Oxysterols

We adopted a charge-tagging approach utilising “enzyme-assisted derivatisation for sterol analysis” (EADSA) to enhance liquid chromatography (LC) separation and mass spectrometry (MS) detection of oxysterols [17 (link),25 (link),26 (link)]. This involves the stereospecific enzymatic oxidation of the 3β-hydroxy-5-ene function in oxysterols (and sterols) to a 3-oxo-4-ene group and subsequent reaction with the cationic Girard P (GP) hydrazine reagent to give charged GP-hydrazones compatible with chromatographic separation using reversed phase solvents and high-sensitivity analysis by electrospray ionisation (ESI)-MS and MS with multistage fragmentation (MSⁿ) (Fig. S1). GP-derivatives give intense [M]⁺ ions in ESI and informative MS² and MS³ spectra. As some oxysterols naturally contain a oxo group they give GP-derivatives even in the absence of oxidising enzyme. However, oxysterols containing a native 3-oxo group are readily differentiated from oxysterols oxidised to contain one by dividing each sample in two and performing derivatisation without oxidising enzyme on one portion of the sample (Fraction B) and performing derivatisation with added enzyme on the second portion (Fraction A), and by exploiting differentially isotope labelled GP reagents to allow discrimination by mass (Fig. S1).
Unless otherwise indicated, materials and methods are as described by Griffiths and co-workers [17 (link),25 (link),26 (link)]. Quantification was achieved using isotope dilution mass spectrometry. No hydrolysis or solvolysis steps were performed. 3β,7α-Dihydroxycholest-5-en-(25S)26-oic acid was supplied as a mixture with 3β,7α-dihydroxycholest-5-en-(25R)26-oic acid (1:3, mole:mole) as the [3α,7β-²H₂] compounds by Avanti Polar Lipids (Alabaster, AL, USA).

Incorporation of [¹⁴C] oleate into cholesteryl [¹⁴C] oleate in cultured cells after oxysterol addition was determined using previously described methods (Goldstein et al., 1983 (link)).

All cell culture steps were carried out in an incubator with 5% CO₂ at the indicated temperatures. On day 0, Huh7.5 or Huh7.5^ΔACAT cells were set up in medium F at a density of 8x10⁴ cells per well of a 24-well plate. On day 1, the media was removed and replaced with 1 ml of medium F supplemented with 5 µM of the indicated oxysterol. After 4 hr at 37 °C, the media was removed and replaced with 200 µl of either hCoV-OC43 virus or ZIKV-MR766-Venus virus in OptiMEM and incubated at 33 °C or 37 °C, respectively. After 1 hr, 800 µl of medium F was added to each well and maintained at either 33 °C or 37 °C as indicated above. After 24 hr, cells were harvested with Accumax, mixed with a 4% (v/v) stock solution of PFA in PBS (1% final concentration), and incubated for 10 min at RT, following which cells were analyzed by FACS either immediately or stored at 4 °C and processed at a later time.
Zika-infected cells were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatants were removed, and the cell pellets were resuspended in 150 µl of buffer E prior to FACS analysis.
OC43-infected cells were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatants were removed, and the cell pellets were resuspended in 50 µl of BD Cytofix/Cytoperm Solution. After incubation for 20 min at RT, 150 µl of BD Wash/Perm Solution was added to each sample, after which samples were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cell pellets were resuspended in 200 µl of BD Wash/Perm Solution, and subjected to centrifugation at 1500 x g for 5 min at RT. After removing the supernatant, cell pellets were resuspended in 50 µl of anti-coronavirus group antigen antibody (1:50). After incubation for 30 min at RT, 150 µl of BD Wash/Perm Solution was added, and samples were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cell pellets were resuspended in 200 µl of BD Wash/Perm Solution and subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cell pellets were resuspended in 50 µl of Goat anti-mouse AlexaFluor488 (1:1000). After incubation in the dark (wrapped in aluminum foil) for 30 min at RT, 150 µl of BD Wash/Perm Solution was added and samples were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cells were resuspended in 200 µl of BD Wash/Perm Solution and subjected to centrifugation at 1500 x g for 5 min at RT. After this final step, the supernatant was removed, and cells were resuspended in 150 µl of buffer E and analyzed on a Stratedigm S1000 EX Flow Cytometer using the GFP channel. Infection levels were determined as the percentage of Venus-positive cells (indicating Zika infection) or AlexaFluor488-positive cells (indicating OC43 infection) from at least 7500 single cells per replicate, as assessed by FlowJo software.