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

Abstract

<jats:p>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.</jats:p>

In-Cell NMR of Intact Mammalian Cells Preserved with the Cryoprotectants DMSO and Glycerol Have Similar DNP Performance

“…The most common cryoprotectant for cultured mammalian cells is dimethylsulfoxide (DMSO) at a concentration of 10% (v/v). Indeed, the handful of studies that examine preparations of mammalian cells using DNP NMR use DMSO ( Albert et al, 2018 ; Narasimhan et al, 2019 ; Schlagnitweit et al, 2019 ; Overall et al, 2020 ), although the sample composition—including the choice of cryoprotectant—and post-experiment cellular viability, have only very recently been considered ( Ghosh et al, 2020 ; Ghosh et al, 2021 ; Overall and Barnes, 2021 ). Given the widespread preference for 10% DMSO over 15% glycerol as the cryoprotectant for cellular cryopreservation, we sought to determine if cryoprotection using 10% DMSO could also support cellular viability throughout the DNP NMR experiments.…”

Biomolecular Perturbations in In-Cell Dynamic Nuclear Polarization Experiments

“…(20% DMSO with 20 mM AMUPol, 60/30/10 glycerol/D 2 O/H 2 O with 20 mM AMUPol or PBS with 20 mM AMUPol), to give a final AMUPol concentration of 10 mM ( Sauvee et al, 2013 ). The cells were then incubated for the indicated times before packing into 3.2 mm sapphire cylindrical rotors (Bruker Biospin) using custom made Teflon filling tools ( Overall et al, 2020 ) by centrifugation for 1 min at 1,500 rpm. Rotors were plunge frozen in liquid nitrogen for 10 min before capping with zirconia drive caps.…”

“…The first demonstration of in-cell DNP within intact mammalian cells showed that significant enhancements could be achieved in the context of intact cells with a range of polarizing agents ( Albert et al, 2019 ), with an added benefit that the DNP enhancement can be targeted to subcellular compartments. Subsequent studies used DNP-NMR to observe exogenously labelled ubiquitin electroporated into HeLa cells ( Narasimhan et al, 2019 ), drug mediated expression of HIV particles ( Overall et al, 2020 ) and antisense RNA drug complexes in HEK 293T cells ( Schlagnitweit et al, 2019 ). However, this potential remains largely untapped.…”

The Physical Chemistry and Chemical Physics (PCCP) Section of the International Journal of Molecular Sciences in Its Publications: The First 300 Thematic Articles in the First 3 Years

“…This area is the largest in terms of the number of related publications and includes 91 articles, namely [ 1 , 2 , 3 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 ,…”