Journal: Retrovirology

Article Title: Mouse mammary tumor virus-based vector transduces non-dividing cells, enters the nucleus via a TNPO3-independent pathway and integrates in a less biased fashion than other retroviruses

doi: 10.1186/1742-4690-11-34

Figure Lengend Snippet: MMTV-based vector system. (A) Schematic representation of the MMTV provirus and the four-plasmid expression system used for generating MMTV-based vectors. For the MMTV provirus the regions encoding structural, enzymatic and accessory proteins are shown. The splice donor (SD) and acceptor (SA) sites with their nucleotide coordinates, the long terminal repeat (LTR), the Rem responsive element (RmRE) and the packaging signal are also indicated. In the transfer vector, pRRpCeGFPWPRE25, the truncated gag gene is followed by the Rev responsive element (RRE) from HIV-1 and the internal CMV promoter that drives the expression of enhanced green fluorescent protein (EGFP). Woodchuck hepatitis virus posttranscriptional element (WPRE) upstream of the 3′ LTR is also indicated. In the packaging construct pCMgpRRE17, the restriction sites used for the construction of this chimera, SD, SA, RRE and the beta globin polyadenylation signal (βg pA) are shown. The Rev-encoding plasmid (pRSV-Rev) and the VSV-G envelope protein-encoding plasmid (pHCMV-G) have been previously described [ , ]. (B) and (C) Infectious titers of VSV-G-pseudotyped vectors (transduction units [TU]/ ml) determined by flow cytometric analysis on transduced HeLa cells three days post transduction using un-concentrated (B) or concentrated (C) supernatant from transfected 293 T cells. The mean values from three infection replicates are shown, together with the standard deviations (SD). (D) Time-course analysis of EGFP positivity after single exposure of HeLa cells to the MMTV- or HIV-1-based vectors. The cells were analysed by FACS at various time points (days 3-48) after transduction. Transductions were performed in triplicate. All values are means ± SD. The nucleotide coordinates are based on the prototypic MMTV strain BR6 (GenBank Acc. no.15122).

Article Snippet: To investigate the genomic distribution of the integrated vectors, we mapped the location of integration sites in the transduced HeLa cells [MMTV and MMTV(SIN)] and γ-irradiated 293 cells [MMTV(SIN)arrest] (3 dpt) by a ligation-mediated polymerase chain reaction (LM-PCR) followed by 454-pyrosequencing.

Techniques: Plasmid Preparation, Expressing, Virus, Construct, Transduction, Transfection, Infection

Journal: Retrovirology

Article Title: Mouse mammary tumor virus-based vector transduces non-dividing cells, enters the nucleus via a TNPO3-independent pathway and integrates in a less biased fashion than other retroviruses

doi: 10.1186/1742-4690-11-34

Figure Lengend Snippet: Effect of TNPO3 knockdown on transduction efficiency of MMTV and HIV-1 vectors. (A) Hela cells transfected with the TNPO3-specific or non-silencing siRNA were transduced with HIV-1 and MMTV vectors 48 h after transfection. At 48 h post transduction cells were harvested and the percentage of EGFP-positive cells was determined by flow cytometry. Transduction efficiency in TNPO3-specific siRNA-treated cells relative to control siRNA-treated cells is shown. Results represent mean values ± standard deviation of three independent experiments. (B) The expression levels of TNPO3 at the time of transduction (48 h post transfection) were determined by immunoblotting with an anti-TNPO3-specific antibody. Equivalent loading and blotting was confirmed by membrane re-probing using an anti-actin-specific antibody.

Article Snippet: To investigate the genomic distribution of the integrated vectors, we mapped the location of integration sites in the transduced HeLa cells [MMTV and MMTV(SIN)] and γ-irradiated 293 cells [MMTV(SIN)arrest] (3 dpt) by a ligation-mediated polymerase chain reaction (LM-PCR) followed by 454-pyrosequencing.

Techniques: Knockdown, Transduction, Transfection, Flow Cytometry, Control, Standard Deviation, Expressing, Western Blot, Membrane

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I from Aster koraiensis inhibits SARS-CoV-2 infection. (A) The effects of various plant extracts on the entry of SARS-CoV-2 pseudovirus (pSARS-CoV-2) into ACE2 + and ACE2/TMPRSS2 + H1299 cells. (B, C) pSARS-CoV-2 entry and cell viability assays for the 70% EtOH extract of Aster koraiensis (B) and triterpenoid saponins isolated from A. koraiensis (C) in ACE2 + and ACE2/TMPRSS2 + H1299 cells. The data from pSARS-CoV-2 entry assay were representative of three independent experiments. The error bars indicate the SEM (n > 3). P values were determined by one-way ANOVA followed by Tukey's post hoc test. *P < 0.05; **P < 0.01, ****P < 0.0001.

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Infection, Isolation

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I isolated from Aster koraiensis blocks viral membrane fusion with the host cell membrane. (A) Schematic illustration of the binding of SARS-CoV-2 spike receptor binding domain (RBD) fused to GFP (RBD-GFP) to ACE2 protein overexpressed in H1299 cells. (B) Examination of the effect of AK extract and AI on the interaction between RBD-GFP and ACE2 on the surface of H1299 cells by flow cytometry after treatment with indicated concentration of AK extract and AI. The grey peaks indicate the control experiments without RBD-GFP addition. (C) ACE2 + H1299 cells were treated with 1 or 5 μM AI for 1 h and washed with fresh media (Wash) or maintained (No wash), followed by the addition of pSARS-CoV-2. The data from pSARS-CoV-2 entry assay were representative of two independent experiments. The error bars indicate the SEM (n > 3). P values were determined by the unpaired, two-tailed Student's t-test (NS, not significant). (D) Schematic illustration of the SARS-CoV-2 S protein-mediated cell fusion. Addition of the cell suspension of H1299 cells stably expressing S protein and GFP (Spike-H1299) to a monolayer of ACE2/TMPRSS2 + H1299 cells with mRuby fluorescence (ACE2/TMPRSS2-H1299) leads to cell-cell fusion. (E) Still images at different time points from time-lapse imaging of S-mediated cell fusion. (F) The fusion between Spike-H1299 and ACE2/TMPRSS2 + H1299 was determined by counting the number of cells double-positive for GFP and mRuby by flow cytometry. H1299 cells expressing only GFP (no spike) were used for control experiment. All compounds were used at the concentration of 10 μM. The data were representative of three independent experiments. (G, H) Filipin cholesterol staining of ACE2 + after treatment with DMSO, AI (5 μM), and other indicated compounds (10 μM) for 1 h (G) and quantification of the intensity of membrane filipin staining using Image J software (n = 20 for each group). Error bars in the graphs indicate the SEM. P values were determined by one-way ANOVA followed by Tukey's post hoc test. ****P < 0.0001; NS not significant (H).

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Isolation, Binding Assay, Flow Cytometry, Concentration Assay, Two Tailed Test, Stable Transfection, Expressing, Fluorescence, Imaging, Staining, Software

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I prevents SARS-CoV-2 S-induced syncytia formation. (A) Schematic illustration of Split-GFP assay. Ectopic expression of SARS-CoV-2 spike protein into cultures of the mixture of ACE2/TMPRSS2 + H1299 cells expressing GFP1-10 or GFP11 leads to cell-cell fusion, generating GFP fluorescence. (B) Images of the S-mediated cell-to-cell fusion using Split-GFP. GFP and blue fluorescence indicate the cell-to-cell fusion and DAPI-stained nuclei, respectively. The nuclei are automatically pseudocolored in white and overlapped with GFP fluorescence by CellReporterXpress software. (C) Examination of effect of AI on the S-mediated cell-cell fusion using Split-GFP assay. Representative images of GFP-positive cell-to-cell fusion. The GFP signal and DAPI nuclei staining are automatically pseudocolored in green and red by CellReporterXpress software. (D) Quantitative evaluation for cell-cell fusion using Split-GFP assay. Images of GFP and nuclei stained with DAPI were obtained in five random fields per well. The percentage of fusion cells were calculated by dividing number of nuclei in GFP-positive cells by total number of nuclei. The data were representative of three independent experiments. The error bars indicate the SEM (n > 3). P values were determined by one-way ANOVA followed by Tukey's post hoc test. *P < 0.05; ****P < 0.0001; NS not significant. (E) Protein lysates from co-culture experiments were assessed by Western blot. GAPDH was used as a loading control.

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Split GFP Assay, Expressing, Fluorescence, Staining, Software, Co-Culture Assay, Western Blot

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I equally inhibits the infection of SARS-CoV-2 WT and D614G mutant. (A) pSARS-CoV-2 entry assay in ACE2 + and ACE2/TMPRSS2 + H1299 cells. The data were representative of two independent experiments. The error bars indicate the SEM (n > 3). P values were determined by the unpaired, two-tailed Student's t-test (****P < 0.0001). (B) The effects of AI on the infection of WT and D614G mutant of pSARS-CoV-2 in ACE2 + and ACE2/TMPRSS2 + H1299 cells. The data were representative of three independent experiments. The error bars indicate the SEM (n > 3).

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Infection, Mutagenesis, Two Tailed Test

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I inhibits the infection of SARS-CoV-2 and its variants with a similar efficiency. (A, C) Dose-response curves for AI (A) and chloroquine (C) against ancestral SARS-CoV-2 (A, black circle) and SARS-CoV-2 variants Alpha (B.1.1.7, red square), Beta (B.1.351, orange triangle), Delta (B.1.617.2, pink inverted triangle), and Omicron (B.1.1.529, green diamond) in Vero cells. The blue diamond represents cell viability. The mean ± SEM was calculated from duplicate experiments. (B, D) Confocal images of SARS-CoV-2 N protein (green) and cell nuclei (de Vries et al.) at concentrations near the IC 50 of AI (B) and chloroquine (D) in Vero cells. (E) Dose-response curve for AI against replicable SARS-CoV-2 recombinant viruses expressing Nanoluciferase (Nluc) into A549 cells that overexpress ACE2 and TMPRSS2. The blue diamond represents cell viability.

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Infection, Recombinant, Expressing