Visualization of HIV-1 RNA Transcription from Integrated HIV-1 DNA in Reactivated Latently Infected Cells
Obiaara B. Ukah,
Maritza Puray-Chavez,
Philip R. Tedbury,
Alon Herschhorn,
Joseph G. Sodroski,
Stefan G. Sarafianos
Affiliations
Obiaara B. Ukah
CS Bond Life Sciences Center, University of Missouri, Columbia, MO 65201, USA
Maritza Puray-Chavez
CS Bond Life Sciences Center, University of Missouri, Columbia, MO 65201, USA
Philip R. Tedbury
Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65212, USA
Alon Herschhorn
Division of Infectious Diseases and International Medicine, Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
Joseph G. Sodroski
Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA
Stefan G. Sarafianos
Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65212, USA
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.
