title: Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency.
abstract: The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.
authors.affiliation: Department of Cell Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: Olaf
authors.initials: O
authors.lastname: Kutsch
authors.forename: Etty N
authors.initials: EN
authors.lastname: Benveniste
authors.forename: George M
authors.initials: GM
authors.lastname: Shaw
authors.forename: David N
authors.initials: DN
authors.lastname: Levy
mesh_terms: D002453:Cell Cycle; D002461:Cell Line, Transformed; D005434:Flow Cytometry; D005470:Fluorometry; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity*; D006801:Humans; D019169:Jurkat Cells / Q000821:virology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000821:virology; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000821:virology; D014775:Virus Activation; D017735:Virus Latency
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins