106 supt1 cells  (Lonza)

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: (A) Outline of the infection and Nanopore sequencing protocols . SupT1 T cells or primary CD4 + T cells were infected with HIV NL4-3 pseudotyped with VSV-G to enhance viral infection. At 20 or 30 hours post-infection (pi) for HIV NL4-3 -infected SupT1 cells and at 48 h or 72 h pi for HIV NL4-3 -infected primary CD4 + T cells, we collected a fraction of the cell culture. The viability and percentages of HIV-Gag + and CD4 + cells were assessed by flow cytometry. During HIV infection, cells start expressing HIV-Gag protein and progressively lose their membrane expression of CD4 due to expression of the HIV accessory proteins Nef and Vpu, and HIV-Env. The cells were harvested and polyA RNA was purified and sequenced with Nanopore technology. Reads were mapped onto the HIV NL4-3 genome sequence. Created with BioRender. Moris, A. (2025) https://BioRender.com/o40u811 (B) Analysis of the infection rate of HIV NL4-3 -infected CD4 + SupT1 cells. After 20 h or 30 h, cells infected with HIV NL4-3 or mock-treated were labeled with a viability dye and stained with antibodies specific for CD4 and HIV-Gag. Left panels: the percentage viable cells. Middle and right panels: percentages of CD4 - HIV-Gag + and CD4 + HIV-Gag + cells among mock-treated and HIV NL4-3 -infected CD4 + SupT1 cells, respectively. (C) Analysis of the infection rate of HIV NL4-3 -infected primary CD4 + T cells. After 48 h or 72 h, cells from donors 864 and 880 were harvested and stained as in (B). Left panels: the percentage viable cells. Middle and right panels: percentages of CD4 - HIV-Gag + and CD4 + HIV-Gag + cells among mock-treated and HIV NL4-3 -infected primary CD4 + T cells, respectively, from donors 864 (top panels) and 880 (bottom panels). Numbers indicated the percentage of cells within the quadrant. MOCK: cells mock-treated without infection used as a negative control.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Infection, Nanopore Sequencing, Cell Culture, Flow Cytometry, Expressing, Membrane, Purification, Sequencing, Labeling, Staining, Negative Control

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Detection of m 6 A sites in all HIV-1 transcripts. (A) The IGV screenshot shows the methylation rate measured in the cluster 1 region of the HIV-1 genome including sites A8436 to A8632. Line 1, methylation rate measured directly during the m 6 A base-call of SupT1_20h_pi rep1. Line 2, methylation rate measured directly for the in vitro sample in the same region. Line 3, Difference (line 1) - (line 2); red arrows indicate the eight sites detected; negative values are set to zero; sites with rates < 3% were not retained (black arrow). Vertical lines, positions of As in the five DRACH sequences of the region. (B) Methylation profile of all HIV-1 transcripts in the 3’ region (8360–9700). Closed red circles, SupT1_20h_pi rep1; empty red circles, SupT1_30h_pi rep1; closed green circles, SupT1_20h_pi rep2; empty green circles, SupT1_30h_pi rep2; closed black circles, CD4_48h_pi; empty black circles, CD4_72h_pi. Vertical lines, positions of As in the 33 DRACH sequences of the region.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation, In Vitro

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Methylation rates for the 14 m 6 A sites detected in SupT1_20h_pi rep1. (A) Unsupervised heatmap of the methylation rates of HIV-1 transcripts (Materials and Methods). Isoforms are clustered based on the methylation rates of the 14 m 6 A sites. The clusters essentially correspond to US, PS and CS transcripts. (B) Methylation rates of m 6 A sites in the CS, PS and US transcripts of the SupT1_20h_pi rep1 sample.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: m 6 A sites detected in splicing isoforms. (A) SupT1_20h_pi rep 1 sample; (B) SupT1_30h_pi rep1 sample; (C) SupT1_20h_pi rep2 sample; (D) SupT1_30h_pi rep2 sample; (E) CD4_48h_pi sample; (F) CD4_72h_pi sample. Only positions with p -values ≤ 1 x 10 -5 and a number of modified reads ≥ 30 are displayed.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Modification

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Position and methylation rates of the four isoform-specific m 6 A sites. (A) The A5613, A5654 and A5703 sites are common to the Vif and Vpr isoforms; A5887 is common to the Vif, Vpr, Tat and SORF1 isoforms. (B) Methylation rates in SupT1_20h_pi rep1, calculated for the four sites in the CS (-intron 4, Vif1, Vpr1, Vpr2), PS (+ intron 4, Vif2, Vpr3, Vpr4) and US isoforms. (C) Methylation rates calculated for the A5887 site for the CS (Tat1, 2, 3, 4, SORF1), PS (Tat5) and US isoforms.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet:

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Single-molecule analysis of methylated m 6 A sites. (A) Cluster 1. (B) Cluster 2. (C-E) Correlation between the number of methylated sites in cluster 1 and in cluster 2 of the same transcript from the SupT1_20h_pi rep1 sample. The intensity of the color on the heatmaps indicates the number of reads showing the indicated numbers of methylated As in cluster 1 (0 to 8) and in cluster 2 (0 to 3) (Materials and Methods).

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Single-molecule analysis of methylated m 6 A sites. (A) Cluster 1 and (B) cluster 2 of the indicated SupT1 samples. (C) Cluster 1 and (D) cluster 2 of the indicated CD4 samples.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Transcription and methylation of the 2-LTR transcripts. (A) Diagram of the 2-LTR circles and of the transcripts joining the RU5 segment of the 3’ LTR to the U3R segment of the 5’ LTR. Several examples of insertion variants at the U5-U3 junction are shown. (B) Sequence of the 2-LTR transcripts. The successive transcribed regions — R, U5, U3, R — are indicated; m 6 A sites are shown in bold typeface and underlined and are named according to the position of the methylated A in the 2-LTR transcript; in brackets, the homologous position in the HIV-1 reference genome; for example, for 2L125(578), 125 is the position of the m 6 A in the 2-LTR transcript and (578) is the homologous position in the HIV-1 reference genome; * indicates that the sequence context of 2L183 in the 2-LTR transcript (AG A CT, see B) differs from that at the homologous position 9074 in the reference genome (GG A CT). (C) Correspondence table for a position in the 2-LTR transcript and the homologous position in the reference genome. (D) Sites detected in the 2-LTR transcripts of infected SupT1 and CD4 + T-cell samples (Materials and Methods). Large red circles, SupT1_20h_pi rep1; orange circles, SupT1_30h_pi rep1; blue circles, SupT1_20h_pi rep2; green circles, SupT1_30h_pi rep2; yellow circles, CD4_48h_pi; black circles, CD4_72h_pi. Vertical lines indicate the positions of the As in all the DRACH motifs of the transcript.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation, Sequencing, Infection