Multiparametric characterization of rare HIV-infected cells using an RNA-flow FISH technique.

TitleMultiparametric characterization of rare HIV-infected cells using an RNA-flow FISH technique.
Publication TypeJournal Article
Year of Publication2017
AuthorsBaxter AE, Niessl J, Fromentin R, Richard J, Porichis F, Massanella M, Brassard N, Alsahafi N, Routy J-P, Finzi A, Chomont N, Kaufmann DE
JournalNat Protoc
Volume12
Issue10
Pagination2029-2049
Date Published10/17/2017
ISSN1750-2799
KeywordsCD4-Positive T-Lymphocytes, Flow Cytometry, Fusion Proteins, gag-pol, HIV Infections, Humans, In Situ Hybridization, Fluorescence, Limit of Detection, RNA, Messenger, RNA, Viral
Abstract

Efforts to cure HIV are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in individuals receiving antiretroviral therapy (ART). We describe a protocol for flow cytometric identification of viral reservoirs, based on concurrent detection of cellular HIV Gagpol mRNA by in situ RNA hybridization combined with antibody staining for the HIV Gag protein. By simultaneously detecting both HIV RNA and protein, the CD4 T cells harboring translation-competent virus can be identified. The HIV(RNA/Gag) method is 1,000-fold more sensitive than Gag protein staining alone, with a detection limit of 0.5-1 Gagpol mRNA(+)/Gag protein(+) cells per million CD4 T cells. Uniquely, the HIV(RNA/Gag) assay also allows parallel phenotyping of viral reservoirs, including reactivated latent reservoirs in clinical samples. The assay takes 2 d, and requires antibody labeling for surface and intracellular markers, followed by mRNA labeling and multiple signal amplification steps.

DOI10.1038/nprot.2017.079
Alternate JournalNat Protoc
PubMed ID28880280
Grant ListR01 HL092565 / HL / NHLBI NIH HHS / United States
UM1 AI100663 / AI / NIAID NIH HHS / United States
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