Non essential amino acids (neaa)

ZIKV strain MR766 was originally provided by Dr. Andrew Oberst (University of Washington, Seattle, WA, USA). ZIKV-DAKAR-MA was first generated [7 (link)] and generously provided by Dr. Michael Diamond (Washington University, St. Louis, MO, USA). Viral stocks were generated by infecting Vero cells (MOI 0.01) and harvesting supernatants at 72hpi. Viral titers of stocks were determined via plaque assay on Vero cells (ATCC, #CCL-81). Cells were maintained in DMEM (Corning #10-013-CV) supplemented with 10% Heat Inactivated FBS (Gemini Biosciences #100-106), 1% Penicillin–Streptomycin-Glutamine (Gemini Biosciences #400-110), 1% Amphotericin B (Gemini Biosciences #400–104), 1% Non-Essential Amino Acids (Cytiva #SH30238.01), and 1% HEPES (Cytiva SH30237.01). Plaque assay basal media was 10X EMEM (Lonza # 12-684F) adjusted to 1X and supplemented with 2% Heat Inactivated FBS (Gemini Biosciences #100-106), 1% Penicillin–Streptomycin-Glutamine (Gemini Biosciences #400-110), 1% Amphotericin B (Gemini Biosciences #400-104), 1% Non-Essential Amino Acids (Cytiva #SH30238.01), and 1% HEPES (Cytiva SH30237.01), 0.75% Sodium Bicarbonate (VWR #BDH9280) and 0.5% Methyl Cellulose (VWR #K390). Plaque assays were developed 4dpi by removal of overlay media and staining/fixation using 10% neutral buffered formalin (VWR #89370) and 0.25% crystal violet (VWR #0528).

Two human SHH-MB cell lines, Daoy and ONS76, were originally obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Daoy cells were cultured in Eagle’s minimum essential medium (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Cytiva) and 1% penicillin-streptomycin (PS) (Cytiva). ONS76 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% PS. HEK293FT cells obtained from Invitrogen (ThermoFisher Scientific, Waltham, MA, USA) were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) (Cytiva) supplemented with 10% FBS, 1% PS, and 1% non-essential amino acids (Cytiva). Cells were incubated at 37 °C in 5% CO₂ and were routinely passaged upon 90% confluence.

Bone marrow-derived macrophages (BMDMs) were generated from 6- to 10-week-old female C57BL/6 mice (Jackson Laboratories) as previously described (55 (link)). Cells recovered from murine femurs were incubated for 5 days in Dulbecco’s modified Eagle medium (DMEM) with 4.5 g/L glucose (Corning) containing 10% heat-inactivated fetal bovine serum (FBS) (R&D Systems), 20 ng/mL recombinant mouse macrophage colony-stimulating factor (MCSF) (Peprotech), 1 mM l-glutamine (Gibco), 1 mM HEPES (Gibco), and 1 mM nonessential amino acids (Cytiva) (complete DMEM [cDMEM]). On day 2, nonadherent cells were spun down and reseeded in new cDMEM. On day 4, cells were refed with fresh cDMEM and seeded on day 5 for infections. BMDMs were routinely checked for macrophage markers, with >90% of cells being positive for F4/80 and CD11b and negative for CD11c (data not shown). Mouse embryonic fibroblasts (MEFs) were maintained in DMEM with 4.5 g/L glucose and 10% heat-inactivated FBS. Wild-type and AMPK-deficient (AMPK^−/−) MEFs were a generous gift from Nathanial Moorman from the University of North Carolina—Chapel Hill, Chapel Hill, NC. J774A.1 (ATCC TIB-67) macrophages were maintained in DMEM supplemented with 4.5 g/L glucose, 10% heat-inactivated FBS, 2 mM l-glutamine, and 1 mM sodium pyruvate (Cytiva). All tissue culture lines were maintained at 37°C with 5% CO₂.