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Nat Commun. 2017 Dec 1;8(1):1882. doi: 10.1038/s41467-017-01693-z.

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

Puray-Chavez M1,2, Tedbury PR1,2,3, Huber AD1,4, Ukah OB1,2, Yapo V1,2, Liu D1,2, Ji J1, Wolf JJ1,2, Engelman AN5,6, Sarafianos SG7,8,9,10,11.

Author information

1
CS Bond Life Sciences Center, University of Missouri, Columbia, MO, 65211, USA.
2
Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO, 65212, USA.
3
Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, 30332, USA.
4
Department of Veterinary Pathobiology, University of Missouri, Columbia, MO, 65211, USA.
5
Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA.
6
Department of Medicine, Harvard Medical School, Boston, MA, 02115, USA.
7
CS Bond Life Sciences Center, University of Missouri, Columbia, MO, 65211, USA. sarafianoss@missouri.edu.
8
Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO, 65212, USA. sarafianoss@missouri.edu.
9
Department of Veterinary Pathobiology, University of Missouri, Columbia, MO, 65211, USA. sarafianoss@missouri.edu.
10
Department of Biochemistry, University of Missouri, Columbia, MO, 65201, USA. sarafianoss@missouri.edu.
11
Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, 30332, USA. sarafianoss@missouri.edu.

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.

PMID:
29192235
PMCID:
PMC5709414
DOI:
10.1038/s41467-017-01693-z
[Indexed for MEDLINE]
Free PMC Article

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