Transcriptionally Active HIV Reservoirs Enriched for Interferon-Inducible APOBEC3-Related Mutational Signatures Associate with Arterial Inflammation

Despite suppressive antiretroviral therapy (ART), people with HIV (PWH) remain at increased risk of cardiovascular disease (CVD), likely driven, at least in part, by persistent immune activation and vascular inflammation. Mechanistic pathways may include ongoing low-level HIV transcription within immune cells, particularly peripheral blood mononuclear cells (PBMCs), which may contribute to chronic inflammatory signaling. Persistent viral transcription and inflammatory signaling may also be associated with an interferon-responsive cellular environment conducive to APOBEC3 activation and mutational editing of viral transcripts. APOBEC3 enzymes, which are interferon (IFN)-inducible cytidine deaminases, generate characteristic premature stop codons (PSCs) within retroviral genomes and may reflect interferon-associated inflammatory states relevant to vascular pathology. We investigated whether APOBEC3-associated PSC signatures and reservoir-associated drug resistance mutations (RA-DRMs) in viral reservoirs, as markers of persistent reservoir transcriptional activity, are associated with vascular inflammation in ART-suppressed individuals. PBMCs from ART-suppressed PWH (n=36) were analyzed for long HIV-1 gag/pol transcripts (>4.2 kb) and PSCs as a surrogate marker of APOBEC3 activity, with RA-DRMs assessed by Oxford Nanopore sequencing. Arterial inflammation was quantified using PET imaging of the aorta and carotid arteries. Additionally, an exploratory panel of >60 cardiometabolic, clinical, and inflammatory biomarkers was profiled to identify host immune signatures associated with reservoir PSC burden, with focused analyses on circulating inflammatory biomarkers sCD14, sCD163, and IP-10. Long HIV-1 transcripts were detected in 67% (24/36) despite ART. Higher PSC frequency within these transcripts was significantly associated with increased aortic inflammation, but not carotid inflammation, in univariable analysis ({beta} = 0.00834, p = 0.013). RA-DRMs were detected in 50% (12/24), including triple-class resistance, but were not associated with vascular inflammation or soluble biomarkers. In multivariable models, PSCs remained independently associated with aortic inflammation after adjustment for sCD14 and sCD163 ({beta} = 0.0075, p = 0.023), and remained the only significant predictor when IP-10 was included ({beta} = 0.0073, p = 0.036). These findings suggest that APOBEC3-associated PSC signatures in long HIV transcripts identify transcriptionally active HIV reservoir states associated with aortic inflammation during suppressive ART. Rather than reflecting ongoing mutational accumulation or direct pathogenic effects of PSCs themselves, these signatures may serve as biomarkers of an interferon-associated inflammatory reservoir state related to cardiovascular risk. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/729898v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@6a4f9forg.highwire.dtl.DTLVardef@1917e18org.highwire.dtl.DTLVardef@ed9802org.highwire.dtl.DTLVardef@96cb94_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights: Interferon-inducible APOBEC3-associated PSC signatures in HIV reservoirs and vascular inflammationO_LIAPOBEC3-associated premature stop codon (PSC) signatures were identified in transcriptionally active HIV reservoir-derived RNA despite suppressive ART. C_LIO_LIHigher PSC burden was significantly associated with increased aortic vascular inflammation measured by FDG-PET. C_LIO_LIReservoir-associated drug resistance mutations (RA-DRMs) showed no association with arterial inflammation. C_LIO_LIPSC burden remained independently associated with aortic vascular inflammation in multivariable analyses. C_LIO_LIPSC-enriched reservoir states were associated with inflammatory biomarker patterns consistent with monocyte-macrophage activation and vascular immune activation. C_LIO_LIThese findings are consistent with an interferon-inducible APOBEC3-edited reservoir state associated with persistent arterial inflammation during suppressive ART. C_LIO_LIAPOBEC3-associated mutational signatures may represent a candidate biomarker of vascular inflammation and cardiovascular risk in people with HIV. C_LI