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, Figure 1: The nature of M-36 residue impacts WNV infectious cycle by potentially 725 disrupting the M protein 3-dimensional structure

, Sequence comparison of M protein ectodomain and TM1 from different Flavivirus

, Residue 36 location is indicated in red and that of residue 43 in blue

, YFV 730 17D strain accession number MN708489.1, Zika virus accession number MG827392.1, WNV 731 accession number AF481864.1 and JEV accession number KF907505.1. (B): WNV 732 membrane protein precursor (prM) organization showing ectodomain (ectoM) and part of 733 transmembrane domain 1 (TM1) sequences. Residue at position 36 is indicated in red for WT 734 virus, black for M-I36F or grey M-I36A mutant viruses, DV3 accession number MK506265.1, YFV Asibi strain accession number AY640589, p.735

, C6/36 cell supernatants at times indicated and titrated by foci-forming assay (FFA) in Vero 736 cells. No statistical difference was observed, Foci morphology of wild-type WNV, p.737

M. I36f, mutated viral stocks collected from C6/36 supernatants, observed on Vero 738 cells. Vero cells were infected with the indicated virus and foci were observed

, SK-N-SH cells were 740 infected with the indicated virus at a MOI of 1, cell supernatants were collected at indicated 741 times for quantitation of virus titers by FFA using Vero cells. (F): Structure of M-E mature 742 heterodimers (PDB accession number 5wsn), Growth curves of wild-type, M-I36F and M-I36A mutant WNV, p.743

, The F36 aromatic ring clashes (in red) with 744 the side chain of the A43 located in the TMD-1. (G): Same as (H) with alanine at position M-745 36. The insert zooms into the A36-A43 contact, with A36 highlighted in pink and A43 in 746 green, No clash between A36 and A43 was observed. The image was generated using, p.747

. Pymol, The data are representative of 3 independent experiments and error bars indicate 748 standard deviation (SD). * p-value < 0, p.5

. **-p,

, Phenotypical characterization of WNV M-I36F and/or M-A43G mutant 752, vol.2