Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency

Olaf Kutsch, Etty N. Benveniste, George M. Shaw, David Levy

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)8776-8786
Number of pages11
JournalJournal of Virology
Volume76
Issue number17
DOIs
StatePublished - 2002

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Single-Cell Analysis
Human immunodeficiency virus 1
HIV-1
green fluorescent protein
Virus Latency
viruses
cells
Fluorescence
fluorescence
Alphavirus
Viruses
Virus Activation
Fluorometry
fluorometry
terminal repeat sequences
Terminal Repeat Sequences
Highly Active Antiretroviral Therapy
Virus Replication
fluorescence microscopy
Fluorescence Microscopy

ASJC Scopus subject areas

  • Immunology

Cite this

Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency. / Kutsch, Olaf; Benveniste, Etty N.; Shaw, George M.; Levy, David.

In: Journal of Virology, Vol. 76, No. 17, 2002, p. 8776-8786.

Research output: Contribution to journalArticle

Kutsch, Olaf ; Benveniste, Etty N. ; Shaw, George M. ; Levy, David. / Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency. In: Journal of Virology. 2002 ; Vol. 76, No. 17. pp. 8776-8786.
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