Human immunodeficiency virus (HIV) is the primary cause of AIDS, affecting over 80 million people and causing 42.3 million deaths worldwide. Despite the success of combined antiretroviral therapy (cART), lifelong therapy is required due to the persistence of latent reservoirs in resting CD4+ T cells. The “kick and kill” strategy aims to reactivate latent HIV-1 using latency-reversing agents (LRAS) followed by immune clearance under cART in people living with HIV (PLWH). However, clinical success has been limited due to insufficient clearance and potency.
A recent study published in Nature aims to identify endogenous circulating factors capable of reactivating latent HIV-1 by systematically screening a hemofiltrate-derived peptide library using J-Lat latency models. Retinol-binding protein 4 (RBP4), carrying retinol, strongly reactivated HIV-1 J-Lat clones and patient-derived cells, highlighting RBP4 as a potential mediator of viral latency reversal.
In this study, hemofiltrate was fractionated into peptide libraries (<30 kDa) from patients with chronic renal failure using cation-exchange and reversed-phase liquid chromatography. Fractions were screened in J-Lat green fluorescent protein (GFP) reporter cells to identify HIV-1 activators, with further purification yielding RBP4, which was confirmed by sequence analysis and mass spectrometry (MALDI-TOF MS). Latently infected J-Lat and other HIV-1 cell models were cultured under standard conditions. These cells were treated with purified or recombinant RBP4 and inhibitors (4-Diethylaminobenzaldehyde, bis-diamine, signal transducer and activator of transcription 5 [STAT5] inhibitors, rottlerin, selumetinib, c-Jun-N-terminal kinase (JNK) inhibitor II, B022, U0216, NF-κB-inducing kinase inhibitor [NIK] SM1), as well as with retinoids like retinoic acid, retinol, and all-trans retinal.
Reactivation was assessed by GFP or p24 expression through flow cytometry. Proteins were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ligand-binding assays. RNA-seq and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to determine the transcriptional responses, while enzyme-linked immunosorbent assay (ELISA) and single-molecule array (Simoa) were employed to quantify viral and RBP4 proteins. Ex vivo cluster of differentiation 4 positive T lymphocytes (CD4+ T) cells from ART-treated individuals were tested, and an intact proviral deoxyribonucleic acid (DNA) assay (IPDA) quantified latent reservoirs.
Results showed that RBP4 levels in fractions strongly correlated with HIV-1 reactivation in vitro (R2=0.667 and p < 0.0001). Several fractions induced strong GFP expression, which was comparable to that of positive controls, including tumor necrosis factor alpha (TNF-α) and phorbol 12-myristate 13-acetate (PMA). RBP4 dose-dependently reactivated latent HIV-1 in 10.6, 11.1, A1, A2, and H2 cell lines, whereas other subclones showed minimal response.
RBP4 transports retinol from the liver to tissues as holo-RBP4 or apo-RBP4. Removal of retinol abolished HIV-1 reactivation in J-Lat cells, whereas free retinoids or retinoic acid had minimal effect. Therefore, reactivation of latent HIV-1 needs circulating holo-RBP4 but is independent of intracellular retinoic acid production.
Holo-RBP4 specifically reactivates HIV-1 in J-Lat 10.6 but not 8.4 cell lines, including those involved in NF-κB and vital signaling pathways. This reactivation was independent of being stimulated by retinoic acid 6 (STRA6) or toll-like receptor 4 (TLR4) expression and relies on canonical NF-κB, JNK, and JAK/STAT5 pathways. Inhibition of these pathways led to a reduction in the induction of HIV-1. Levels of holo-RBP4 in plasma of ART-treated PLWH were sufficient to trigger latent HIV-1, although they were not correlated with reservoir size, supporting its potential as an LRA.
In ex vivo, RBP4 significantly (p < 0.05) reactivated latent HIV-1 in peripheral blood mononuclear cells (PBMCs) from seven ART-suppressed PLWH (males) without notable toxicity. p24 induction correlated with defective and total proviruses, demonstrating that physiological concentrations of RBP4 can reverse latency in individuals with viral suppression.
In conclusion, this study revealed that holo-RBP4 reactivates latent HIV-1 through multiple signaling pathways but independent of retinoic acid production. As the primary retinol carrier, RBP4 may act in lymphoid tissues, offering a novel target for latent reservoirs. Screening protein/peptide libraries from body fluids suggests a strategy for discovering natural LRAs.
References: Pastorio C, Richard K, Usmani S, et al. Retinol Binding Protein 4 reactivates latent HIV-1 by triggering canonical NF-κB, JAK/STAT5, and JNK signalling. Signal Transduct Target Ther. 2025;10:326. doi:10.1038/s41392-025-02424-3
