title: Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique.
abstract: HIV-1 establishes a pool of latently infected cells early following infection. New therapeutic approaches aiming at diminishing this persisting reservoir by reactivation of latently infected cells are currently being developed and tested. However, the reactivation kinetics of viral mRNA and viral protein production, and their respective consequences for phenotypical changes in infected cells that might enable immune recognition, remain poorly understood. We adapted a novel approach to assess the dynamics of HIV-1 mRNA and protein expression in latently and newly infected cells on the single-cell level by flow cytometry. This technique allowed the simultaneous detection of gagpol mRNA, intracellular p24 Gag protein, and cell surface markers. Following stimulation of latently HIV-1-infected J89 cells with human tumor necrosis factor alpha (hTNF-α)/romidepsin (RMD) or HIV-1 infection of primary CD4(+) T cells, four cell populations were detected according to their expression levels of viral mRNA and protein. gagpol mRNA in J89 cells was quantifiable for the first time 3 h after stimulation with hTNF-α and 12 h after stimulation with RMD, while p24 Gag protein was detected for the first time after 18 h poststimulation. HIV-1-infected primary CD4(+) T cells downregulated CD4, BST-2, and HLA class I expression at early stages of infection, proceeding Gag protein detection. In conclusion, here we describe a novel approach allowing quantification of the kinetics of HIV-1 mRNA and protein synthesis on the single-cell level and phenotypic characterization of HIV-1-infected cells at different stages of the viral life cycle.
  
  
  
  Early after infection, HIV-1 establishes a pool of latently infected cells, which hide from the immune system. Latency reversal and immune-mediated elimination of these latently infected cells are some of the goals of current HIV-1 cure approaches; however, little is known about the HIV-1 reactivation kinetics following stimulation with latency-reversing agents. Here we describe a novel approach allowing for the first time quantification of the kinetics of HIV-1 mRNA and protein synthesis after latency reactivation or de novo infection on the single-cell level using flow cytometry. This new technique furthermore enabled the phenotypic characterization of latently infected and de novo-infected cells dependent on the presence of viral RNA or protein.
authors.affiliation: Department of Virus Immunology, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Martrus
authors.affiliation: Department of Virus Immunology, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Niehrs
authors.affiliation: Department of Virus Immunology, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Cornelis
authors.affiliation: Department of Virus Immunology, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany University Medical Center Hamburg-Eppendorf, 1st Department of Medicine, Hamburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Rechtien
authors.affiliation: Ragon Institute of MGH, MIT, and Harvard, Cambridge, Massachusetts, USA.
authors.forename: W
authors.initials: W
authors.lastname: García-Beltran
authors.affiliation: Department of Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Lütgehetmann
authors.affiliation: ICH Study Center, Hamburg, Germany Department of Medicine II, University of Schleswig-Holstein, Campus Kiel, Kiel, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Hoffmann
authors.affiliation: Department of Virus Immunology, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany marcus.altfeld@hpi.uni-hamburg.de.
authors.forename: M
authors.initials: M
authors.lastname: Altfeld
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D002478:Cells, Cultured; D003584:Cytological Techniques / Q000379:methods*; D015497:HIV-1 / Q000502:physiology*; D054298:Human Immunodeficiency Virus Proteins / Q000032:analysis; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D013997:Time Factors; D014773:Virology / Q000379:methods*; D014775:Virus Activation
chemical_list: D054298:Human Immunodeficiency Virus Proteins; D012333:RNA, Messenger; D012367:RNA, Viral