Figures and data
Single Cell Transcriptomic Analysis of HIV-1 RNA+ cells from Early Infected Patients
(a) UMAP plot displays the distribution of PBMCs in early infected patients (n=9), clustered based on transcriptome signatures post-removing batch effects.
(b) The UMAP plot displays the distribution of 4,435 CD4 T cells from early infected patients.
(c) UMAP of CD4 T cells identifies 3,450 uninfected cells and 985 HIV-1 RNA+ cells.
(d) Volcano plot reveals differentially expressed genes (DEGs) in HIV-1 RNA+ CD4 T cells compared to uninfected cells; red dots signify significant DEGs (adj P-value < 0.01).
(e) Bar plot reveals enriched biological processes and immune pathways in HIV-1 RNA+ CD4 T cells (P-value < 0.01) using upregulated DEGs; significance presented as −log(P-value).
(f) Heatmap exhibits signaling pathways enriched in CD4 T cell types through GSEA; positive scores (dark orange) denote enrichment in HIV-1 RNA+ cells (Nominal P-val < 0.05 and < 0.25) are marked with stars.
(g) GSEA plots show significant immune pathways in CD4 naïve, Th2 and Th17 cells, presenting enrichment score (ES), normalized enrichment score (NES), and false discovery rate (FDR).
Exploring The Epigenetic Characteristics of HIV-1 RNA+ Cells Through Single-Cell Multiome Data
(a) UMAP plot illustrates PBMC distribution in early infected patients (n=8) post-clustering based on transcriptome and epigenome signatures, with batch effects removed.
(b) Visualization of differential DNA accessibility across 15 cell clusters. Tracks display normalized chromatin accessibility at promoter regions of cluster-specific marker genes.
(c) Heatmap displays differentially chromatin accessible regions (DARs) in HIV-1 RNA+ and uninfected cells of Naïve and Memory CD4 T cell types. Right heatmap presents enriched gene ontology for DAR-associated genes.
(d) Volcano plot shows DARs between HIV-1 RNA+ CD4 T cells and uninfected cells; red dots signify significant DARs, with associated genes displayed.
(e) Tracks display normalized chromatin accessibility at the promoter regions of key genes (PSMB7, CDK9, NR4A2, TRIM62) in HIV-1 RNA+ and uninfected cells.
Identifying Key Regulators of HIV-1 RNA+ CD4 T Cells Through Integrated Analysis
(a) Heatmap depicts relative regulon activity in each CD4 T cell subtype between HIV-1 RNA+ and uninfected cells. Positive values indicate increased activity in HIV-1 RNA+ cells, while negative values represent a decrease.
(b) Heatmap displays regulon activity levels in each CD4 T cell subtype, with color indicating scaled regulon activity.
(c) Dot plot displays gene expression of upregulated TFs in HIV-1 RNA+ cells, with dot size representing the percentage of cells expressing the gene.
(d) Volcano plot reveals target genes of upregulated TFs in HIV-1 RNA+ cells; pink dots represent DEGs, and red dots indicate target genes of enriched TFs.
(e) Violin plot illustrates expression of TF-encoding genes in uninfected and HIV-1 RNA+ CD4 T cells across subtypes (*P < 0.01).
(f) DNA sequences for overrepresented TF binding motifs in HIV-1 RNA+ T cells compared to uninfected cells.
(g) Violin plot displays chromVAR motif activity score for enriched motifs.
(h) TF footprinting profiles show the increased chromatin accessibility near three representative motifs in HIV-1 RNA+ cells.
(i) Left panel: Tracks display normalized chromatin accessibility at KLF2 gene locus for HIV-1 RNA+ and uninfected cells, with peak-to-gene links bottom of the coverage plot. Right panel: Expression level of KLF2 in each group.
Functional Insights into KLF2 Upregulation in HIV-1 RNA+ CD4 T Cells
(a) Bar plot displays gene ontologies associated with upregulated KLF2 TF target genes in HIV-1 RNA+ cells (P-value < 0.01); significance expressed as −log(P-value).
(b) Violin plot represents expression of upregulated KLF2 TF target genes in uninfected cells and HIV-1 RNA+ CD4 T cells (*P < 0.01).
(c) Tracks display normalized chromatin accessibility at promoter loci of PIM1, IL32, TXNIP, and LTB for HIV-1 RNA+ and uninfected cells, with peak-to-gene links bottom of the coverage plot.
(d) Dot plot displays ligand-receptor pairs detected between CD4 and CD8 T cells; red-labeled gene symbols are KLF2 target genes. Circle size indicates P-values, while color represents mean average expression levels.
(e) Interactions between KLF2 target genes and HIV-1 proteins identified using STRING database. Yellow dots represent human proteins, and red dot represents HIV-1 protein; line thickness indicates interaction strength.
Epigenetic analysis in HIV-1 DNA+ CD4 T cells
(a) Bar plot presents scATAC-seq reads aligned to the HIV-1 genome, showing coverage for individual genomic windows (log-scaled, max value: 245).
(b) UMAP of CD4 T cells based on chromatin accessibility identifies 5,535 uninfected cells and 39 HIV-1 DNA+ cells.
(c) Heatmap displays chromatin accessibility levels of DARs enriched in HIV-1 DNA+ CD4 T cell cluster.
(d) Bar plot represents gene ontologies associated with genes near DARs of HIV-1 DNA+ CD4 T cell cluster; significance expressed as −log(P-value).
(e) DNA sequences for overrepresented transcription factor binding motifs in HIV-1 DNA+ CD4 T cell cluster compared to the rest of CD4 T cells.
(f) Violin plot displays chromVAR motif activity score for enriched motifs.
(g) Bar plots on the left show normalized ATAC reads for individual genes in HIV-1 DNA+ CD4 T cells and uninfected cells. Bottom shows peak-to-gene links; loop color strength signifies significance of links between peak and gene promoter. Right violin plot displays gene expression levels.
Single-cell transcriptomic analysis identifies the cell types of PBMCs and subclustered CD4 T cells from early HIV-1 infected patients.
(A) Dot plot illustrates the gene expression of cell type-specific marker genes in all clusters, where the dot size represents the percentage of cells expressing the marker gene, and the color intensity indicates the average expression of the marker gene.
(B) The proportion of cell types are depicted for each donor.
(C) UMAP plots of subclustered CD4+ T cells from single-cell RNA-seq data. Each color represents a different donor. The right panel shows the distribution of cells after batch correction using the fastMNN algorithm.
(D) Dot plot illustrates the gene expression of cell type-specific marker genes in all CD4 subclusters.
(E) The proportion of HIV-1 RNA+ cells to uninfected cells are depicted for each cell type. The box plot includes the counts of both HIV-1 RNA+ cells and uninfected cells
Single-cell transcriptomic analysis identifies the characteristics of HIV-1 RNA+ cells.
(A-B) Upregulated (red) and downregulated (blue) genes are shown on the map of KEGG PI3K-AKT signaling pathway.
Single-cell multi-omics analysis identifies epigenetic characteristics of scATAC-seq dataset and upregulated transcription factors in HIV-1 RNA+ cells
(A) Violin plot displays the gene expression of marker genes in scATAC-seq dataset.
(B) The bar plot represents the gene ontologies associated with the target genes of the upregulated TFs in HIV-1 RNA+ cells (P-value < 0.01). The significance of these enrichments is expressed as −log(P-value).
KLF2 target gene expression in HIV-1-infected CD4+ T cells
(A) Heatmap showing the expression (Z-score normalized) of KLF2 target genes across healthy controls and HIV-1-infected individuals with high and low viral loads. Gene expression data were obtained from a published dataset (GSE157829)
(B) Violin plots showing the expression levels of FOXP1 and GATA3 in HIV-1 RNA+ versus uninfected CD4+ T cells (top, current study). Bottom panel shows expression levels of KLF2 in published dataset (GSE242997), comparing HIV-infected cells treated with latency-reversing agents (LRA) and DMSO controls.
Cell-cell interaction in HIV-1-infected CD4+ T cells
(A) Circos plot show cell–cell interactions that are upregulated in HIV-1-infected CD4+ T cells. Line thickness represents interaction strength, and arcs are colored by cell types.
(B) Circos plot shows downregulated interactions in HIV-1 infected CD4+ T cells.
Single-cell epigenetic analysis identifies HIV-1 DNA+ cells are mostly represented by Th17 cell type.
(A) The ATAC UMAP plot displays the distribution of 5,608 CD4 T cells from early infected patients.
(B-C) The Feature Plot and violin plot indicate gene expression (RORC, STAT3) in CD4 T cells. *P < 0.01
(D) The tracks display the normalized chromatin accessibility levels at the promoter regions of RORC and STAT3 in all cluster of CD4 T cells.
Epidemiological and Virological Characteristics, and Sequencing Data Information for 9 Acute HIV-infected Patients at Baseline
Differentially expressed genes overlapped with KLF2 target gene in CD4 T cell. (HIV-1 RNA+ vs. Uninfected)
