Multiplex single-cell visualization of nucleic acids and protein during HIV infection

Maritza Puray-Chavez, Philip R. Tedbury, Andrew D. Huber, Obiaara B. Ukah, Vincent Yapo, Dandan Liu, Juan Ji, Jennifer J. Wolf, Alan N. Engelman and Stefan G. Sarafianos ()
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Maritza Puray-Chavez: University of Missouri
Philip R. Tedbury: University of Missouri
Andrew D. Huber: University of Missouri
Obiaara B. Ukah: University of Missouri
Vincent Yapo: University of Missouri
Dandan Liu: University of Missouri
Juan Ji: University of Missouri
Jennifer J. Wolf: University of Missouri
Alan N. Engelman: Dana-Farber Cancer Institute
Stefan G. Sarafianos: University of Missouri

Nature Communications, 2017, vol. 8, issue 1, 1-11

Abstract: Abstract Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. To address this need we develop a microscopy approach, multiplex immunofluorescent cell-based detection of DNA, RNA and Protein (MICDDRP), which is based on branched DNA in situ hybridization technology. MICDDRP enables simultaneous single-cell visualization of HIV (a) spliced and unspliced RNA, (b) cytoplasmic and nuclear DNA, and (c) Gag. We use MICDDRP to visualize incoming capsid cores containing RNA and/or nascent DNA and follow reverse transcription kinetics. We also report transcriptional “bursts” of nascent RNA from integrated proviral DNA, and concomitant HIV-1, HIV-2 transcription in co-infected cells. MICDDRP can be used to simultaneously detect multiple viral nucleic acid intermediates, characterize the effects of host factors or drugs on steps of the HIV life cycle, or its reactivation from the latent state, thus facilitating the development of antivirals and latency reactivating agents.

Date: 2017
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DOI: 10.1038/s41467-017-01693-z

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