supt1 cell  (ATCC)

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

Journal: Nature Communications

Article Title: Non-AUG HIV-1 uORF translation elicits specific T cell immune response and regulates viral transcript expression

doi: 10.1038/s41467-025-56772-3

Figure Lengend Snippet: A Schematic representation of the procedure to monitor transcript abundance and translation in HIV-1 infected cells (Created in BioRender. Ricci, E. (2022) BioRender.com/k47z556). Briefly, SupT1 cells were infected or not (Mock) with HIV-1 (NL4.3 strain) at MOI 5. At 0, 1, 12, 24 and 36 hours post infection (hpi), cells were lysed to recover the cytoplasmic fraction and prepare ribosome profiling and RNA-seq libraries subjected to high-throughput sequencing on the Illumina Hiseq platform ( n = 4 independent experiments). Total cell lysates were recovered for mass spectrometry analysis ( n = 4 independent experiments). B Scatter-plot of the fold-change (log2) in cytoplasmic RNA-seq and Ribo-Seq of the Mock-infected and HIV-1 infected cells at each time point of infection. Orange dots (“Transcript-level only”) corresponds to genes exclusively regulated at the transcript abundance level. Blue dots (“Translation only”) correspond to genes which display differences in ribosome occupancy while transcript abundance remains unchanged. Green dots (“Translationally regulated”) correspond to genes with significant changes in transcript abundance and significantly further changes, in the same direction, in ribosome occupancy upon infection. Red dots (“Translationally buffered”) correspond to transcripts displaying significant changes in transcript abundance but for which there is compensation at the translational level to maintain unchanged ribosome occupancy levels upon infection. C Gene ontology analysis of differentially expressed genes at each time point.

Article Snippet: SupT1 cells were obtained from ATCC and cultured in RPMI medium supplemented with 10% fetal calf serum (FCS).

Techniques: Infection, RNA Sequencing Assay, Next-Generation Sequencing, Mass Spectrometry

Journal: Nature Communications

Article Title: Non-AUG HIV-1 uORF translation elicits specific T cell immune response and regulates viral transcript expression

doi: 10.1038/s41467-025-56772-3

Figure Lengend Snippet: A (Left panel) Scheme describing nascent protein labeling using O-propargyl-puromycin (OPP). Briefly, cells are incubated with OPP and fixed in paraformaldehyde before a fluorophore is covalently linked through a click-reaction. Cells are then analyzed by flow cytometry to monitor signal intensity at a single-cell level. B Flow cytometry analysis ( n = 3 independent experiments) of OPP signal in SupT1 cells infected with HIV-1, 12, 24 and 36 hpi. C OPP signal in SupT1 cells infected with single-round recombinant HIV-1 virions coding for GFP at 24, 48, 72 and 96 hpi ( n = 3 independent experiments). D OPP signal in primary Human CD4 + T cells infected with HIV-1, 48 and 72 hpi and either positive or negative for p24 expression (p24+ or p24-). Results from four independent donors are displayed separately for each time point tested ( n = 5 independent experiments). B – D Multiple paired t tests were performed to compare OPP incorporation signals between control and infected samples, * corresponds to a p -value < 0.05. Barplots represent the average value of all biological replicates and error bars correspond to the standard-deviation. Source data are provided as a Source Data file.

Article Snippet: SupT1 cells were obtained from ATCC and cultured in RPMI medium supplemented with 10% fetal calf serum (FCS).

Techniques: Labeling, Incubation, Flow Cytometry, Infection, Recombinant, Expressing, Control, Standard Deviation

Journal: Nature Communications

Article Title: Non-AUG HIV-1 uORF translation elicits specific T cell immune response and regulates viral transcript expression

doi: 10.1038/s41467-025-56772-3

Figure Lengend Snippet: A (Left panel) Relative cytoplasmic amount of viral transcripts (the sum of all transcripts at any given time point corresponding to 100%) in SupT1 cells, 1, 12, 24 and 36 hpi. (Right panel) Overall abundance of viral transcripts within RNA-seq libraries displayed in transcripts per million (TPM) ( n = 4 independent experiments). B Relative (left panel) and overall (right panel) abundance of viral transcripts in U937 cells bearing a latent HIV-1 provirus integrated in their genome, 3, 6, 9, 12, 15, 18 and 24 hours after induction of proviral DNA expression using PMA and ionomycin ( n = 3 independent experiments). Error bars in figures correspond to the Standard Error of the Mean (SEM).

Article Snippet: SupT1 cells were obtained from ATCC and cultured in RPMI medium supplemented with 10% fetal calf serum (FCS).

Techniques: RNA Sequencing Assay, Expressing

Journal: Nature Communications

Article Title: Non-AUG HIV-1 uORF translation elicits specific T cell immune response and regulates viral transcript expression

doi: 10.1038/s41467-025-56772-3

Figure Lengend Snippet: A HIV-1 genomic structure. B Distribution of RNA-seq (red) and ribosome profiling (blue) reads across the HIV-1 genome in SupT1 cells at 1, 12, 24 and 36 hpi ( n = 4 independent experiments). C Translation efficiency of the genomic RNA (across both Gag and Pol coding sequences) at each time point of infection ( n = 4 independent experiments). Boxplots are defined as minima, 1st quartile, median, 3th quartile and maxima. D Translation of incoming viral genomic RNAs as tested by infecting cells pre-incubated or not with cycloheximide or puromycin with a recombinant replication-competent HIV-1 virus bearing a GFP sequence within Gag ( n = 4 independent experiments). Barplots correspond to the mean value of all biological replicates. A one tailed, paired t test was performed to compare samples. Source data are provided as a Source Data file (Partially created in BioRender. Ricci, E. (2022) BioRender.com/k47z556 and using an Illustration from NIAID NIH BIOART Source https://bioart.niaid.nih.gov/bioart/160 ). E Translation efficiency of canonical viral mRNAs, 12, 24 and 36 hpi ( n = 4 independent experiments). Points correspond to the mean value of all biological replicates and error bars correspond to the Standard Error of the Mean (SEM). F Percentage of Gag-Pol ribosome frameshifting at each time point of infection ( n = 4 independent experiments). Boxplots are defined as minima, 1st quartile, median, 3th quartile and maxima.

Article Snippet: SupT1 cells were obtained from ATCC and cultured in RPMI medium supplemented with 10% fetal calf serum (FCS).

Techniques: RNA Sequencing Assay, Infection, Incubation, Recombinant, Virus, Sequencing, One-tailed Test

Journal: Nature Communications

Article Title: Non-AUG HIV-1 uORF translation elicits specific T cell immune response and regulates viral transcript expression

doi: 10.1038/s41467-025-56772-3

Figure Lengend Snippet: A (Top panel) Distribution of ribosome profiling reads across the HIV-1 genome from infected U937 (Green, n = 3 independent experiments), SupT1 (Red, n = 4 independent experiments) and primary CD4 + T lymphocytes (Blue, n = 2 independent experiments). All y axis correspond to reads per million values - RPM. (Bottom panel) Close-up view of the unspliced HIV-1 5’UTR showing the distribution of ribosome profiling reads and the position of putative non-AUG start codons as well as the open-reading frames of putative peptides produced from non-AUG start codons. The y- axis corresponds to reads per million of sequenced reads (RPM). B Distribution of FLOSS (Fragment Length Distribution Score) values for cellular and viral transcripts computed from ribosome profiling reads from SupT1 (left panel), U937 (middle panel) or primary CD4 T cells (right panel).

Article Snippet: SupT1 cells were obtained from ATCC and cultured in RPMI medium supplemented with 10% fetal calf serum (FCS).

Techniques: Infection, Produced

Journal: Nature Communications

Article Title: Non-AUG HIV-1 uORF translation elicits specific T cell immune response and regulates viral transcript expression

doi: 10.1038/s41467-025-56772-3

Figure Lengend Snippet: A Ribosome profiling reads across the 5′UTR of unspliced viral mRNAs in U937 lysates incubated with or without 1 M KCl, Ribolace experiments in SupT1 cells, and RPL7a IPs from HEK293T cells ( n = 2 independent experiments). Reads are expressed as RPM. B Distribution of ribosome profiling reads on luciferase coding reporter transcripts bearing the wild-type HIV-1 5’UTR (WT 5’UTR), a mutant version in which all non-AUG uORF start codons were mutated to UAA stop codons (no uORFs 5’UTR; uORFs with mutated initiation codons are labeled as discontinued lines), a mutant in which all non-AUG uORF start codons were mutated into AUG codons (AUG uORFs 5’UTR; uORFs with mutated initiation codons are labeled as bold lines) ( n = 2 independent experiments). Reads are expressed as RPM. C Relative renilla luciferase activity (normalized against the Globin 5’UTR reporter mRNA) of the different 5’UTR reporter mRNAs upon in vitro translation in the rabbit reticulocyte lysate ( n = 3 independent experiments) ( D ), Western-blot analysis of β-actin (left panel) and DDX3 (right panel) expression in HEK293T cells transfected with plasmids coding for the Cas9 and a sgRNA targeting the GFP sequence (sgGFP) or the DDX3 coding sequence (sgDDX3). ( n = 3 independent experiments with similar results). E Relative luciferase activity of 5′UTR reporters in sgDDX3 or sgGFP-transfected HEK293T cells (left), and fold-change in luciferase activity upon DDX3 knockdown (right) ( n = 3 independent experiments). F Relative renilla luciferase activity (normalized against the Globin 5’UTR reporter mRNA) from viral reporter mRNAs in which the TAR loop is present or absent, transfected into HEK293T cells in which DDX3 expression was knocked-down (sgDDX3) or not (sgYFP) using CRISPR-Cas9 ( n = 3 independent experiments). For panels ( C , E and F ), a two-tailed Student t-test was performed to assess the differences between the mean values of compared conditions. Barplot values correspond to the mean value of all biological replicates and error bars correspond to the Standard Error of the Mean (SEM). Source data are provided as a Source Data file.

Article Snippet: SupT1 cells were obtained from ATCC and cultured in RPMI medium supplemented with 10% fetal calf serum (FCS).

Techniques: Incubation, Luciferase, Mutagenesis, Labeling, Activity Assay, In Vitro, Western Blot, Expressing, Transfection, Sequencing, Knockdown, CRISPR, Two Tailed Test

Journal: bioRxiv

Article Title: CPSF6 Promotes HIV-1 Preintegration Complex Function

doi: 10.1101/2025.01.28.635394

Figure Lengend Snippet: (A) Schematic of human CPSF6 ; the FG motif is encoded by DNA sequences within exon 7. Exon 6, in red, is most usually removed via alternative splicing. (B) Sanger sequencing results of PCR amplicons derived from parental SupT1 cells (upper), transfected cells prior to limited dilution cloning (middle), and the clonally expanded CKI17 cell line. Targeted nucleotides are underlined. (C) Immunoblot of CPSF6 expression levels in parental SupT1 cells versus clonally-expanded CKI7 and CKI19 cell lines.

Article Snippet: Approximately 1×10 6 SupT1 cells were transfected with 10 μL of 15 μM crRNPs and 10 μL of 100 μM ssODN using the nucleofection kit and Nucleofector I device (Lonza), following the manufacturer’s protocol.

Techniques: Alternative Splicing, Sequencing, Derivative Assay, Transfection, Clone Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: CPSF6 Promotes HIV-1 Preintegration Complex Function

doi: 10.1101/2025.01.28.635394

Figure Lengend Snippet: SupT1 cells were compared amongst each other in terms of live and total cell count to assess relative cell viability. Cells (10 6 ) were counted prior to seeding in culture plate for 24 h. Following incubation, cells were once again counted and assessed for total cell count, live cell count (A) and cell viability (B) . Dashed line represents the number of cells seeded prior to seeding. Black patterned bar is representative of an average of 3 total cell counts whereas the white patterned bar is representative of 3 live cell counts. Both values were normalized to 10 6 cells. (B) Shows cell viability (live cell count/total cell count) as a percentage of total cells counted. Error bars are representative of SEM. The p-values (*) represents statistical significance (p< 0.05) compared to WT1. NS represents non-significant difference as compared to WT1.

Article Snippet: Approximately 1×10 6 SupT1 cells were transfected with 10 μL of 15 μM crRNPs and 10 μL of 100 μM ssODN using the nucleofection kit and Nucleofector I device (Lonza), following the manufacturer’s protocol.

Techniques: Cell Counting, Incubation

Journal: bioRxiv

Article Title: CPSF6 Promotes HIV-1 Preintegration Complex Function

doi: 10.1101/2025.01.28.635394

Figure Lengend Snippet: (A) Parental WT SupT1 cells or CKI cells were spinoculated with high titer infectious HIV-1 particles (1500 ng p24/mL) and cultured for 5 h followed by extraction of PIC-containing cytoplasmic extracts. In vitro assays using these PICs as the source of integration activity and quantification of viral DNA integration into an exogenous target DNA by nested PCR were carried out with appropriate controls. (B) In vitro integration assays of PICs extracted from WT1 and from CKI cells were carried out and the copy numbers of integrated viral DNAs were determined. (C) Viral DNA copy numbers were quantified by qPCR. (D) The specific activity of the PIC-mediated integration was determined by calculating the ratio of integrated viral DNA copy numbers (from panel B) to the corresponding PIC-associated viral DNA copy numbers (from panel C). Mean values from two independent experiments, each conducted in triplicate are shown with error bars representing the SEM. White circles present in graphs represent individual data points. The p-values (*) represents statistical significance (p< 0.05) between the control and CKI PICs.

Article Snippet: Approximately 1×10 6 SupT1 cells were transfected with 10 μL of 15 μM crRNPs and 10 μL of 100 μM ssODN using the nucleofection kit and Nucleofector I device (Lonza), following the manufacturer’s protocol.

Techniques: Cell Culture, Extraction, In Vitro, Activity Assay, Nested PCR, Control

Journal: bioRxiv

Article Title: CPSF6 Promotes HIV-1 Preintegration Complex Function

doi: 10.1101/2025.01.28.635394

Figure Lengend Snippet: WT cell lines WT1 (A-D) , WT2 (E-H) and WT3 (I-L) and CKI SupT1 cell lines CKI7 (A-B, E-F, and I-J) , and CKI19 (C-D, G-H, and K-L) were spinoculated with 450 ng p24 of pseudotyped HIV-1.Luc reporter particles. The total infection timecourse was 24 h, and luciferase activity was measured in the cellular lysates as an indicator of infectivity (Panels-A, C, E, G, I, and K). Infectivity data were also plotted as percent infectivity relative to respective control clones (Panels-B, D, F, H, J, and L). Data shown are mean values from three independent experiments, each conducted in triplicates. (M) Infectivity values sorted by editing treatment (CPSF6 non-edited vs edited). Colored circles individual data point from corresponding cell line. (N) Displays aggregate infectivity as a percent relative to WT. Error bars represent the standard error of the mean (SEM) and the p-values (*) represents statistical significance (p< 0.05) between the control and CKI cells.

Article Snippet: Approximately 1×10 6 SupT1 cells were transfected with 10 μL of 15 μM crRNPs and 10 μL of 100 μM ssODN using the nucleofection kit and Nucleofector I device (Lonza), following the manufacturer’s protocol.

Techniques: Infection, Luciferase, Activity Assay, Control, Clone Assay