Journal: Cells

Article Title: Further Characterization of the Antiviral Transmembrane Protein MARCH8

doi: 10.3390/cells13080698

Figure Lengend Snippet: MARCH8 cannot impede cell-to-cell HIV-1 infection from MDMs to CD4-positive T cells. ( A ) Schematic representation of the cell-free infectivity assays. Lentiviral CRISPR-mediated knockout of MARCH8 expression was performed in monocyte-derived macrophages (MDMs) obtained from two donors. Control and MARCH8-depleted MDMs were infected with HIV-1 Env-intact firefly luciferase (fLuc)-reporter viruses, washed, and cultured in fresh media. Infection of MDMs was performed using VSV-G-pseudotyped luc-reporter viruses that carry intact CXCR4-tropic HIV-1 Env, enabling the collection of viruses produced during single-round replication from MDMs without reinfection. Progeny viruses from control and MARCH8-depleted MDMs were normalized for p24 antigen and used to infect MAGIC cells, which were subjected to luc assays. ( B ) Cell-free infectivity of virions produced from control (CRISPR-Ctrl) and MARCH8-depleted (CRISPR-MARCH8) MDMs obtained from two donors (Donor #1 and Donor #2). Data are shown as the fold increase in viral infectivity relative to that of viruses produced from CRISPR-Ctrl MDMs (mean ± S.D. from three independent experiments). * p < 0.005, ** p < 0.001 compared with the CRISPR-Ctrl using two-tailed unpaired t -tests. ns, not significant. ( C ) Schematic representation of the cell-to-cell infectivity assays. Transduction with lentiviral CRISPR and HIV-1 infection were similarly conducted as in ( A ). After infection, MDMs were washed, incubated, and cocultured with MT4 cells that were transduced with renilla luciferase (rLuc)-expressing lentiviruses. MT4 cells were washed, cultured in the presence of nelfinavir (NFV) to block multiple replications, and subjected to dual luc assays. ( D ) Cell-to-cell infectivity in MT4 cells cocultured with CRISPR-Ctrl and CRISPR-MARCH8 MDMs obtained from Donor #1 and Donor #2. Data are shown as the ratio of fLuc/rLuc activity (mean ± S.D. from three independent experiments). The p value was calculated using a two-tailed unpaired Student’s t -test. ns, not significant.

Article Snippet: The lentiviral renilla luciferase expression plasmid pWPI-hRL-neoR was generated by inserting the NheI/SpeI fragment of phRL-TK (Promega, E6241) into SpeI-digested pWPI-MCS-neoR.

Techniques: Infection, CRISPR, Knock-Out, Expressing, Derivative Assay, Luciferase, Cell Culture, Produced, Two Tailed Test, Transduction, Incubation, Blocking Assay, Activity Assay

Journal: PLoS Pathogens

Article Title: HTLV-1 Tax-Mediated Inhibition of FOXO3a Activity Is Critical for the Persistence of Terminally Differentiated CD4 + T Cells

doi: 10.1371/journal.ppat.1004575

Figure Lengend Snippet: Briefly, activated T cells were transduced by lentiviral particles (LVP) expressing or not Tax. (A) Tax expression at 48 h on transduced T cells in response to LVP Tax concentrations (5–160 ng/10 6 cells; n = 3). (B) At 24 and 48 h post-transduction, total RNA was extracted, and subjected to Biomark analyses. Heatmaps of genes significantly modulated following Tax expression. (C) Table showing fold changes and P -values for several modulated FOXO3a targets genes on Tax-transduced T cells (n = 5; paired t test).

Article Snippet: The lentiviral vector pWPI (empty vector), packaging plasmid psPAX2 and envelope plasmid pMD2G were generously provided by VGTI-Florida, whereas pCLXSN-Tax vector was purchased from Addgene (ref: 44038; MA, USA).

Techniques: Expressing, Transduction

Journal: PLoS Pathogens

Article Title: HTLV-1 Tax-Mediated Inhibition of FOXO3a Activity Is Critical for the Persistence of Terminally Differentiated CD4 + T Cells

doi: 10.1371/journal.ppat.1004575

Figure Lengend Snippet: Briefly, activated T cells were transduced by lentiviral particles (LVP) expressing or not Tax for 2–28 days. (A) FOXO3a signaling on transduced T cells expressing or not Tax at 48 h determined by immunoblotting (n = 3). (B) Densitometric quantification of specific bands was performed using ImageJ software. Results shown represent the mean relative expression ± SD of 3 independent experiments. (C–E) Persistence and stepwise differentiation of activated CD4 + T cells transduced with LVP expressing Tax in the presence or absence of AKT i (n = 5). (C) Absolute numbers of total viable CD3 + T cells were determined by trypan blue exclusion. Results are expressed in log 2 scale. P values were determined based on the comparison with LVP empty -transduced cells. The underlined numbers represent the half-life of cultured LVP empty (black) and LVP Tax +AKT i (green) conditions. (D) Differentiation status of transduced T cells subsets at 7–28 dpt. Pie charts are representative of raw data from five independent experiments. (E) Correlation between the absolute numbers of viable transduced T cells at 28 days and the levels of expression of FOXO3a-related proteins at 48 h are also shown (n = 9; Spearman test).

Article Snippet: The lentiviral vector pWPI (empty vector), packaging plasmid psPAX2 and envelope plasmid pMD2G were generously provided by VGTI-Florida, whereas pCLXSN-Tax vector was purchased from Addgene (ref: 44038; MA, USA).

Techniques: Expressing, Western Blot, Software, Transduction, Cell Culture

Journal: Cells

Article Title: NSP4 and ORF9b of SARS-CoV-2 Induce Pro-Inflammatory Mitochondrial DNA Release in Inner Membrane-Derived Vesicles

doi: 10.3390/cells11192969

Figure Lengend Snippet: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins NSP4 and ORF9b synergistically induce the extracellular release of mitochondrial DNA (mtDNA). ( a ) Heatmap showing the integrated density of mtDNA associated with the mitochondria in A549 cells transfected with 29 SARS-CoV-2 proteins. A549 cells were individually transfected with the constructs encoding the proteins and probed with the anti-TFAM antibodies. Images were acquired at 24 h post-transfection and subjected to densitometric analysis. The combination of actinomycin D, ABT-737, and zVD (ActD + ABT + zVD) (treatment for 2 h) was used as a positive control for the mtDNA release experiments. ( b ) Quantitative analysis of images of the indicated groups probed with the anti-TFAM (representing mtDNA inside mitochondria) antibodies (n = 9). A549 cells were transduced (48 h) with lentiviral-encoded shortlisted SARS-CoV-2 protein plasmids (as shown in ( a )) and probed with the anti-TFAM antibodies. Images were subjected to densitometric analysis. Corresponding representative images are shown in . Data are plotted as integrated density. ( c ) Quantification of Cyt c levels in the mitochondria. A549 cells transduced with the selected plasmids were subsequently probed with the anti-Cyt c antibodies and imaged after 48 h. Images were subjected to densitometric analysis (n = 9). Data are plotted as integrated density. ( d , e ) Representative flow cytometry histograms of cells stained with tetramethylrhodamine, ethyl ester (TMRE) (representing ΔΨm), and mitoSOX (representing mitochondrial reactive oxygen species (mtROS)) under the conditions mentioned in ( b , c ). ( f ) Heatmap of mtDNA and Cyt c release in A549 cells transduced with the selected protein-encoding constructs, which upregulated mtROS and downregulated ΔΨm. The values were calculated from the analysis data shown in ( b – e ). ( g ) Quantification of mtDNA release (n = 9) from cells co-transduced with various plasmids in the images. The combination of NSP4 and ORF9b robustly promoted the release of mtDNA. Data are plotted as integrated density. ( h ) Representative confocal images showing the localization of mtDNA (probed with anti-TFAM antibodies; magenta) with the mitochondria (probed with the anti-TOM20 antibodies; green) in cells transduced with the combination of NSP4 and ORF9b (N4 + 9b). Insets show the association of mtDNA with the mitochondria in vector (VEC)-transduced cells ( blue arrowheads ). In infected cells, mtDNA is mostly localized outside the mitochondria ( yellow arrowheads ) or the mitochondria are devoid of mtDNA ( red arrowheads ). ( i ) Representative immunofluorescence images showing Cyt c (red) localization with the mitochondria (probed with the anti-TOM20 antibodies; green) in cells transduced with VEC or N4 + 9b. ( j ) Quantitative analysis of images shown in ( i ) (n = 10). Data are plotted as integrated density. ( k ) Immunoblot of Cyto c in the cytosolic extract prepared from cells after transduction with N4+9b or VEC. GAPDH was used as a loading control. ( l ) Representative immunoblot showing the expression of TFAM in the cytosol. GAPDH served as the reference control. The cytosolic extract was prepared from cells transduced with VEC or N4 + 9b (48 h). The combination of ActD + ABT + zVD (treatment for 2 h) was used as the positive control for the experiments evaluating mtDNA release. ( m ) Representative immunoblots of concentrated cell supernatant from the cells treated as indicated in ( k ). E-Cadherin (E-Cad) was used as the reference control. ( n ) Densitometry analysis of proteins in the immunoblots shown in ( k , l ) (n = 4). Data are plotted as the ratio of TFAM and the reference control. ( o ) Representative immunoblot showing TFAM (pulled down using the anti-TFAM antibodies; mtDNA marker) expression in the immunoprecipitated samples. Chromatin immunoprecipitation (ChIP) was performed using the concentrated cell supernatant (Sup) with three different volumes as the starting materials (2, 4, and 6 mL). IgG was used as the isotype control and tested only at a higher concentration (6 mL). ( p ) Quantification of mtDNA copy number (calculated using polymerase chain reaction and represented as copies/mL). mtDNA was extracted from various supernatant volumes and subjected to ChIP with the anti-TFAM antibodies (n = 6). ( q ) Quantitative analysis of IL1B expression in normal human bronchial epithelial (NHBE) cells incubated with various mtDNA concentrations (24 h). Total RNA was extracted from the cells and subjected to quantitative real-time PCR (n = 8). ( r ) Quantitative analysis of IL-1β levels in the total protein samples extracted from NHBE cells, which were treated as described in ( p ). ( s , t ) Representative flow cytometry histogram plots showing NHBE cell death (determined using Sytox dye) induced by different mtDNA concentrations (n = 6). Data are plotted as percentage of cell death. All data are represented as mean ± standard error of mean from three independent experiments. Statistical analyses (one-way analysis of variance ( b , c , h , n , p ) and unpaired t -tests ( j , q , r , t )) were performed using Graphpad Prism software. * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant. Scale bar: 10 μm.

Article Snippet: A549 cells were transduced with lentiviral particles derived from the ACE2/TMPRSS2-expressing vector (Addgene #154987), and cells stably expressing ACE2/TMPRSS2 were selected using puromycin selection.

Techniques: Transfection, Construct, Positive Control, Transduction, Flow Cytometry, Staining, Plasmid Preparation, Infection, Immunofluorescence, Western Blot, Control, Expressing, Marker, Immunoprecipitation, Chromatin Immunoprecipitation, Concentration Assay, Polymerase Chain Reaction, Incubation, Real-time Polymerase Chain Reaction, Software