Fig 1.
DNA and RNA were extracted from CD4+ T cells enriched from cryopreserved peripheral blood mononuclear cells (PBMCs) from 191 ART-suppressed people with HIV (PWH). Extracted DNA was used to perform HIV reservoir quantification (total DNA by quantitative PCR, intact DNA by digital droplet PCR) and host DNA exome sequencing [37]. Extracted RNA was used to perform HIV reservoir quantification (unspliced RNA by qPCR) and host bulk RNA sequencing for the current study. Created with BioRender.com.
Table 1.
Descriptive statistics for the study population of 191 HIV-infected ART-suppressed non-controllers.
Table 2.
Differentially expressed host genes in relation to HIV total DNA.
Results shown for the overall cohort of 191 participants using a Benjamini-Hochberg false discovery rate (FDR) cutoff value of q<0.05.
Fig 2.
Intracellular P3H3 and NBL1 protein levels from peripheral CD4+ T cells.
The distribution of normalized protein levels for P3H3 (A) and NBL1 (C) among a subset of 40 study participants. Correlation scatterplots are shown for P3H3 (B) and NBL1 (D), excluding outliers (>2 standard deviations).
Table 3.
Differentially expressed host genes in relation to log10copies of HIV unspliced RNA (usRNA).
Results shown for the overall cohort of 191 participants (top panel) as well as the European ancestry subgroup (bottom panel) using a Benjamini-Hochberg false discovery rate (FDR) cutoff value of q<0.05. Additional genes meeting an FDR cut-off of q<0.25 are shown in S3 Table.
Fig 3.
Clustering of top differentially expressed host genes associated with HIV unspliced RNA.
Given the large number of statistically significant host genes (Tables 3 and S3 Table), we used network analysis to group the top-ranked genes (q<0.25) into biologically interpretable clusters (ClueGo Network software). Host genes related to NRLP3 inflammasome activation (e.g., IL-1β), Th2 cell cytokine production (e.g., IL-10), and bacterial translocation (e.g., TLR4, lipopolysaccharide) signaling were significantly associated with HIV usRNA. A Benjamini-Hochberg false discovery rate (FDR) of q<0.05 was used to generate nodes (circles) based on kappa scores ≥0.4. The size of the nodes reflects the enrichment significance of the terms, and the different colors represent distinct functional groups. Created with https://apps.cytoscape.org/apps/cluego.
Fig 4.
Unbiased gene set enrichment analyses (GSEA) of the entire transcriptome (rank-ordered by q-value) in association with HIV unspliced RNA.
Host gene sets involving interferon, IL-10, TNF, NLRP3 inflammasome activation (e.g., IL-1β, IL-6), and bacterial translocation (e.g., lipopolysaccharide-mediated) signaling were significantly associated with HIV usRNA. A Benjamini-Hochberg false discovery rate (FDR) of q<0.05 was used to generate nodes (circles) based on kappa scores ≥0.4. The size of the nodes reflects the enrichment significance of the terms, and the different colors represent distinct functional groups. Created with https://apps.cytoscape.org/apps/cluego.
Fig 5.
Plasma cytokine expression of IL-10 (A), TNF-α (B), IL-1β (C), G-CSF (D), IP-10 (E), TNFAIP5 (F), and sTLR4 (G).
The association between plasma protein expression among 175 study participants in relation to measures of HIV unspliced RNA.
Fig 6.
Intracellular Kir2.1 (KCNJ2) and connexin 26 (GJB2) protein levels from peripheral CD4+ T cells.
The distribution of normalized protein levels for Kir2.1 (A) and connexin 26 (C) among a subset of 40 study participants. Correlation scatterplots are shown for Kir2.1 (B) and connexin 26 (D), excluding outliers (>2 standard deviations).
Fig 7.
Proposed model based on host bulk RNA-sequencing and HIV reservoir quantification from peripheral CD4+ T cells of 191 ART-suppressed people with HIV.
Increased expression of host tumor suppressor genes NBL1 and P3H3 were observed among individuals with smaller HIV total DNA reservoir size in our cross-sectional study (q<0.05). The model depicts host genes from HIV-uninfected cells influencing the subset of CD4+ T cells harboring provirus. Created with BioRender.com.
Fig 8.
Proposed model for the inverse association between HIV unspliced RNA and host gene expression in our cross-sectional study of 191 ART-suppressed people living with HIV.
Within bulk peripheral CD4+ T cells, higher transcriptional reservoir activity (HIV usRNA) from HIV+ cells (left) may chronically lead to downregulation of host proinflammatory gene expression in bystander cells (right) in an attempt to suppress persistent inflammation and innate immune activation. Created with BioRender.com.