In situ detection of endogenous HIV activation by dynamic nuclear polarization nmr and flow cytometry

Sarah A. Overall, Lauren E. Price, Brice J. Albert, Chukun Gao, Nicholas Alaniva, Patrick T. Judge, Erika L. Sesti, Paul A. Wender, George B. Kyei, Alexander B. Barnes

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

We demonstrate for the first time in-cell dynamic nuclear polarization (DNP) in conjunction with flow cytometry sorting to address the cellular heterogeneity of in-cell samples. Utilizing a green fluorescent protein (GFP) reporter of HIV reactivation, we correlate increased15N resonance intensity with cytokine-driven HIV reactivation in a human cell line model of HIV latency. As few as 10% GFP+ cells could be detected by DNP nuclear magnetic resonance (NMR). The inclusion of flow cytometric sorting of GFP+ cells prior to analysis by DNP-NMR further boosted signal detection through increased cellular homogeneity with respect to GFP expression. As few as 3.6 million15N-labeled GFP+ cells could be readily detected with DNP-NMR. Importantly, cell sorting allowed for the comparison of cytokine-treated GFP+ and GFP− cells in a batch-consistent way. This provides an avenue for normalizing NMR spectral contributions from background cellular processes following treatment with cellular modulators. We also demonstrate the remarkable stability of AMUPol (a nitroxide biradical) in Jurkat T cells and achieved in-cell enhancements of 46 with 10 mM AMUPol, providing an excellent model system for further in-cell DNP-NMR studies. This represents an important contribution to improving in-cell methods for the study of endogenously expressed proteins by DNP-NMR.

Original languageEnglish
Article number4649
Pages (from-to)1-13
Number of pages13
JournalInternational journal of molecular sciences
Volume21
Issue number13
DOIs
StatePublished - Jul 1 2020

Keywords

  • Dynamic nuclear polarization
  • HIV
  • In-cell NMR
  • Solid-state NMR
  • Viral latency

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