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Open AccessArticle

In Situ Detection of Endogenous HIV Activation by Dynamic Nuclear Polarization NMR and Flow Cytometry

1
Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland
2
Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA
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Department of Biochemistry, Biophysics and Structural Biology, Washington University in St. Louis, St. Louis, MO 63110, USA
4
Departments of Chemistry and Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA
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Department of Medicine, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA
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Noguchi Memorial Institute for Medical Research, University of Ghana, Accra 00233, Ghana
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2020, 21(13), 4649; https://doi.org/10.3390/ijms21134649
Received: 4 June 2020 / Revised: 20 June 2020 / Accepted: 23 June 2020 / Published: 30 June 2020
(This article belongs to the Section Physical Chemistry and Chemical Physics)
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 increased 15N 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 million 15N-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. View Full-Text
Keywords: HIV; viral latency; dynamic nuclear polarization; in-cell NMR; solid-state NMR HIV; viral latency; dynamic nuclear polarization; in-cell NMR; solid-state NMR
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Overall, S.A.; Price, L.E.; Albert, B.J.; Gao, C.; Alaniva, N.; Judge, P.T.; Sesti, E.L.; Wender, P.A.; Kyei, G.B.; Barnes, A.B. In Situ Detection of Endogenous HIV Activation by Dynamic Nuclear Polarization NMR and Flow Cytometry. Int. J. Mol. Sci. 2020, 21, 4649.

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