Amino acids

E. coli K 12 strain RP437 (CGSC 12122) with a tetracysteine tag (TC tag) insertion (RP437-TC-FliC strain) was used in this study for flagellar growth measurement. The RP437-TC-FliC strain was constructed by inserting the optimized TC tag (FLNCCPGCCMEP) with a chloramphenicol resistance gene and ccdB under control of the rhamnose promoter into the chromosomal wild-type fliC gene using the lamda-Red system. Specifically, the TC tag was incorporated after the proline residue at amino acid position 285 in the D-loop domain of the FliC protein^{25 (link)}. To eliminate the antibiotic resistance and ccdB, the construct was selected by survival on rhamnose minimal medium (100 mM potassium phosphate (pH 7.0) (11.2 g/l K₂HPO₄, 4.8 g/l KH₂PO₄), 15 mM (NH₂)₂SO₄, 1 mM MgSO₄, 2 mM Fe₂ (SO₄)₃) with 25 mM rhamnose (Sigma Aldrich) and MEM vitamins (×100, Invitrogen) and amino acids (×50, Invitrogen))^{41 (link), 42 (link)}. For the flagellin overexpression assay, TC-FliC was expressed on the plasmid pBAD18 in the strain RP437-TC-FliC under the control of the arabinose-inducible promoter, maintained by the addition of ampicillin (50 µg/ml) to the growth medium. The presence of the tetracysteine tag in the fliC gene was verified by colony PCR and sequencing. Strains and primers used are listed in Supplementary Tables 1 and 2.

Two cell cultures were used in the study. The culture of rat astroglial cells was obtained with the modified method of McCarthy and de Vellis [40 (link)] from the brain of two-to three-day-old rats. Experiments were carried out using outbred animals kept in a vivarium on a standard diet. The experiments were approved by the Bioethics Committee of the A.N.Belozersky Research Institute of Physico-Chemical Biology of Moscow State University. A commercial C6 glioma cell line from the collection of cell lines of the Belozersky Institute was used as the second cell culture. Cells were cultivated in the complete medium containing DMEM/F-12 (1:1), 10% of a fetal bovine (PAA Laboratories, USA) supplemented with 1% glutamine, 1% vitamins (Paneco, Russia), and 2% amino acids (Gibco, Waltham, MA, USA).

African green monkey Vero cells were grown in MEM 1× + Glutamax (Gibco, Billings, MT, USA) supplemented with 10% foetal bovine serum, antibiotics and aminoacids (Gibco, Billings, MT, USA). ZIKV H/PF/2013 strain of the Asian genotype (Baronti et al., 2014) was grown in Vero cells and titrated by plaque assay. AmphB and Methyl-β-Cyclodextrin (MβCD) were purchased by Gibco and Sigma-Aldrich (St. Louis, MO, USA) respectively.