctions

doi: 10.1186/s11658-025-00758-y

Figure Lengend Snippet: Subcellular localization of the Envelope protein and subversion of ZO-1 protein during SARS-CoV-2 infection in an ALI Transwell model. A Schematic view of the MucilAir reconstructed human bronchial epithelium. The model exhibits an air–liquid interface with different cell types (ciliated, goblet, and basal cells). B , C Kinetics of viral replication of the recombinant viruses in apical B and basal C compartment of the ALI Transwell model. Cells were infected apically with recombinant SARS-CoV-2 viruses with 250,000 pfu (~ MOI of 0.5), and viral release was monitored over time. Apical washes were collected at 2, 4, and 7 dpi and basal medium was simultaneously harvested from the lower chamber to evaluate trans-epithelial viral passage. Viral titers in both apical and basal samples were determined TCID 50 assay on Vero-E6 cells. The “Day 0” time point corresponds to viral quantification from the third apical wash performed after the inoculum removal to assess residual input virus. Dots correspond to medians with interquartile range and dashed lines indicate the limit of detection ( n = 3 independent replicates/time-point, nd: not detected). D , E Immunofluorescence assay showing E protein localization within apical ciliated cells D and basal cells E in the MucilAir model. Staining was performed at 4 dpi on a 250,000 pfu infected epithelium (~ MOI of 0.5) with recombinant E-WT (top panel), E-ΔPBM (middle panel), or E-MutPBM (bottom panel) (scale bar = 10 µm). Images are reconstituted in 3D from Z stack acquisitions. Hoechst: nuclei (blue); GM130: Golgi apparatus (red); Envelope: recombinant SARS-CoV-2 envelope protein (green); Phalloidin: actin (cyan). Arrowheads indicate E-Golgi colocalizations. Images correspond to multiple representative fields from three independent experiments. F Visualization of ZO-1 protein network correlated with E protein localization in the MucilAir model using immunofluorescence staining. Staining was performed on either non-infected epithelium or on epithelium fixed at 4 days post-infection (dpi) with 250,000 pfu (~ MOI of 0.5) of E-WT, E-∆PBM or E-MutPBM recombinant viruses. Arrowheads indicate areas of E-ZO-1 colocalization. Images are reconstituted in 3D from Z stack acquisitions (scale bar = 20 µm). Hoechst: nuclei (blue); ZO-1: zonula occludens 1 (red); Envelope: recombinant SARS-CoV-2 envelope protein (green). Images correspond to mulitple representative fields from 3 independent experiments. G. Western blot of GFP pull-down. GFP or GFP-ZO-1 from transfected HEK293 cell lysates were immobilised on GFPtrap resin and incubated with Vero-E6 cell lysates non infected or infected 48 h at a 0.01 MOI with recombinant E-WT (top panel) or E-MutPBM viruses (bottom panel). The bound fractions were analysed by immunoblotting using anti-GFP (top panel) and anti-E (bottom panel) antibodies

Article Snippet: Lung sections immunohistochemistry was performed on a Bond RX ( Leica TM ) using a rabbit polyclonal antibody against SARS Nucleocapsid Protein antibody (1:500, NB100-56576, Novus Biologicals ) and biotinylated goat anti-rabbit Ig secondary antibody (1:600, E0432, Dako ® , Agilent ).

Techniques: Infection, Recombinant, Virus, Immunofluorescence, Staining, Western Blot, Transfection, Incubation

Journal: Cellular & Molecular Biology Letters

Article Title: The SARS-CoV-2 envelope PDZ binding motif acts as a virulence factor disrupting host’s epithelial cell–cell junctions

doi: 10.1186/s11658-025-00758-y

Figure Lengend Snippet: Clinical profile and viral metrics of hamsters infected with recombinant wild-type SARS-CoV-2 (E-WT) and PBM lacking viruses (E-ΔPBM and E-MutPBM). A Schematic representation of the in vivo experimental design. Male golden hamsters were intranasally infected with 60,000 PFU per animal of each recombinant virus ( n = 4/group). Animals were monitored and euthanized at defined time points post-infection for sample collection and analysis. Mock group received a control inoculation with vehicle solution. Tissues and organs were harvested for downstream virological, histopathological, and transcriptomic assessments. B Body weight at four days post-infection (4 dpi). C Clinical score at 4 dpi. The clinical score is based on a cumulative 0–4 scale: ruffled fur; slow movements; apathy; and absence of exploration activity. D Lung-to-body weight ratio measured at 4 dpi. Horizontal lines indicate median and the interquartile range ( n = 8/group). Kruskal–Wallis test followed by the Dunn’s multiple comparisons test (the adjusted p value is indicated when significant). E Olfactory performance loss measured at 3 days post-infection (dpi). The olfaction test is based on the hidden (buried) food finding test. Bars represent the percentage of anosmic animals ( n = 4/group). Chi-square test for trend. F , G Infectious viral titers in nasal turbinates F and lung G at 4 days post-infection (dpi) expressed as TCID 50 per 100 mg of tissue. Horizontal lines indicate median and the interquartile range ( n = 4/group). Kruskal–Wallis test followed by the Dunn’s multiple comparisons test (the adjusted p value is indicated when significant). H , I Viral genomic and subgenomic RNA load detected in nasal turbinates H and lung I at 4 dpi. Horizontal lines indicate median and the interquartile range. Lines connect symbols from the same animals ( n = 4/group). Kruskal–Wallis test followed by the Dunn’s multiple comparisons test (the adjusted p value is indicated when significant). ( n = 4/group)

Article Snippet: Lung sections immunohistochemistry was performed on a Bond RX ( Leica TM ) using a rabbit polyclonal antibody against SARS Nucleocapsid Protein antibody (1:500, NB100-56576, Novus Biologicals ) and biotinylated goat anti-rabbit Ig secondary antibody (1:600, E0432, Dako ® , Agilent ).

Techniques: Infection, Recombinant, In Vivo, Virus, Control, Activity Assay

Journal: Cellular & Molecular Biology Letters

Article Title: The SARS-CoV-2 envelope PDZ binding motif acts as a virulence factor disrupting host’s epithelial cell–cell junctions

doi: 10.1186/s11658-025-00758-y

Figure Lengend Snippet: Histopathology and immunohistochemical study of lungs from hamsters infected with WT SARS-CoV-2 recombinant virus (E-WT) and PBM lacking viruses (E-ΔPBM and E-MutPBM) at 4 dpi. A Representative images of Hematoxylin and Eosin (H&E) stained-whole lung sections (upper panels), alveoli (middle panels) and bronchiolar epithelium (bottom panels). Arrows indicate inflammation and arrowhead corresponds to epithelial damages. B Representative images of whole lung sections (upper panels), alveoli (middle panels) and bronchiolar epithelium (bottom panels) immuno-stained with SARS-CoV-2 Nucleocapsid antibody. Scale bars correspond to 2 mm for the whole-organ sections and 50 µm for the close-up views ( n = 4/group)

Article Snippet: Lung sections immunohistochemistry was performed on a Bond RX ( Leica TM ) using a rabbit polyclonal antibody against SARS Nucleocapsid Protein antibody (1:500, NB100-56576, Novus Biologicals ) and biotinylated goat anti-rabbit Ig secondary antibody (1:600, E0432, Dako ® , Agilent ).

Techniques: Histopathology, Immunohistochemical staining, Infection, Recombinant, Virus, Staining

Journal: Journal of Virology

Article Title: The furin cleavage site is required for pathogenesis, but not transmission, of SARS-CoV-2

doi: 10.1128/jvi.00467-25

Figure Lengend Snippet: Generation and in vitro characterization of SARS-CoV-2 PQQAR mutant. ( A ) Alignment of the S1/S2 cleavage site of SARS-CoV-2 WA1 and series of mutant viruses generated for evaluation including deletion of the furin cleavage site (ΔFCS), truncation of the extended loop (ΔQTQTN), and disruption of the furin cleavage site motif (PQQAR). ( B ) SARS-CoV-2 spike trimer structure (gray) highlighting the S1/S2 cleavage loop. WT (left) and PQQAR mutant (right) are zoomed with mutated residues (Q682 and Q683) in orange to disrupt the furin cleavage site. ( C ) Schematic of SARS-CoV-2 spike with PQQAR substitutions identified. ( D ) Viral titer from Vero E6 infected with WT (black) or PQQAR (orange) SARS-CoV-2 at an MOI of 0.01 ( n = 3). ( E ) Viral titer from Calu-3 2B4 infected with WT or PQQAR SARS-CoV-2 at an MOI of 0.01 ( n = 3). Data are mean ± SD. Statistical analysis was conducted using a two-tailed Student’s t test. * P ≤ 0.05; ** P ≤ 0.01; and *** P ≤ 0.001.

Article Snippet: Membranes were stripped and reprobed with SARS-CoV N -specific antibodies (Novus Biologicals #NB100-56576) and the HRP-conjugated anti-rabbit secondary IgG to measure loading.

Techniques: In Vitro, Mutagenesis, Generated, Disruption, Infection, Two Tailed Test

Journal: Journal of Virology

Article Title: The furin cleavage site is required for pathogenesis, but not transmission, of SARS-CoV-2

doi: 10.1128/jvi.00467-25

Figure Lengend Snippet: SARS-CoV-2 PQQAR mutant attenuated in golden Syrian hamsters. ( A and B ) Schematic of golden Syrian hamster infection with WT (black) or PQQAR mutant (orange) SARS-CoV-2. Three- to four-week-old male hamsters were infected with 10 5 pfu and monitored for ( B ) weight loss and disease for 7 days post-infection. ( C through E ) Viral titers were measured at days 2 and 4 from ( C ) infected lung, ( D ) nasal wash, and ( E ) trachea. Data are representative of mean ± SEM. Statistical analysis was conducted using a two-tailed Student’s t test. * P ≤ 0.05; ** P ≤ 0.01; and *** P ≤ 0.001. Experimental schematic was made in BioRender.

Article Snippet: Membranes were stripped and reprobed with SARS-CoV N -specific antibodies (Novus Biologicals #NB100-56576) and the HRP-conjugated anti-rabbit secondary IgG to measure loading.

Techniques: Mutagenesis, Infection, Two Tailed Test

Journal: Journal of Virology

Article Title: The furin cleavage site is required for pathogenesis, but not transmission, of SARS-CoV-2

doi: 10.1128/jvi.00467-25

Figure Lengend Snippet: Reduced inflammation and damage in PQQAR-infected lungs. ( A through G ) Representative H&E staining of the left lung of hamsters infected with 10 5 pfu of either WT or PQQAR SARS-CoV-2 at ( A and B ) 2 days, ( C and D ) 4 days, or ( E and F ) 7 days post-infection or ( G ) mock. ( H ) WT (black), PQQAR (orange), or PBS (gray) lung sections from each day were scored for histopathological analysis, with sections from individual animals averaged and representing a single point. Statistical analysis was conducted using a two-tailed Student’s t test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: Membranes were stripped and reprobed with SARS-CoV N -specific antibodies (Novus Biologicals #NB100-56576) and the HRP-conjugated anti-rabbit secondary IgG to measure loading.

Techniques: Infection, Staining, Two Tailed Test

Journal: Journal of Virology

Article Title: The furin cleavage site is required for pathogenesis, but not transmission, of SARS-CoV-2

doi: 10.1128/jvi.00467-25

Figure Lengend Snippet: Disruption of FCS alters spike processing and protease usage. ( A ) Schematic of SARS-CoV-2 virion sucrose cushion purification approach. ( B ) Lysates from sucrose cushion purified WT, PQQAR, and ΔFCS virions grown in Vero E6 were probed with α-Spike and α-Nucleocapsid (N) antibodies by Western blot. Full-length spike (FL) and S1/S2 cleavage product are indicated. ( C ) Quantification of densitometry of the proportion between FL (black) and S1/S2 (red) of the total spike shown (lower). ( D ) Schematic of SARS-CoV-2 entry and protease usage, including knockout of TMPRSS2-mediated entry. ( E ) Viral titer from Calu-3 TMPRSS2 knock-out cells infected with WT (black) or PQQAR (orange) SARS-CoV-2 at an MOI of 0.01 ( n = 3). ( F ) Viral titer at 48 hpi from Calu3 WT and Calu3 TMPRSS2 -/- cells. Statistical analysis was conducted using a two-tailed Student’s t test. * P ≤ 0.05; ** P ≤ 0.01; and *** P ≤ 0.001. Entry schematic was made in BioRender.

Article Snippet: Membranes were stripped and reprobed with SARS-CoV N -specific antibodies (Novus Biologicals #NB100-56576) and the HRP-conjugated anti-rabbit secondary IgG to measure loading.

Techniques: Disruption, Purification, Western Blot, Knock-Out, Infection, Two Tailed Test

Journal: Journal of Virology

Article Title: The furin cleavage site is required for pathogenesis, but not transmission, of SARS-CoV-2

doi: 10.1128/jvi.00467-25

Figure Lengend Snippet: The FCS is not required for SARS-CoV-2 transmission. ( A ) Schematic of transmission experiment in golden Syrian hamsters. Three- to four-week-old male donor hamsters were intranasally infected with 10 5 pfu of WT or PQQAR SARS-CoV-2 and individually housed. Donors were subsequently paired 1:1 with recipients 24 hpi and cohoused for 8 h before separating and nasal washing donors. ( B through F ) Nasal washes and lungs were collected at 2 days post-infection for donors (dpi) ( B through D ) and post-contact for recipients ( E and F ). Viral titers were measured using focus-forming assays for donor and recipient samples. Statistical analysis was conducted using a two-tailed Student’s t test. * P ≤ 0.05; ** P ≤ 0.01; and *** P ≤ 0.001. Experimental schematic was made in BioRender.

Article Snippet: Membranes were stripped and reprobed with SARS-CoV N -specific antibodies (Novus Biologicals #NB100-56576) and the HRP-conjugated anti-rabbit secondary IgG to measure loading.

Techniques: Transmission Assay, Infection, Two Tailed Test

Journal: Journal of Virology

Article Title: The furin cleavage site is required for pathogenesis, but not transmission, of SARS-CoV-2

doi: 10.1128/jvi.00467-25

Figure Lengend Snippet: The furin cleavage site impacts SARS-CoV-2 transmission efficiency. ( A ) Schematic of transmission competition experiment in golden Syrian hamsters. Three- to four-week-old male donor hamsters were intranasally infected with 10 5 pfu of WT:PQQAR SARS-CoV-2 in a 1:1 ratio and were individually housed. ( B through F ) After 24 hpi, donors were paired with recipients and cohoused for 12 h before separating and nasal washing on donors. Nasal washes and lungs were collected at 2 and 4 days post-infection for donors (dpi) and post-contact for recipients (dpc). Next-generation sequencing (NGS) was performed on extracted RNA to measure the percentage of WT (gray) and PQQAR (orange) present in the nasal wash and lung of donors ( B through D ) and recipients ( E and F ). The expected distribution (B–F, top bar) based on NGS percentage mutant/WT observed in the inoculating dose (two inoculum preparations with RNA sequenced twice from each) if no differences between WT and mutant virus were observed. (G) Level of PQQAR mutation detection in recipient animals (orange) versus background mutations (black). Statistical analysis was conducted using a two-tailed Student’s t test. * P ≤ 0.05; ** P ≤ 0.01; and *** P ≤ 0.001. Experimental schematic was made in BioRender.

Article Snippet: Membranes were stripped and reprobed with SARS-CoV N -specific antibodies (Novus Biologicals #NB100-56576) and the HRP-conjugated anti-rabbit secondary IgG to measure loading.

Techniques: Transmission Assay, Infection, Next-Generation Sequencing, Mutagenesis, Virus, Two Tailed Test