rhifn γ  (R&D Systems)

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) Schematic of MPRAs tiling through HIV genomes, saturation mutagenesis, and evaluation of different isolates. (B) Saturation mutagenesis MPRA experiments in unstimulated Jurkat cells for four tiles in the HIV-1 LTR from the clade B REJO strain that show transcriptional activity. Region coordinates are provided using standardized HXB2 genomic coordinates. TF motifs that contribute to activity are outlined. Inset shows MPRA activity tiling through the HIV-1 LTR. Blue indicates activity of tiles in the sense, and red in the antisense (opposite orientation) strand. (C) CASCADE-derived motifs for different cofactors at various regions of the HIV-1 LTR. (D) Regional distribution of HIV-1 isolates tested by MPRAs. (E) Violin plots showing the distribution of activity in unstimulated Jurkat cells across 5,569 isolates for different regions of the HIV-1 LTR across clades. The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. The dots heatmaps below indicate the fold activation by stimulation of Jurkat cells with αCD3+PMA, TNFα, or IFNγ. The color indicates the median fold activation across isolates, whereas the size of the dots reflects the fold activity differences between the 5th and 95th percentiles. (F) Pearson correlation between the activity of different regions of the HIV-1 LTR across isolates. (G) Schematic of chimeric proviral constructs and reactivation experimental approach. (H-I) Fold change in GFP+ cells relative to DMSO control (H) or geometric mean fluorescence intensity of GFP+ cells (I) in Jurkat cells infected with chimeric proviruses carrying the U3 region of the indicated isolates from clades A, B, and C, and stimulated with TNFα for 2 days. (J) Comparison between fold activation of MPRA data from tile HXB2:250-431 in Jurkat cells activated with TNFα, and provirus reactivation by TNFα measured as fold change in GFP+ cells relative to DMSO control or geometric mean fluorescence intensity of GFP+ cells. Pearson correlation coefficients and one-tailed p-values are indicated for each comparison to MPRA data. (K) Dot heatmaps of recombinant HIV-1 viruses derived from clades B and C. Median baseline activity in tile HXB2:250-431 is shown in shades of green, whereas the median fold activation by αCD3+PMA, TNFα, or IFNγ is shown blue-white-red gradient. The size for the dots reflects the fold activity differences between the 5th and 95th percentiles.

Article Snippet: Cells were then left unstimulated or were stimulated with 20 ng/mL of IFNγ (Invivogen cat# rcyec-hifng) for 6 hours, 20 ng/mL of TNFα (Invivogen cat# rcyc-htnfa) for 1 hour, or 10 ng/mL Phorbol 12-myristate 13-acetate (Millipore Sigma, Cat# P8139-1MG) and 1ug/mL Anti-CD3 (Fisher Scientific, Cat# 16-0037-8 5 ) for 1 hour.

Techniques: Mutagenesis, Activity Assay, Derivative Assay, Activation Assay, Construct, Control, Fluorescence, Infection, Comparison, One-tailed Test, Recombinant

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) Distribution of the number of TF binding sites across HIV-1 isolates in tiles HXB2:250-431 and HXB2:581-780. Each pie chart shows the proportion of isolates with different number of binding sites for the indicated TFs. (B-C) Violin plots of baseline activity or fold activation by αCD3+PMA or TNFα for tile HXB2:250-431 across HIV-1 isolates based on the number of NF-κB (B) or SP/KLF sites (C). The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. (D, H) Activity distribution in Jurkat cells across HIV-1 isolates with different TF configurations in tiles HXB2:250-431 (D) and HXB2:581-780 (H). The left boxes represent the TF configurations based on aligned TF positions. The distribution of isolates from different clades across TF configurations is shown as pie chats. Violin plots indicate the distributions of activity in unstimulated Jurkat cells (baseline), and cells stimulated with αCD3+PMA, TNFα, or IFNγ. In the case tile HXB2:250-431 only configurations with at least 10 isolates are shown. (E) Alphafold3 model of the HIV-1 REJO LTR including three SP1 and two sets of NF-κB (p65 and p50) proteins. (F-G) Violin plots of baseline activity or fold activation by IFNγ for tile HXB2:581-780 across HIV-1 isolates based on the number of IRF (F) or SP/KLF sites (G). The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. (I) Violin plots of F-statistic showing the variability in activity for each isolate for tile HXB2:581-780 across four donors in CD4+ T cells. The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. (J) Activity distribution and donor variability (F-statistic) in CD4+ T cells in Jurkat cells across HIV-1 isolates with different TF configurations in tile HXB2:581-780. The left boxes represent the TF configurations based on aligned TF positions. The distribution of isolates from different clades across TF configurations is shown as pie charts. (K) Distribution of donor variability (F-statistic) in tile HXB2:581-780 for isolates that contain or lack an SP/KLF site across four CD4+T cell donors or five replicates of Jurkat cells. Statistical significance determined by two-tailed Brunner-Munzel test.

Article Snippet: Cells were then left unstimulated or were stimulated with 20 ng/mL of IFNγ (Invivogen cat# rcyec-hifng) for 6 hours, 20 ng/mL of TNFα (Invivogen cat# rcyc-htnfa) for 1 hour, or 10 ng/mL Phorbol 12-myristate 13-acetate (Millipore Sigma, Cat# P8139-1MG) and 1ug/mL Anti-CD3 (Fisher Scientific, Cat# 16-0037-8 5 ) for 1 hour.

Techniques: Binding Assay, Activity Assay, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) MPRA activity in Jurkat cells tiling through the LTRs of HIV-1 and HIV-2. (B, G) Saturation mutagenesis MPRA experiments in unstimulated Jurkat cells for tiles in the LTR (B), gag/env (G) regions of HIV-2 ROD strain that show transcriptional activity. Region coordinates are provided using standardized SIVmac239 genomic coordinates. TF motifs that contribute to activity are outlined. (C) Saturation mutagenesis MPRA experiments in unstimulated and stimulated Jurkat cells for tile SIVmac239:379-571 in the HIV-2 LTR. (D) Violin plots showing the distribution of LTR activity in unstimulated Jurkat cells across 41 HIV-2 isolates with full-length sequences in NCBI. The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. The dots heatmaps below indicate the fold activation by stimulation of Jurkat cells with αCD3+PMA, TNFα, or IFNγ. The color indicates the median fold activation across isolates, whereas the size of the dots reflects the fold activity differences between the 5th and 95th percentiles. (E) CASCADE-derived motifs for different cofactors in the LTR and gag regions of HIV-2 ROD strain. (F) MPRA activity map across the genome of HIV-2 ROD strain in unstimulated Jurkat cells. The genome organization is shown below. (H-I) Los Alamos National Laboratory (LANL) genome alignments of HIV-2 strains using the SIVmac239 genome as a reference. (H) Transcriptional activity predicted using CREST is shown in shades of green. (I) Transcription start sites predicted using Puffin trained on FANTOM CAGE data are shown in shades of red.

Article Snippet: Cells were then left unstimulated or were stimulated with 20 ng/mL of IFNγ (Invivogen cat# rcyec-hifng) for 6 hours, 20 ng/mL of TNFα (Invivogen cat# rcyc-htnfa) for 1 hour, or 10 ng/mL Phorbol 12-myristate 13-acetate (Millipore Sigma, Cat# P8139-1MG) and 1ug/mL Anti-CD3 (Fisher Scientific, Cat# 16-0037-8 5 ) for 1 hour.

Techniques: Activity Assay, Mutagenesis, Activation Assay, Derivative Assay