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.