*4.1. Materials*

Chromatography columns HiTrap Heparin (1 and 5 mL), Sephadex S200 and the chromatography equipment AKTA-explorer were from GE Healthcare (Little Chalfont, UK). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) reagents were from BioRad (Hercules, CA, USA). Unless otherwise stated, other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

#### *4.2. Flavivirus C Proteins Primary, Secondary and Tertiary Structural Predictions*

For primary structure alignments we used the 16 non-DENV *Flavivirus* polyprotein sequences identified in reference [14], plus the four DENV reference sequences from NCBI, namely: DENV serotype 1, strain 45AZ5, NCBI ID NP\_059433.1; DENV serotype 2, strain New Guinea C, NCBI ID NP\_056776.2; DENV serotype 3, strain D3/H/IMTSSA-SRI/2000/1266, NCBI ID YP\_001621843.1; and, DENV serotype 4, strain rDEN4, NCBI ID NP\_073286.1. For the phylogenetic trees, both the entire polyproteins and the C protein regions were used. For the alignments and subsequent data analysis, the residues next to the NS2B-NS3 protease cleavage site [64,65] were excluded, leaving only the C protein sequences. Alignments and the derived phylogenetic trees were performed via Clustal Omega web tool (http://www.ebi.ac.uk/Tools/msa/clustalo/) [66,67].

Statistical comparison of the disordered N-terminal plus flexible fold regions with the conserved fold region of *Flavivirus* C proteins, for G and P content, as well as charged amino acid residues, was performed via a paired *t*-test, using GraphPad Prism v5 software. *p*-values were always lower than 0.001.

Predictions of hydrophobicity and α-helix propensity were done using ProtScale server (http://web.expasy.org/protscale/) [26,27], tertiary structure predictions were performed via I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) [28–30], following previous approaches [15]. Briefly, *Flavivirus* C protein sequences from our previous work were employed [14]. DENV and WNV (serotype Kunjin) C structures were excluded, not serving as templates for the tertiary structure prediction. ZIKV C protein structure was also not included, as it was not yet determined when the modeling was conducted. This avoids a bias towards known homologous protein structures. Five I-TASSER models were obtained for each C protein sequence. These were superimposed with DENV C experimental structure (PDB ID 1R6R, model 21) [12] after root-mean-square deviation (RMSD) minimization in UCSF Chimera v1.9 software [68]. Clusters were formed based on the visual similarity between predictions. The number of N-terminal amino acid residues with backbone clashes with the other monomer backbone was calculated for each model. In our previous work [15], a DENV C predicted structure was excluded from further analysis if it had 6 clashes or more, as it would not be viable as an homodimer [15]. Here we excluded models with more than 5 clashes (28 models rejected). These would preclude homodimer formation and, thus, were not considered in the clusters analysis (Table 1 excluded models column).

#### *4.3. Structure Comparison Between DENV C and Influenza NS1*

Protein structures coordinates were extracted from the Protein Data Bank (PDB, www.pdb.org). PDB identification codes are specified ahead after each protein name. The protein structures were superimposed through UCSF Chimera 1.13.1 software MatchMaker tool. After that, we carefully analyzed the superposition visually. Then, using the Match-Align tool of UCSF Chimera, which returns a sequence alignment based on the regions and taking into account the structure superimposition, we identified the residues simultaneously similar in structure and sequence. Protein structure figures were obtained using UCSF Chimera 1.13.1 version [68].
