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Reactivation of the latent viral reservoirs is crucial for a cure of HIV/AIDS. However, current latency reversing agents are inefficient, and the endogenous factors that have the potential to reactivate HIV in vivo remain poorly understood. To identify natural activators of latent HIV-1, we screened a comprehensive peptide/protein library derived from human hemofiltrate, representing the entire blood peptidome, using J-Lat cell lines harboring transcriptionally silent HIV-1 GFP reporter viruses. Fractions potently reactivating HIV-1 from latency contained human Retinol Binding Protein 4 (RBP4), the carrier of retinol (Vitamin A). We found that retinol-bound holo-RBP4 but not retinol-free apo-RBP4 strongly reactivates HIV-1 in a variety of latently infected T cell lines. Functional analyses indicate that this reactivation involves activation of the canonical NF-¦ÊB pathway and is strengthened by JAK/STAT5 and JNK signalling but does not require retinoic acid production. High levels of RBP4 were detected in plasma from both healthy individuals and people living with HIV-1. Physiological concentrations of RBP4 induced significant viral reactivation in latently infected cells from individuals on long-term antiretroviral therapy with undetectable viral loads. As a potent natural HIV-1 latency-reversing agent, RBP4 offers a novel approach to activating the latent reservoirs and bringing us closer to a cure. Subject terms: Preclinical research, Infectious diseases

IntroductionCurrent antiretroviral therapy (ART) for HIV infection reduces plasma viral loads to undetectable levels and has increased the life expectancy of people with HIV (PWH). However, this increased lifespan is accompanied by signs of accelerated aging and a higher prevalence of age-related comorbidities. Tat (Trans-Activator of Transcription) is a key protein for viral replication and pathogenesis. Tat is encoded by 2 exons, with the full-length Tat ranging from 86 to 101 aa (Tat101). Introducing a stop codon in position 73 generates a 1 exon, synthetic 72aa Tat (Tat72). Intracellular, full-length Tat activates the NF-¦ÊB pro-inflammatory pathway and increases antiapoptotic signals and ROS generation. These effects may initiate a cellular senescence program, characterized by cell cycle arrest, altered cell metabolism, and increased senescence-associated secretory phenotype (SASP) mediator release However, the precise role of HIV-Tat in inducing a cellular senescence program in CD4+ T-cells is currently unknown.MethodsJurkat Tetoff cell lines stably transfected with Tat72, Tat101, or an empty vector were used. Flow cytometry and RT-qPCR were used to address senescence biomarkers, and 105 mediators were assessed in cell supernatants with an antibody-based membrane array. Key results obtained in Jurkat-Tat cells were addressed in primary, resting CD4+ T-cells by transient electroporation of HIV-Tat-FLAG plasmid DNA.ResultsIn the Jurkat cell model, expression of Tat101 increased the levels of the senescence biomarkers BCL-2, CD87, and p21, and increased the release of sCD30, PDGF-AA, and sCD31, among other factors. Tat101 upregulated CD30 and CD31 co-expression in the Jurkat cell surface, distinguishing these cells from Tat72 and Tetoff Jurkats. The percentage of p21+, p16+, and ¦Ã-H2AX+ cells were higher in Tat-expressing CD4+ T-cells, detected as a FLAG+ population compared to their FLAG- (Tat negative) counterparts. Increased levels of sCD31 and sCD26 were also detected in electroporated CD4+ T-cell supernatants.DiscussionIntracellular, full-length HIV-Tat expression increases several senescence biomarkers in Jurkat and CD4+ T-cells, and SASP/Aging mediators in cell supernatants. Intracellular HIV-Tat may initiate a cellular senescence program, contributing to the premature aging phenotype observed in PWH. Graphical Abstract

Background:A primary barrier to curing HIV is the HIV reservoir. The leading infectious cause of death worldwide for people living with HIV is tuberculosis (TB), but we do not know how TB impacts the HIV reservoir.Methods:Participants in identification and validation cohorts were selected from previously enrolled studies at Groupe Ha?tien d'?tude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) in Port au Prince, Haiti. Intact and non-intact proviral DNA were quantified using droplet digital PCR of peripheral blood mononuclear cell (PBMC)-derived CD4+ T cells. Kruskal-Wallis tests were used to compare medians with tobit regression for censoring.Results:In the identification cohort, we found that people living with HIV with a history of active pulmonary TB (n=19) had higher levels of intact provirus than people living with HIV without a history of active TB (n=47) (median 762; IQR, 183-1173 vs 117; IQR, 24-279 intact provirus per million CD4, respectively; P=0.0001). This difference also was seen in the validation cohort (n=31), (median 102; IQR, 0-737 vs 0; IQR, 0-24.5 intact provirus per million CD4, P=0.03) for TB vs no-TB history groups, respectively. The frequencies of CD4+ T cells with any detectable proviral fragment was directly proportional to the levels of interleukin-1 beta (r=0.524, P= 0.0025) and interleukin-2 (r=0.622, P=0.0002).Conclusions:People living with HIV with a history of active pulmonary TB have more HIV pro-virus in their circulating CD4+ T cells, even years after TB cure. We need to characterize which CD4+ T cells are harboring intact provirus to consider the impact of T cell-targeting HIV cure interventions for people living in TB-endemic areas.