Journal: Signal Transduction and Targeted Therapy

Article Title: Inducible CD147 up-regulation boosts extended SARS-CoV-2 infection triggering severe COVID-19 independent of ACE2

doi: 10.1038/s41392-025-02551-x

Figure Lengend Snippet: SARS-CoV-2-induced CD147 up-regulation causes extended virus infection and pathological injury. a A flow chart for establishing hCD147 mice model of COVID-19. b The virus loads in the lung tissues of hCD147 mice were detected by Taqman-based RT-PCR at 3 and 7 dpi, *** p < 0.001. c , d ,The expression of CD147 was evaluated by western blot in the lung tissues of virus-infected hCD147 mice at 3 and 7 dpi, and the lung tissues of healthy hCD147 mice were used as the control ( c ). The relative expression level of CD147 was analyzed by Image Lab software, *** p < 0.001 ( d ). e The scRNA-seq identified the expression of CD147 in virus-infected ( n = 4,203) and virus-uninfected ( n = 13,354) cells of lung tissues from hCD147 mice. Wilcoxon test, p < 0.0001. f The scRNA-seq identified the expression of CD147 in virus-infected and virus-uninfected AT2 cells of lung tissues from hCD147 mice. g –i Infected, border, and uninfected regions in the lung tissues of virus-infected hCD147 mice were defined using spatial transcriptomics ( g ). The total expression of CD147 was quantified in three defined regions ( h ), and the expression of CD147 in AT2, M2 macrophages, and fibroblasts was determined ( i ). The number of spots used for data analysis in uninfected, border, and infected group were 312, 3,554, and 1,833, respectively. Wilcoxon test, p < 0.05, p < 0.001. j The BEAS-2B cells with SARS-CoV-2 pseudovirus infection in the upper chamber were co-cultured with the BEAS-2B cells in the lower chamber for 48 h, and the expression of CD147 in cells collected from the lower chamber were detected by western blot. k , l The expression of CD147 was determined by western blot in BEAS-2B cells with SARS-CoV-2 pseudovirus infection ( k ) or RBD incubation ( l ). m The relative luciferase signals in pseudovirus-infected BEAS-2B cells were determined by dual-luciferase reporter assays with RBD incubation, *** p < 0.001. n The relative luciferase signals in pseudovirus-infected BEAS-2B cells were determined by dual-luciferase reporter assays, ns, not significant, *** p < 0.001. o The virus loads in authentic virus-infected BEAS-2B cells were determined by Taqman-based RT-PCR, ns, not significant, ** p < 0.01, *** p < 0.001. p A flow chart for establishing a virus-infected hCD147 mouse model by primarily inoculating with RBD protein followed by authentic virus infection. q The expression of CD147 was detected in the lung tissues of hCD147 mice with His-RBD ( n = 4) or His-tag ( n = 4) inoculation and hCD147 mice with His-RBD ( n = 4) or His-tag ( n = 4) inoculation followed by authentic virus infection. r The virus loads in the lung tissues of virus-infected hCD147 mice were determined, * p < 0.05. s , t The infiltration of F4/80+iNOS+ M1 macrophages and Ly6G+ neutrophils in lung tissues of virus-infected hCD147 mice was evaluated by multicolor immunofluorescence staining, Ly6G, yellow; F4/80, green; iNOS, red, scale bar, 100 μm ( s ), and their infiltrated percentages were quantified, ** p < 0.01, *** p < 0.001 ( t ). u The level of cytokines and chemokines in lung tissues of virus-infected hCD147 mice was performed by RT-PCR, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The mobile phases contained the mutants of CD147 (His-CD147-R54A, His-CD147-E84A, His-CD147-E92A, His-CD147-Q100A, His-CD147-S112A, our laboratory), the mutants of RBD (His-RBD-G413A, His-RBD-K417A, His-RBD-K424A, His-RBD-G447A, His-RBD-Y489A, Sino Biological), His-RBD (WT) (40592-V08H, Sino Biological), His-RBD (Beta) (40592-V08H85, Sino Biological), His-RBD (Gamma) (40592-V08H86, Sino Biological), His-RBD (JN.1) (40592-V08H155, Sino Biological), rhesus macaque CD147 (Sino Biological), human ACE2 (Sino Biological), and rhesus macaque ACE2 (Sino Biological).

Techniques: Virus, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Control, Software, Cell Culture, Incubation, Luciferase, Multicolor Immunofluorescence Staining

Journal: mSphere

Article Title: The pan-variant potential of light: 425 nm light inactivates SARS-CoV-2 variants of concern and non-cytotoxic doses reduce viral titers in human airway epithelial cells

doi: 10.1128/msphere.00230-25

Figure Lengend Snippet: 425 nm light reduces SARS-CoV-2 Beta, Delta, and Omicron titers at non-cytotoxic doses in ALI HAE. Well-differentiated human large airway epithelial cells ( A ) were illuminated twice daily (BID) with 16 or 32 J/cm 2 of 425 nm light ( B ). At 72 hpi (six total doses), cell viability was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay ( C ). Data presented are the mean viability ± SEM relative to 0 J/cm 2 ( n = 6). ( D ) Plates coated with synthetic human ACE-2 were illuminated with 425 nm light and inoculated with recombinant spike trimers derived from WA1 and Omicron. Data presented are the mean percent binding to ACE-2 relative to 0 J/cm 2 ± SEM ( n = 5). ( E, F ) ALI HAE cells were infected with SARS-CoV-2 Beta, Delta, or Omicron variants, treated, and illuminated twice daily for 3 days with 32 J/cm 2 of 425 nm light starting at 3 hpi (MOI 0.1 [ E ]) or 24 hpi (MOI 0.001 [ F ]). Apical rinses were collected daily and enumerated via plaque assay. Data presented are the mean viral titer (PFU/mL) ± SEM ( n = 5–6). Statistical significance was determined with the Mann-Whitney rank-sum test and is indicated by * ( P < 0.05) and ** ( P < 0.01). For panels C and D, statistical significance was determined relative to the 0 J/cm 2 group. For panels E and F, statistical significance was determined relative to the 0 J/cm 2 group at the corresponding timepoint.

Article Snippet: Plates were blocked with 200 μL blocking buffer (5% FBS in PBST) and incubated at 37°C/5% CO 2 for 1 h. His-tagged recombinant WA1 S1 (Sino Biological #40591-V08H), Omicron B.1.1.529 S1 (Sino Biological #40591-V08H41), MERS-CoV S1 (Sino Biological #40069-V08H), SARS-CoV-1 S1+S2 (Sino Biological #40634-V08B), NL63 S1+S2 (Sino Biological #40604-V08B), MERS-CoV S1+S2 (Sino Biological #40069-V08B), WA1 spike trimer (Sino Biological #40589-V08H4), Alpha spike trimer (Sino Biological #40589-V08H12), Beta spike trimer (Sino Biological #40589-V08H13), Gamma spike trimer (Sino Biological #40589-V08H23), Delta spike trimer (Sino Biological #40589-V08H10), Lambda spike trimer (Sino Biological #40589-V08H24), Omicron B.1.1.529 spike trimer (Sino Biological #40589-V08H26), Omicron BA.2 spike trimer (Sino Biological #40589-V08H28), Omicron XBB.1.5 spike trimer (40589-V08H45), and Omicron JN.1 spike trimer (Sino Biological #40589-V08H59) were diluted in viral diluent.

Techniques: MTT Assay, Recombinant, Derivative Assay, Binding Assay, Infection, Plaque Assay, MANN-WHITNEY

Journal: mSphere

Article Title: The pan-variant potential of light: 425 nm light inactivates SARS-CoV-2 variants of concern and non-cytotoxic doses reduce viral titers in human airway epithelial cells

doi: 10.1128/msphere.00230-25

Figure Lengend Snippet: The RD-X19 reduces spike binding to ACE-2, inactivates SARS-CoV-2, and reduces viral titers at non-cytotoxic doses. ( A ) Recombinant spike trimers derived from WA1 and Omicron JN.1 were assessed for receptor binding following illumination with the active RD-X19 or sham. Data presented are the mean percent binding to ACE-2 relative to unilluminated ± SEM ( n = 4). ( B ) SARS-CoV-2 Beta and Gamma were diluted in cell culture media and illuminated with the active RD-X19 or sham. Data presented are the mean viral titer (PFU/mL) ± SEM ( n = 8). ( C ) ALI HAE cell cultures were illuminated twice daily (BID) with the Active RD-X19 or Sham for 3 days (six total doses). Cell viability was determined via MTT assay. Data presented are the mean viability ± SEM relative to unilluminated ( n = 4). ( D ) ALI HAE cell cultures were infected with SARS-CoV-2 WA1 illuminated twice daily for 3 days with the active RD-X19 or sham starting at 3 or 24 hpi. Apical rinses were collected daily and enumerated via plaque assay. Data presented are the mean viral titer (PFU/mL) ± SEM ( n = 10). Statistical significance was determined with the Mann-Whitney rank-sum test and is indicated by * ( P < 0.05), ** ( P < 0.01), and *** ( P < 0.001). For panel A, statistical significance was determined relative to the 0 J/cm 2 group. For panel B, statistical significance was determined relative to the inoculum. For panel D, statistical significance was determined relative to the unilluminated group at the corresponding timepoint.

Article Snippet: Plates were blocked with 200 μL blocking buffer (5% FBS in PBST) and incubated at 37°C/5% CO 2 for 1 h. His-tagged recombinant WA1 S1 (Sino Biological #40591-V08H), Omicron B.1.1.529 S1 (Sino Biological #40591-V08H41), MERS-CoV S1 (Sino Biological #40069-V08H), SARS-CoV-1 S1+S2 (Sino Biological #40634-V08B), NL63 S1+S2 (Sino Biological #40604-V08B), MERS-CoV S1+S2 (Sino Biological #40069-V08B), WA1 spike trimer (Sino Biological #40589-V08H4), Alpha spike trimer (Sino Biological #40589-V08H12), Beta spike trimer (Sino Biological #40589-V08H13), Gamma spike trimer (Sino Biological #40589-V08H23), Delta spike trimer (Sino Biological #40589-V08H10), Lambda spike trimer (Sino Biological #40589-V08H24), Omicron B.1.1.529 spike trimer (Sino Biological #40589-V08H26), Omicron BA.2 spike trimer (Sino Biological #40589-V08H28), Omicron XBB.1.5 spike trimer (40589-V08H45), and Omicron JN.1 spike trimer (Sino Biological #40589-V08H59) were diluted in viral diluent.

Techniques: Binding Assay, Recombinant, Derivative Assay, Cell Culture, MTT Assay, Infection, Plaque Assay, MANN-WHITNEY

Journal: Vaccines

Article Title: Mucosal and Serum Neutralization Immune Responses Elicited by COVID-19 mRNA Vaccination in Vaccinated and Breakthrough-Infection Individuals: A Longitudinal Study from Louisville Cohort

doi: 10.3390/vaccines13060559

Figure Lengend Snippet: Omicron BA.1 is more resistant to neutralization by sera from individuals vaccinated with two doses of Moderna or Pfizer vaccine. 50% SARS-CoV-2 neutralization titers (VNT50) of sera collected from 18–40 age group ( A ), 41–60 age group ( B ), >60 age group ( C ), vaccine recipients collected within or after 6 months post two doses of either Pfizer or Moderna vaccine. Sera from participants were tested for neutralization against ancestral Wuhan Strain, Beta, Delta, and Omicron BA.1. following 1 h incubation with indicated SARS-CoV-2 VOCs before adding to VeroE6/TMPRSS2 cell monolayers. Each serum was tested in duplicate, and geometric mean 50% SARS-CoV-2 virus neutralization titers (VNT50) or 50% inhibitory dilutions were plotted. Values above the dots represent group GMTs and fold reduction in neutralization of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan strain. Descriptive statistical analysis was performed using two-sided Friedman test with Dunn’s multiple comparison using 3 variables (age group, vaccine duration, and different virus types) and 2 comparisons (age group vs. virus type and vaccine duration vs. virus type). The asterisks (*** p < 0.001, **** p < 0.0001) above the dots represents significant fold reduction in neutralization compared with ancestral Wuhan strain. All statistical results have been shown in .

Article Snippet: We tested the vaccine-elicited antibodies neutralization capabilities against SARS-CoV-2 VOCs including Beta, Delta, and Omicron BA.1 from sera samples of two-dose vaccinated Pfizer- or Moderna-vaccinated individuals ( ).

Techniques: Neutralization, Incubation, Virus, Comparison

Journal: Vaccines

Article Title: Mucosal and Serum Neutralization Immune Responses Elicited by COVID-19 mRNA Vaccination in Vaccinated and Breakthrough-Infection Individuals: A Longitudinal Study from Louisville Cohort

doi: 10.3390/vaccines13060559

Figure Lengend Snippet: Reduced IgG binding titers against RBD protein of mutant SARS-CoV-2 VOCs from individuals vaccinated with the Moderna or Pfizer vaccine. ELISA IgG binding endpoint titers against different RBD of SARS-CoV-2 VOCs in sera collected from 18–40 age group ( A ), 41–60 age group ( B ), >60 age group ( C ), vaccine recipients collected within or after 6 months post two doses of either Pfizer or Moderna vaccine. Sera from participants were tested for binding interactions between RBD protein of different SARS-CoV-2 VOCs and antibody using endpoint titer ELISA. Serially diluted serum was added in duplicates to pre-coated RBD 96 well plates in duplicates, detected using anti-human IgG, and endpoint titers were calculated and plotted based on the cutoff value of naïve human sera (at 1:100 dilution). The fold reduction in neutralization of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan strain. Descriptive statistical analysis was performed using two-sided Friedman test with Dunn’s multiple comparison using 3 variables (age group, vaccine duration, and virus type) and 2 comparisons (age group vs. virus type and vaccine duration vs. virus type). The asterisks (*** p < 0.001, ** p < 0.01) above the bars represent significant fold reduction in IgG binding titers to RBD protein compared with ancestral Wuhan strain. All statistical results have been shown in .

Article Snippet: We tested the vaccine-elicited antibodies neutralization capabilities against SARS-CoV-2 VOCs including Beta, Delta, and Omicron BA.1 from sera samples of two-dose vaccinated Pfizer- or Moderna-vaccinated individuals ( ).

Techniques: Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Neutralization, Comparison, Virus

Journal: Vaccines

Article Title: Mucosal and Serum Neutralization Immune Responses Elicited by COVID-19 mRNA Vaccination in Vaccinated and Breakthrough-Infection Individuals: A Longitudinal Study from Louisville Cohort

doi: 10.3390/vaccines13060559

Figure Lengend Snippet: Breakthrough infection of previously vaccinated (2 doses) individuals induced broad neutralization of mutant SARS-CoV-2 VOCs including Omicron BA.1. 50% SARS-CoV-2 neutralization titers ( A ) and ELISA IgG binding endpoint titers ( B ) against different RBD of SARS-CoV-2 VOCs in sera collected from Modena or Pfizer Vaccinated and naturally COVID-19 infected individuals. ( A ) Values above the dots represents group GMTs, fold reduction, and significant difference in neutralization of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan Strain by serum samples. ( B ) IgG endpoint titers were calculated and plotted based on the cutoff value against naïve human sera (at 1:100 dilution). Values above the bars indicate GMTs, fold reduction, and significant difference in IgG binding titers to RBD protein of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan strain. For both experiments, the serum was tested in duplicates, and in each panel, data are mean from 2 independent experiments. A two-sided Friedman test with Dunn’s multiple comparisons was performed (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: We tested the vaccine-elicited antibodies neutralization capabilities against SARS-CoV-2 VOCs including Beta, Delta, and Omicron BA.1 from sera samples of two-dose vaccinated Pfizer- or Moderna-vaccinated individuals ( ).

Techniques: Infection, Neutralization, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Vaccines

Article Title: Mucosal and Serum Neutralization Immune Responses Elicited by COVID-19 mRNA Vaccination in Vaccinated and Breakthrough-Infection Individuals: A Longitudinal Study from Louisville Cohort

doi: 10.3390/vaccines13060559

Figure Lengend Snippet: Omicron BA.1 is more resistant to neutralization by sera from individuals vaccinated with two doses of Moderna or Pfizer vaccine. 50% SARS-CoV-2 neutralization titers (VNT50) of sera collected from 18–40 age group ( A ), 41–60 age group ( B ), >60 age group ( C ), vaccine recipients collected within or after 6 months post two doses of either Pfizer or Moderna vaccine. Sera from participants were tested for neutralization against ancestral Wuhan Strain, Beta, Delta, and Omicron BA.1. following 1 h incubation with indicated SARS-CoV-2 VOCs before adding to VeroE6/TMPRSS2 cell monolayers. Each serum was tested in duplicate, and geometric mean 50% SARS-CoV-2 virus neutralization titers (VNT50) or 50% inhibitory dilutions were plotted. Values above the dots represent group GMTs and fold reduction in neutralization of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan strain. Descriptive statistical analysis was performed using two-sided Friedman test with Dunn’s multiple comparison using 3 variables (age group, vaccine duration, and different virus types) and 2 comparisons (age group vs. virus type and vaccine duration vs. virus type). The asterisks (*** p < 0.001, **** p < 0.0001) above the dots represents significant fold reduction in neutralization compared with ancestral Wuhan strain. All statistical results have been shown in .

Article Snippet: We tested the vaccine-elicited antibodies neutralization capabilities against SARS-CoV-2 VOCs including Beta, Delta, and Omicron BA.1 from sera samples of two-dose vaccinated Pfizer- or Moderna-vaccinated individuals ( ).

Techniques: Neutralization, Incubation, Virus, Comparison

Journal: Vaccines

Article Title: Mucosal and Serum Neutralization Immune Responses Elicited by COVID-19 mRNA Vaccination in Vaccinated and Breakthrough-Infection Individuals: A Longitudinal Study from Louisville Cohort

doi: 10.3390/vaccines13060559

Figure Lengend Snippet: Reduced IgG binding titers against RBD protein of mutant SARS-CoV-2 VOCs from individuals vaccinated with the Moderna or Pfizer vaccine. ELISA IgG binding endpoint titers against different RBD of SARS-CoV-2 VOCs in sera collected from 18–40 age group ( A ), 41–60 age group ( B ), >60 age group ( C ), vaccine recipients collected within or after 6 months post two doses of either Pfizer or Moderna vaccine. Sera from participants were tested for binding interactions between RBD protein of different SARS-CoV-2 VOCs and antibody using endpoint titer ELISA. Serially diluted serum was added in duplicates to pre-coated RBD 96 well plates in duplicates, detected using anti-human IgG, and endpoint titers were calculated and plotted based on the cutoff value of naïve human sera (at 1:100 dilution). The fold reduction in neutralization of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan strain. Descriptive statistical analysis was performed using two-sided Friedman test with Dunn’s multiple comparison using 3 variables (age group, vaccine duration, and virus type) and 2 comparisons (age group vs. virus type and vaccine duration vs. virus type). The asterisks (*** p < 0.001, ** p < 0.01) above the bars represent significant fold reduction in IgG binding titers to RBD protein compared with ancestral Wuhan strain. All statistical results have been shown in .

Article Snippet: We tested the vaccine-elicited antibodies neutralization capabilities against SARS-CoV-2 VOCs including Beta, Delta, and Omicron BA.1 from sera samples of two-dose vaccinated Pfizer- or Moderna-vaccinated individuals ( ).

Techniques: Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Neutralization, Comparison, Virus

Journal: Vaccines

Article Title: Mucosal and Serum Neutralization Immune Responses Elicited by COVID-19 mRNA Vaccination in Vaccinated and Breakthrough-Infection Individuals: A Longitudinal Study from Louisville Cohort

doi: 10.3390/vaccines13060559

Figure Lengend Snippet: Breakthrough infection of previously vaccinated (2 doses) individuals induced broad neutralization of mutant SARS-CoV-2 VOCs including Omicron BA.1. 50% SARS-CoV-2 neutralization titers ( A ) and ELISA IgG binding endpoint titers ( B ) against different RBD of SARS-CoV-2 VOCs in sera collected from Modena or Pfizer Vaccinated and naturally COVID-19 infected individuals. ( A ) Values above the dots represents group GMTs, fold reduction, and significant difference in neutralization of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan Strain by serum samples. ( B ) IgG endpoint titers were calculated and plotted based on the cutoff value against naïve human sera (at 1:100 dilution). Values above the bars indicate GMTs, fold reduction, and significant difference in IgG binding titers to RBD protein of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan strain. For both experiments, the serum was tested in duplicates, and in each panel, data are mean from 2 independent experiments. A two-sided Friedman test with Dunn’s multiple comparisons was performed (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: We tested the vaccine-elicited antibodies neutralization capabilities against SARS-CoV-2 VOCs including Beta, Delta, and Omicron BA.1 from sera samples of two-dose vaccinated Pfizer- or Moderna-vaccinated individuals ( ).

Techniques: Infection, Neutralization, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Journal of Virology

Article Title: PLSCR1 suppresses SARS-CoV-2 infection by downregulating cell surface ACE2

doi: 10.1128/jvi.02085-24

Figure Lengend Snippet: PLSCR1 was identified as a crucial restriction factor against SARS-CoV-2 infection by screening ISG-knockout cell lines, and it can be induced by type I interferon. ( A ) 109 ISG-knockout cell lines were infected with SARS-CoV-2 at an MOI = 0.2 for 24 h. Cells were then harvested, and total RNA was extracted. SARS-CoV-2 RNA levels were quantified using quantitative real-time PCR. ( B ) A549-ACE2 cells and HeLa-ACE2 cells were treated with 500 U/mL of recombinant human IFN-β for 0, 2, 6, and 24 h. PLSCR1 expression, STAT1 level, and the phosphorylation of STAT1 were detected by western blot analyses. ( C ) A549-ACE2 cells and HeLa-ACE2 cells were treated as described in panel ( B ). Cells were fixed and stained with indicated antibodies. Scale bar, 10 µm. All experiments were performed at least twice, and one representative is shown.

Article Snippet: The following plasmids were utilized and stored at the Christophe Merieux Laboratory: Myc-PLSCR1, pCMV6-entry, eGFP-C1, ACE2, pLenti-CRISPRv2, pCAGGS-S (SARS-CoV-2 Wuhan, Alpha, Beta, Gamma, Delta, and Omicron), psPAX2, pVSVG, pLenti-GFP, pLenti-GFP/luc, pMDIG, RSV-REV, and pCDH-CMV-MCS-EF1-Puro.

Techniques: Infection, Knock-Out, Real-time Polymerase Chain Reaction, Recombinant, Expressing, Phospho-proteomics, Western Blot, Staining

Journal: Journal of Virology

Article Title: PLSCR1 suppresses SARS-CoV-2 infection by downregulating cell surface ACE2

doi: 10.1128/jvi.02085-24

Figure Lengend Snippet: SARS-CoV-2 infection induces the redistribution of PLSCR1. ( A ) A549-ACE2 and ( B ) HeLa-ACE2 cells were infected with SARS-CoV-2 at an MOI = 0.5. At the indicated time points (0, 2, 6, 18, and 24 h post-infection), the cells were fixed and stained to visualize SARS-CoV-2 N (red), PLSCR1 (green), and nuclei (blue). Scale bar, 10 µm. All experiments were performed at least twice; one representative is shown.

Article Snippet: The following plasmids were utilized and stored at the Christophe Merieux Laboratory: Myc-PLSCR1, pCMV6-entry, eGFP-C1, ACE2, pLenti-CRISPRv2, pCAGGS-S (SARS-CoV-2 Wuhan, Alpha, Beta, Gamma, Delta, and Omicron), psPAX2, pVSVG, pLenti-GFP, pLenti-GFP/luc, pMDIG, RSV-REV, and pCDH-CMV-MCS-EF1-Puro.

Techniques: Infection, Staining

Journal: Journal of Virology

Article Title: PLSCR1 suppresses SARS-CoV-2 infection by downregulating cell surface ACE2

doi: 10.1128/jvi.02085-24

Figure Lengend Snippet: PLSCR1 restricts SARS-CoV-2 infection. ( A ) PLSCR1-KO cells were generated by CRISPR-Cas9 genome editing. The upper panel shows PLSCR1 expression in WT and KO cells. The lower panel displays the sequences of the targeted region. ( B ) A549-ACE2 and A549-ACE2-PLSCR1-KO cells were infected with SARS-CoV-2 at an MOI = 0.5. At the indicated time points (0, 2, 4, 6, 18, and 24 h), cell lysates were collected and analyzed by western blot. ( C ) The cells treated identically to those in panel ( B ) was analyzed by qRT-PCR to measure viral RNA levels. ( D ) A549-ACE2 and A549-ACE2-PLSCR1-KO cells were infected with SARS-CoV-2 at an MOI = 0.5 for 24 h. Supernatants were collected, and the virus titers were determined by TCID 50 assay. ( E-F ) HeLa-ACE2 and 293T-ACE2 cells were transfected with siRNA targeting PLSCR1. After 48 h, the cells were infected with SARS-CoV-2 at an MOI = 0.5. SARS-CoV-2 replication was assessed by western blot at the indicated time points. ( G ) HeLa-ACE2 and HeLa-ACE2-PLSCR1-OE (overexpressing PLSCR1) cells were infected with SARS-CoV-2 at an MOI = 0.5. Whole-cell lysates were collected at 18 and 24 h post-infection and analyzed by western blot. ( H ) Total RNA extracted from cells treated as in panel ( G ) was extracted and analyzed by qRT-PCR to measure viral RNA levels. ( I ) HeLa-ACE2-PLSCR1-KO cells were transfected with either a vector plasmid or a plasmid expressing PLSCR1. After 24 h of transfection, the cells were infected with SARS-CoV-2 at an MOI = 0.5 for an additional 24 h. Cell lysates were collected and analyzed by western blot. All data are mean value ± SD. These experiments were repeated at least twice. P < 0.05 (*), P < 0.01(**) and P < 0.001(***).

Article Snippet: The following plasmids were utilized and stored at the Christophe Merieux Laboratory: Myc-PLSCR1, pCMV6-entry, eGFP-C1, ACE2, pLenti-CRISPRv2, pCAGGS-S (SARS-CoV-2 Wuhan, Alpha, Beta, Gamma, Delta, and Omicron), psPAX2, pVSVG, pLenti-GFP, pLenti-GFP/luc, pMDIG, RSV-REV, and pCDH-CMV-MCS-EF1-Puro.

Techniques: Infection, Generated, CRISPR, Expressing, Western Blot, Quantitative RT-PCR, Virus, Transfection, Plasmid Preparation

Journal: Journal of Virology

Article Title: PLSCR1 suppresses SARS-CoV-2 infection by downregulating cell surface ACE2

doi: 10.1128/jvi.02085-24

Figure Lengend Snippet: PLSCR1 inhibits SARS-CoV-2 spike pseudovirus infectivity. ( A ) The A549-ACE2 and A549-ACE2-PLSCR1-KO cells were infected with SARS-CoV-2/VSV pseudovirus carrying a GFP reporter gene. Infectivity was indicated by GFP expression, and cell nuclei were stained with DAPI (blue). The histogram on the right displays the percentage of GFP-positive cells. Scale bar, 50 µm. ( B ) Similar to panel ( A ), but the pseudovirus carries a luciferase reporter gene. Infectivity in the A549-ACE2 and A549-ACE2-PLSCR1-KO cells was quantified using a luciferase assay. ( C ) This panel is similar to panel ( B ), except PLSCR1 was knockdown (KD) in A549-ACE2 cells. ( D ) HEK293T cells were transfected with plasmids encoding SARS-CoV-2 S and GFP. After 24 h of transfection, the cells were co-cultured with A549-ACE2 or A549-ACE2-PLSCR1-KO cells for 24 h. Syncytia formation (cell fusion) was observed under a microscope. Scale bar, 100 µm. The histogram on the right displays the area of syncytia, which was measured using Image J. ( E ) A549- ACE2 and A549-ACE2-PLSCR1-KO cells were infected with pseudoviruses-bearing SARS-CoV-S protein. ( F ) A549-ACE2 and A549-ACE2-PLSCR1-KO cells were infected with pseudoviruses bearing S proteins from various SARS-CoV-2 variants, including Wuhan, Alpha, Beta, Gamma, Delta, and Omicron. Infectivity was measured using a luciferase assay. ( G ) Similar to panel ( F ), but HeLa-ACE2 and HeLa-ACE2-PLSCR1-OE cells were used. Infectivity of pseudoviruses was again measured by luciferase assay. ( H ) HeLa-ACE2 and HeLa-ACE2-PLSCR1-KO cells were infected with SARS-CoV-2 Omicron strain at an MOI = 0.5. After 0, 6, 18, and 24 h post-infection, whole cell lysates were collected and subjected to western blot analyses using antibodies specific for PLSCR1, SARS-CoV-2 N protein, and β-actin. ( I ) Total RNA extracted from cells treated as described in ( H ) were analyzed by qRT-PCR. All data are mean value ± SD. These experiments were done in triplicates and repeated at least twice. P < 0.05 (*), P < 0.01(**) and P < 0.001(***).

Article Snippet: The following plasmids were utilized and stored at the Christophe Merieux Laboratory: Myc-PLSCR1, pCMV6-entry, eGFP-C1, ACE2, pLenti-CRISPRv2, pCAGGS-S (SARS-CoV-2 Wuhan, Alpha, Beta, Gamma, Delta, and Omicron), psPAX2, pVSVG, pLenti-GFP, pLenti-GFP/luc, pMDIG, RSV-REV, and pCDH-CMV-MCS-EF1-Puro.

Techniques: Infection, Expressing, Staining, Luciferase, Knockdown, Transfection, Cell Culture, Microscopy, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: PLSCR1 suppresses SARS-CoV-2 infection by downregulating cell surface ACE2

doi: 10.1128/jvi.02085-24

Figure Lengend Snippet: CRISPR/Cas9-mediated PLSCR1 knockout promotes SARS-CoV-2 entry. ( A ) A549-ACE2 and A549-ACE2-PLSCR1-KO cells were infected with SARS-CoV-2 at an MOI = 5. Cells were incubated on ice for 1 h to allow virus binding without internalization. Cells were then harvested and subjected to qRT-PCR to measure SARS-CoV-2 N mRNA levels. ( B ) Cells were treated as in panel ( A ) and were then incubated at 37°C for 30 min to allow viral internalization. Cells were harvested and subjected to qRT-PCR analysis. These experiments were done in triplicates and repeated at least two twice. P < 0.05 (*), P < 0.01(**), and P < 0.001(***). ( C ) Western blot analysis ACE2 expression levels in A549-ACE2, A549-ACE2-PLSCR1-KO, and HeLa-ACE2-Vector/PLSCR1-OE cells. ( D ) Co-IP assay was performed in 293T cells co-expressing HA-tagged ACE2 and myc-tagged PLSCR1. ( E ) Co-IP assay was performed in 293T cells co-expression flag-tagged TMPRSS2 and myc-tagged PLSCR1. ( F ) Western blot analysis of CTSL expression in A549-ACE2 and A549-ACE2-PLSCR1-KO cells. ( G ) Co-IP assay performed in 293T cells expressing various forms of SARS-CoV-2 S protein (full-length S, S1, S2 subunits, or RBD) along with myc-tagged PLSCR1. These experiments were repeated at least twice.

Article Snippet: The following plasmids were utilized and stored at the Christophe Merieux Laboratory: Myc-PLSCR1, pCMV6-entry, eGFP-C1, ACE2, pLenti-CRISPRv2, pCAGGS-S (SARS-CoV-2 Wuhan, Alpha, Beta, Gamma, Delta, and Omicron), psPAX2, pVSVG, pLenti-GFP, pLenti-GFP/luc, pMDIG, RSV-REV, and pCDH-CMV-MCS-EF1-Puro.

Techniques: CRISPR, Knock-Out, Infection, Incubation, Virus, Binding Assay, Quantitative RT-PCR, Western Blot, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: Journal of Virology

Article Title: PLSCR1 suppresses SARS-CoV-2 infection by downregulating cell surface ACE2

doi: 10.1128/jvi.02085-24

Figure Lengend Snippet: PLSCR1 inhibits SARS-CoV-2 entry independently of its scramblase activity and interferon signaling. ( A ) HeLa-ACE2-PLSCR1-KO cells were transfected with either an empty plasmid or with a plasmid expressing PLSCR1. After 4 h, cells were treated with different concentrations of R5421. After 24 h of transfection, cells were infected SARS-CoV-2 spike pseudovirus for 48 h. Viral infectivity was measured via a luciferase reporter assay. ( B ) This panel is similar to panel ( A ), except cells were infected with VSV-G pseudovirus. ( C ) HeLa-ACE2-PLSCR1-KO cells were treated with increasing concentrations of R5421 for 48 h. Cell viability was assessed by a CCK8 assay. These experiments were done in triplicates and repeated at least two times.

Article Snippet: The following plasmids were utilized and stored at the Christophe Merieux Laboratory: Myc-PLSCR1, pCMV6-entry, eGFP-C1, ACE2, pLenti-CRISPRv2, pCAGGS-S (SARS-CoV-2 Wuhan, Alpha, Beta, Gamma, Delta, and Omicron), psPAX2, pVSVG, pLenti-GFP, pLenti-GFP/luc, pMDIG, RSV-REV, and pCDH-CMV-MCS-EF1-Puro.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Infection, Luciferase, Reporter Assay, CCK-8 Assay

Journal: Journal of Virology

Article Title: PLSCR1 suppresses SARS-CoV-2 infection by downregulating cell surface ACE2

doi: 10.1128/jvi.02085-24

Figure Lengend Snippet: PLSCR1 inhibits the surface distribution of ACE2 on the plasma membrane. ( A-B ) Flow cytometry (FACS) was used to analyze ACE2 expression in A549-ACE2 and A549-ACE2-PLSCR1-KO cells. Panel ( A ) shows the surface expression of ACE2, whereas panel ( B ) shows the total cellular expression of ACE2. ( C-D ) HeLa-ACE2-PLSCR1-KO cells were transfected with either an empty vector or a plasmid expressing PLSCR1 for 24 h. FACS plots display surface ACE2 expression ( C ) and total ACE2 expression ( D ). ( E ) A549-ACE2 and A549-ACE2-PLSCR1-KO cells were fixed and stained to visualize ACE2 (green) and nuclei (blue). Scale bar, 10 µm. ( F ) HeLa-ACE2-PLSCR1-KO cells were transfected with either an empty vector or a plasmid expressing PLSCR1 for 24 h, then fixed and stained to visualize ACE2 (red), PLSCR1 (green), and nuclei (blue). Scale bar, 10 µm. ( G ) A549-ACE2 and A549-ACE2-PLSCR1-KO cells were subjected to plasma membrane (PM) fractionation. Whole cell lysates and PM fractions were analyzed by western blot. Na + /K + -ATPase was used as a plasma-specific marker. ( H ) HeLa-ACE2-PLSCR1-KO cells were transfected with an empty vector or a plasmid expressing PLSCR1 for 24 h. PMs were isolated and analyzed similarly to panel ( G ). These experiments were repeated at least three times.

Article Snippet: The following plasmids were utilized and stored at the Christophe Merieux Laboratory: Myc-PLSCR1, pCMV6-entry, eGFP-C1, ACE2, pLenti-CRISPRv2, pCAGGS-S (SARS-CoV-2 Wuhan, Alpha, Beta, Gamma, Delta, and Omicron), psPAX2, pVSVG, pLenti-GFP, pLenti-GFP/luc, pMDIG, RSV-REV, and pCDH-CMV-MCS-EF1-Puro.

Techniques: Clinical Proteomics, Membrane, Flow Cytometry, Expressing, Transfection, Plasmid Preparation, Staining, Fractionation, Western Blot, Marker, Isolation

Journal: Journal of Virology

Article Title: PLSCR1 suppresses SARS-CoV-2 infection by downregulating cell surface ACE2

doi: 10.1128/jvi.02085-24

Figure Lengend Snippet: Effect of the absence of PLSCR1 functional site on SARS-CoV-2. ( A-B ) The HeLa-ACE2 cells were transfected with an empty vector, plasmids expressing PLSCR1 of WT or H262Y mutant for 24 h. Cells were then infected with pseudovirus ( A ) for 48 h or SARS-CoV-2 ( B ) for 24 h. Viral infectivity was measured using a luciferase assay ( A ). Total RNA was extracted, and viral RNA levels were measured by qRT-PCR ( B ). ( C ) Empty vector, PLSCR1 wild-type and H262Y mutant were transfected into HeLa-ACE2-PLSCR1-KO cell line for 24 h. Cells were fixed and stained to visualize ACE2 (red), PLSCR1 (green), and nuclei (blue). Scale bar, 10 µm. These experiments were repeated at least three times. ( D ) Schematic diagram illustrating the proposed mechanism by which PLSCR1 antagonizes SARS-CoV-2 infection.

Article Snippet: The following plasmids were utilized and stored at the Christophe Merieux Laboratory: Myc-PLSCR1, pCMV6-entry, eGFP-C1, ACE2, pLenti-CRISPRv2, pCAGGS-S (SARS-CoV-2 Wuhan, Alpha, Beta, Gamma, Delta, and Omicron), psPAX2, pVSVG, pLenti-GFP, pLenti-GFP/luc, pMDIG, RSV-REV, and pCDH-CMV-MCS-EF1-Puro.

Techniques: Functional Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Infection, Luciferase, Quantitative RT-PCR, Staining