Fig. 4

Monoclonal UIS accumulation preferentially occurs at HIV proviral integration sites within in-gene regions during the AIDS treatment stage, with functional enrichment analysis revealing the involvement of these host cell genes in key biological processes. a PCA plots distinguishing monoclonal UIS (red dot, n = 164) from other UIS (blue dot), the cluster based on LTR percentage, Richness, Evenness and PMD. The plot shows clear distinction between monoclonal UIS with other UIS by two principal components (Component 1, 26.1%; Component 2, 54.1%). b Comparison of UIS clonality distribution across different genomic regions (x-axis), indicating that monoclonal UIS (red bars) are more likely to integrate into in-genes (Dashed line, right side) than non-monoclonal UIS (blue bars) are more likely to integrate into transcription start site (TSS) upstream (Dashed line, left side) (up panel). The accompanying table indicates that the sites of most monoclonal UIS, 80.5% (132) of integrations occur in in-gene regions, 11.0%¹⁸ of integrations occur in TSS upstream regions, 8.5%¹⁴ of integrations occur in other sites. In contrast, other clonal types, 48.3% (5859) of integrations occur in in-gene regions, with 25.1% (3049) in upstream promoter regions, with 26.6% (3225) in other sites (below panel). c Chromosomal distribution of monoclonal UIS, highlighting a higher frequency of integration into chromosomes 1, 3, 16, 17, 12, and 19 in monoclonal UIS (red bar) and chromosomes 2, 4, 5, 7, 10, 13, 14, 15, 21, and Y in other UIS (blue bar). d, e Ranking analysis of all UIS, grouped into sets of 100. Ranking the top 1000 UIS and dividing them into 100 subsets, each containing 10 UIS further organized into subsets of 100. The percentage of UIS integrated into in-gene regions (red) was higher in the top-ranked subset and gradually decreased across the ranks (d). In the lowest 1000 UIS, nearly 50% were integrated into genes across the ranks (e). f Functional enrichment analysis of in-gene regions from 164 monoclonal UIS, highlighting genes enriched in the top 20 biological processes. The x-axis represents-log₁₀(p-value), with larger values indicating higher statistical significance of enrichment. g Chi-square/Fisher test comparing UIS genes percentage in enriched pathways between the Acute- and AIDS-ART groups, showing differences in the percentage of integrated genes across 20 enriched pathways. Blue dots represent pathways with significant differences in the AIDS-ART group compared to the Acute-ART group, indicated by red dots. Dot size reflects the percentage of integrated genes from each pathway in two groups, with larger dots indicating higher percentages. h Sankey bubble plot for GO-term classification. This chart illustrates enriched genes associated with lymphocyte activation (GO: 0051249), chromosome segregation (GO: 0051983), chromosome organization (GO: 0033044), regulation of cell cycle process (GO: 0010564), and response to biotic stimulus (GO: 0002831) for two groups—PLWH who initiated ART during the acute stage (red) and those who initiated ART during the AIDS stage (blue). Bubble size represents the UIS count percentage relative to each group, and the y-axis indicates ART duration (in months). (Note: p < 0.05 indicates statistical significance; Kruskal-Wallis test for (b, c) Chi-square / Fisher’s exact test for (g, h) the terms ‘Acute’ and ‘AIDS’ in all figures refer to PLWH who initiated ART during the acute stage (also referred to as the Acute-ART group) and those who initiated ART during the AIDS stage (also referred to as the AIDS-ART group), respectively)