Integrating subcellular multi-omics to identify druggable metabolic markers of latent HIV infection in CD4 T-cells
Lead Research Organisation:
University of Bristol
Department Name: Cellular and Molecular Medicine
Abstract
Four decades after its identification, the human immunodeficiency virus (HIV) continues to infect almost 40 million people worldwide, causing hundreds of thousands of deaths each year. Infection by HIV mainly targets white blood cells and, if left untreated, can lead to a severe, potentially fatal, acquired immunodeficiency syndrome (AIDS). Antiviral drugs have been developed and have drastically improved the life expectancy and quality of people living with HIV. However, these drugs require lifelong administration, and do not lead to a cure. Importantly, HIV persists in a dormant (latent) form in a subset of white blood cells (called CD4 T-lymphocytes) for the entire lifespan of infected individuals. This latency allows the virus to elude antiviral drugs and the immune system.
Several studies have been conducted with the aim to identify features of persistently infected cells that could allow us to specifically target these cells for elimination and, thus, potentially cure the infection. Although multiple features have been proposed, few have the specificity required for safe therapeutic application, and most of the studies have failed to decrease the frequency of persistently infected cells. No current treatment to target persistently HIV-infected cells is approved. One of the therapeutic approaches undergoing clinical testing is based on our research showing that persistently infected cells have specific alterations in energy metabolism. Our studies, as well as those of other groups, however, focussed on whole cells. In reality, most cellular energy production is located within highly specialised subcellular compartments.
To allow us to identify functional, structural, and/or spatial features of metabolic regulation that distinguish cells harbouring the virus from those that do not, the proposed project will produce an in-depth characterization (identikit) of persistently HIV-infected cells. We will approach this work both using CD4 T-lymphocytes infected with HIV in vitro and using CD4 T-lymphocytes isolated from the blood of individuals living with HIV. We will first separate cells infected in vitro based on the presence and stage of infection, and then individually study the main cellular sites of energy production and storage (i.e., nucleus/cytoplasm and mitochondria). The data obtained will comprehensively capture the content of proteins and metabolites, as well as the genetic regulation underpinning their production. These data will be computationally combined and complemented by experimental assessments of the functionality of each major cellular metabolic step in order to identify distinguishing features of persistently infected cells that can be explored for their potential to serve as therapeutic targets. We will then use computer models to predict effective drug candidates and put these predictions in practice by testing drugs in the laboratory for their target affinity and ability to selectively eliminate persistently infected cells.
Overall, this study will aim to provide a unified profile of metabolic determinants of HIV persistence and furnish pre-clinical evidence for novel therapeutic strategies to eliminate infected cells resistant to currently available antiviral drugs. Ultimately, this work could lead to a therapeutic approach to remove the virus from its cellular hideout in infected individuals.
Several studies have been conducted with the aim to identify features of persistently infected cells that could allow us to specifically target these cells for elimination and, thus, potentially cure the infection. Although multiple features have been proposed, few have the specificity required for safe therapeutic application, and most of the studies have failed to decrease the frequency of persistently infected cells. No current treatment to target persistently HIV-infected cells is approved. One of the therapeutic approaches undergoing clinical testing is based on our research showing that persistently infected cells have specific alterations in energy metabolism. Our studies, as well as those of other groups, however, focussed on whole cells. In reality, most cellular energy production is located within highly specialised subcellular compartments.
To allow us to identify functional, structural, and/or spatial features of metabolic regulation that distinguish cells harbouring the virus from those that do not, the proposed project will produce an in-depth characterization (identikit) of persistently HIV-infected cells. We will approach this work both using CD4 T-lymphocytes infected with HIV in vitro and using CD4 T-lymphocytes isolated from the blood of individuals living with HIV. We will first separate cells infected in vitro based on the presence and stage of infection, and then individually study the main cellular sites of energy production and storage (i.e., nucleus/cytoplasm and mitochondria). The data obtained will comprehensively capture the content of proteins and metabolites, as well as the genetic regulation underpinning their production. These data will be computationally combined and complemented by experimental assessments of the functionality of each major cellular metabolic step in order to identify distinguishing features of persistently infected cells that can be explored for their potential to serve as therapeutic targets. We will then use computer models to predict effective drug candidates and put these predictions in practice by testing drugs in the laboratory for their target affinity and ability to selectively eliminate persistently infected cells.
Overall, this study will aim to provide a unified profile of metabolic determinants of HIV persistence and furnish pre-clinical evidence for novel therapeutic strategies to eliminate infected cells resistant to currently available antiviral drugs. Ultimately, this work could lead to a therapeutic approach to remove the virus from its cellular hideout in infected individuals.
Technical Summary
Eliminating latently infected CD4 T-lymphocytes is essential to cure HIV-1 infection. Our previous studies-which have advanced to clinical trials-show that redox metabolism imbalance can be exploited to kill latently infected cells with partial selectivity. Improving selectivity further will require expansion over previous studies that were limited by their focus on individual metabolic pathways or whole cell lysates of mixed infected/uninfected cultures. Here, we aim to obtain an integrated picture of metabolic expression and function in subcellular fractions of sorted latently infected CD4 T-cells, to identify the most selective latency markers and test their therapeutic targeting ex vivo.
We will use CD4 T-cells of healthy donors for infection with an engineered HIV-1 construct to sort cells based on the presence and state of infection (productive or latent). Subcellular fractions most relevant for metabolic activity and regulation (nucleus/cytoplasm and mitochondria) will be then isolated and used for omics (mitochondrial and nuclear DNA methylation, transcriptomics, proteomics, and metabolomics) and functional/structural analyses (seahorse XF, western blot, microscopy, FACS, gene modulation). By integrating these results, we will pinpoint selective latency markers and evaluate their druggability in silico, through literature analysis, and through biophysical and physico-chemical analysis of drug binding conformation and stability. The most promising drug candidates will be finally assessed for their potential to disrupt viral latency and selectively eliminate latently infected cells in cultures of CD4 T-cells isolated from people living with HIV.
Overall, by using donor-matched (infected and uninfected) sorted cells, we aim to achieve a high signal-to-noise ratio, potentially identifying rare markers, while multiple layers of validation, culminating in ex vivo tests, will increase the reproducibility and physiologic relevance of the results.
We will use CD4 T-cells of healthy donors for infection with an engineered HIV-1 construct to sort cells based on the presence and state of infection (productive or latent). Subcellular fractions most relevant for metabolic activity and regulation (nucleus/cytoplasm and mitochondria) will be then isolated and used for omics (mitochondrial and nuclear DNA methylation, transcriptomics, proteomics, and metabolomics) and functional/structural analyses (seahorse XF, western blot, microscopy, FACS, gene modulation). By integrating these results, we will pinpoint selective latency markers and evaluate their druggability in silico, through literature analysis, and through biophysical and physico-chemical analysis of drug binding conformation and stability. The most promising drug candidates will be finally assessed for their potential to disrupt viral latency and selectively eliminate latently infected cells in cultures of CD4 T-cells isolated from people living with HIV.
Overall, by using donor-matched (infected and uninfected) sorted cells, we aim to achieve a high signal-to-noise ratio, potentially identifying rare markers, while multiple layers of validation, culminating in ex vivo tests, will increase the reproducibility and physiologic relevance of the results.
