Journal: Microbiology Spectrum

Article Title: Development and characterization of a double-fluorescent HIV-1 reporter cellular model to tackle the Rev-dependent export pathway

doi: 10.1128/spectrum.01903-24

Figure Lengend Snippet: Construction and validation of pHIV-dual vector. ( A ) The mko2 gene was inserted into the intronic region of the pHIV_Intro construct at the SpeI and BstBI restriction sites, positioned just after the truncated gag gene. In the absence of Rev, the unspliced HIV-1 RNA undergoes splicing, resulting in a short, spliced transcript that contains the ecfp gene, leading to the expression of the ECFPskl protein. When Rev is present, it binds to the RRE sequence located within the intronic sequence, facilitating the export of the unspliced transcript from the nucleus and resulting in the expression of the mKO2 protein. ( B ) U2OS cells were transfected with the viral vectors and pTat-101, both in the absence and presence of the pRev-GFP vector. Twenty-four hours post-transfection, total RNA was isolated, and RT-qPCR was conducted to measure levels of unspliced, spliced, and TAR-containing total RNAs (primers are listed in ). Values were normalized using GAPDH mRNA primers and are presented as fold changes relative to the samples without Rev, which were arbitrarily set to a value of 1. Results are displayed as mean values ± standard error of the mean. Statistical analysis was performed using the t -test with Welch correction from three biological replicates ( N = 3), with two technical replicates each. Significance is indicated as follows: ns, not significant and ** P -value < 0.05. ( C ) Immunoblotting was performed to detect mKO2 and ECFP levels in untransfected (mock) cells and cells transfected with the pHIV_dual vector, using two doses of the Tat-expressing plasmid, with or without the plasmid encoding Rev. GAPDH was used as a loading control.

Article Snippet: Immunoblots were performed as described before ( ) with the following antibodies: mKO2 (Medical and Biological Laboratories, PM051M, 1:500), GFP (St. John’s Laboratory, STJ140006, 1:1,000), MATR3 (Sigma-Aldrich, MABN1587, 1:1,000), CRNKL1 (NovoPro Bioscience, #175347-50, 1:1,000), GAPDH (Cell Signaling Technology, 14C10, 1:4,000), anti-rabbit-HRP (Sigma-Aldrich, A0545, 1:20,000), and anti-mouse-HRP (Dako, P0447, 1:20,000).

Techniques: Biomarker Discovery, Plasmid Preparation, Construct, Expressing, Sequencing, Transfection, Isolation, Quantitative RT-PCR, Western Blot, Control

Journal: Microbiology Spectrum

Article Title: Development and characterization of a double-fluorescent HIV-1 reporter cellular model to tackle the Rev-dependent export pathway

doi: 10.1128/spectrum.01903-24

Figure Lengend Snippet: Generation and validation of U2OS HIV-dual clone cell lines. ( A ) Schematic representation of screening protocol by fluorescent-activated cell sorting, fluorescent microscopy, and flow cytometry. See text for details. ( B and C ) Clonal cell lines isolated using the procedure described above were analyzed by flow cytometry to assess the ( B ) percentages and ( C ) mean fluorescent intensities of mKO2 + cells transfected with pTat101 in the absence and presence of pRev-GFP. # indicates clones that were selected based on the following arbitrarily chosen criteria: ≥10% of mKO2 + cells and ≥5,000 MFI of mKO2 + cells. ( D ) Western blotting analysis of 2D5, 4F4, 4G10, 6A12, and 6B12 clonal cell lines to detect the levels of mKO2, Rev-GFP, and ECFP in pTat101-transfected cells in the absence and presence of pRev-GFP. GAPDH is the protein loading control.

Article Snippet: Immunoblots were performed as described before ( ) with the following antibodies: mKO2 (Medical and Biological Laboratories, PM051M, 1:500), GFP (St. John’s Laboratory, STJ140006, 1:1,000), MATR3 (Sigma-Aldrich, MABN1587, 1:1,000), CRNKL1 (NovoPro Bioscience, #175347-50, 1:1,000), GAPDH (Cell Signaling Technology, 14C10, 1:4,000), anti-rabbit-HRP (Sigma-Aldrich, A0545, 1:20,000), and anti-mouse-HRP (Dako, P0447, 1:20,000).

Techniques: Biomarker Discovery, FACS, Microscopy, Flow Cytometry, Isolation, Transfection, Clone Assay, Western Blot, Control

Journal: Microbiology Spectrum

Article Title: Development and characterization of a double-fluorescent HIV-1 reporter cellular model to tackle the Rev-dependent export pathway

doi: 10.1128/spectrum.01903-24

Figure Lengend Snippet: Flow cytometry analysis of 4G10 and 6A12 clones. ( A ) Representative dot plots showing flow cytometry analysis on total cells of clones either untransfected (left panels), transfected with pTat101 alone (middle panels), or in the presence of Rev-GFP expressing vector (right panels). ( B ) Quantitative analysis of mKO2 median fluorescence intensity from ECFP + cells is shown as mean values ± SD from two independent biological repetitions performed in two technical repetitions. Statistical analysis was performed using t -test with Welch correction, *** P -value < 0.001.

Article Snippet: Immunoblots were performed as described before ( ) with the following antibodies: mKO2 (Medical and Biological Laboratories, PM051M, 1:500), GFP (St. John’s Laboratory, STJ140006, 1:1,000), MATR3 (Sigma-Aldrich, MABN1587, 1:1,000), CRNKL1 (NovoPro Bioscience, #175347-50, 1:1,000), GAPDH (Cell Signaling Technology, 14C10, 1:4,000), anti-rabbit-HRP (Sigma-Aldrich, A0545, 1:20,000), and anti-mouse-HRP (Dako, P0447, 1:20,000).

Techniques: Flow Cytometry, Clone Assay, Transfection, Expressing, Plasmid Preparation, Fluorescence

Journal: Microbiology Spectrum

Article Title: Development and characterization of a double-fluorescent HIV-1 reporter cellular model to tackle the Rev-dependent export pathway

doi: 10.1128/spectrum.01903-24

Figure Lengend Snippet: Leptomycin B treatment of 4G10 and 6A12 clones results in decreased Rev efficiency. ( A ) Representative flow cytometry plots showing mKO2 vs ECFP fluorescence of mock-treated (left panels) or LMB-treated (right panels) U2OS_dual clones co-transfected with Tat and Rev-GFP plasmids. ( B ) Western blot analysis of the Rev-GFP, mKO2, and ECFP protein levels in mock-treated or LMB-treated cell lines. GAPDH is a loading control. ( C ) Evaluation of Rev efficiency (% mKO2 + high ECFP + )/(% total ECFP + ) × 100. Data represent averages of five biological repetitions, and error bars indicate the standard error of the mean. Statistics are performed using t -test with Welch correction, **** P -value < 0.000001.

Article Snippet: Immunoblots were performed as described before ( ) with the following antibodies: mKO2 (Medical and Biological Laboratories, PM051M, 1:500), GFP (St. John’s Laboratory, STJ140006, 1:1,000), MATR3 (Sigma-Aldrich, MABN1587, 1:1,000), CRNKL1 (NovoPro Bioscience, #175347-50, 1:1,000), GAPDH (Cell Signaling Technology, 14C10, 1:4,000), anti-rabbit-HRP (Sigma-Aldrich, A0545, 1:20,000), and anti-mouse-HRP (Dako, P0447, 1:20,000).

Techniques: Clone Assay, Flow Cytometry, Fluorescence, Transfection, Western Blot, Control

Journal: Microbiology Spectrum

Article Title: Development and characterization of a double-fluorescent HIV-1 reporter cellular model to tackle the Rev-dependent export pathway

doi: 10.1128/spectrum.01903-24

Figure Lengend Snippet: Depletion of MATR3 decreases Rev activity. Clones 4G10 and 6A12 were transduced with lentiviral vectors containing shRNA against luciferase (shLuc, control) or MATR3 (shMATR3). After 3 days of puromycin (1 ng/µL) selection, cells were co-transfected with pTat-101 and pRev-GFP plasmids. Twenty-four hours after transfection, cells were collected for western blotting ( A ) and flow cytometry ( B–D ) analysis. ( A ) Western blot analysis to detect MATR3, mKO2, Rev-GFP, and ECFP protein levels in shLuc- and shMATR3-transduced cells. GAPDH is a loading control. ( B ) Representative flow cytometry plots showing mKO2 vs ECFP fluorescence of control shLuc-transduced (left panels) or shMATR3-transduced (right panels) U2OS_dual clones. The black dotted line indicates the median mKO2 fluorescence from shLuc cells within the double-positive population, distinguishing two subpopulations: mKO2 high and mKO2 low . ( C ) Comparison of mKO2 fluorescence histograms from double-positive population shown in panel B. The gray dotted line indicates the median fluorescence intensity of mKO2. ( D ) Evaluation of Rev efficiency (% mKO2 + high ECFP + )/(%total ECFP + ) × 100. Data represent averages of three biological repetitions ( N = 3), and error bars indicate the standard error of the mean. Statistics are performed using t -test with Welch correction, **** P -value < 0.0001.

Article Snippet: Immunoblots were performed as described before ( ) with the following antibodies: mKO2 (Medical and Biological Laboratories, PM051M, 1:500), GFP (St. John’s Laboratory, STJ140006, 1:1,000), MATR3 (Sigma-Aldrich, MABN1587, 1:1,000), CRNKL1 (NovoPro Bioscience, #175347-50, 1:1,000), GAPDH (Cell Signaling Technology, 14C10, 1:4,000), anti-rabbit-HRP (Sigma-Aldrich, A0545, 1:20,000), and anti-mouse-HRP (Dako, P0447, 1:20,000).

Techniques: Activity Assay, Clone Assay, Transduction, shRNA, Luciferase, Control, Selection, Transfection, Western Blot, Flow Cytometry, Fluorescence, Comparison

Journal: Microbiology Spectrum

Article Title: Development and characterization of a double-fluorescent HIV-1 reporter cellular model to tackle the Rev-dependent export pathway

doi: 10.1128/spectrum.01903-24

Figure Lengend Snippet: Depletion of CRNKL1 enhances Rev activity. Clones 4G10 and 6A12 were transduced with a lentiviral vector encoding Tat-IRES-Rev transgene. After 3 days of blasticidin (10 µg/µL) selection, cells were transfected with siSCR (scramble control siRNA) or siCRNKL1. At 24 h after siRNA transfections, cells were collected for ( A–C ) flow cytometry and ( D ) western blotting. ( A ) Western blot analysis to detect CRNKL1 and mKO2 protein levels in siSCR- and siCRNKL1-transfected cells. GAPDH is a loading control. ( B ) Representative flow cytometry plots showing mKO2 vs ECFP fluorescence of control (left panels) or CRNKL1-depleted (right panels) U2OS_dual clones. The black dotted line indicates the median mKO2 fluorescence from siSCR-treated cells within the double-positive population, distinguishing two subpopulations: mKO2 high and mKO2 low . ( C ) Comparison of mKO2 fluorescence histograms from double-positive population shown in panel B . The gray dotted line indicates the median fluorescence intensity of mKO2. ( D ) Evaluation of Rev efficiency (% mKO2 + high ECFP + )/(%total ECFP + ) × 100. Data represent averages of three biological repetitions, and error bars indicate the standard error of the mean. Statistics are performed using t -test with Welch correction, ** P -value < 0.01 and **** P -value < 0.0001.

Article Snippet: Immunoblots were performed as described before ( ) with the following antibodies: mKO2 (Medical and Biological Laboratories, PM051M, 1:500), GFP (St. John’s Laboratory, STJ140006, 1:1,000), MATR3 (Sigma-Aldrich, MABN1587, 1:1,000), CRNKL1 (NovoPro Bioscience, #175347-50, 1:1,000), GAPDH (Cell Signaling Technology, 14C10, 1:4,000), anti-rabbit-HRP (Sigma-Aldrich, A0545, 1:20,000), and anti-mouse-HRP (Dako, P0447, 1:20,000).

Techniques: Activity Assay, Clone Assay, Transduction, Plasmid Preparation, Selection, Transfection, Control, Flow Cytometry, Western Blot, Fluorescence, Comparison