We have recently developed the first microscopy-based strategy that enables simultaneous multiplex detection of viral RNA (vRNA), viral DNA (vDNA), and viral protein. Here, we used this approach to study the kinetics of latency reactivation in cells infected with the human immunodeficiency virus (HIV). We showed the transcription of nascent vRNA from individual latently integrated and reactivated vDNA sites appearing earlier than viral protein. We further demonstrated that this method can be used to quantitatively assess the efficacy of a variety of latency reactivating agents. Finally, this microscopy-based strategy was augmented with a flow-cytometry-based approach, enabling the detection of transcriptional reactivation of large numbers of latently infected cells. Hence, these approaches are shown to be suitable for qualitative and quantitative studies of HIV-1 latency and reactivation.
Bibliographical noteFunding Information:
The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: J-Lat Full Length Cells (clone 10.6) from Eric Verdin; HIV-1 LAV infected Jurkat E6 Cells (J1.1) from Thomas Folks; H9 and HIV-1 IIIB infected H9 Cells from Robert Gallo; HIV-1 p24 Gag Monoclonal (#24-2) from Michael H. Malim. Support for the confocal microscopy was provided by Alexander Jurkevich, Molecular Cytology Core, Bond Life Sciences Center, University of Missouri.
Funding: This work was supported by NIH AI120860 and GM103368 to S.G.S., and GM056901 to O.U., and the Gus T. Ridgel Fellowship awarded to O.U. by the University of Missouri Graduate School.
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- Human immunodeficiency virus
- In situ hybridization