FIG 3.

The DENV-2 NS1 protein is highly tolerant of insertions. (A) The insertional mutagenesis profile within the NS1-encoding region of the DENV-2 genome was examined with respect to NS1 protein domains. In the NS1 schematic the β-roll, Wing, and β-ladder domains are colored blue, yellow, and red, respectively, while connector subdomains are depicted in orange and the location of N-glycosylation sites (N-glyc) are indicated in green. The graph shows an overlay of the number of raw reads for each transposon insertion site in pool 0 (cyan), pool 1 (magenta), and pool 2 (yellow) and where they overlap, according to the legend to the right of the graph. The x axis shows the DENV-2 nucleotide positions of the NS1-encoding region (nt 2422 to 3477). (B) The location of the 10 most tolerated transposon insertion sites in the NS1 protein, provided that at least two nucleotides within the given codon preceded tolerated insertions, are shown as green van der Waals spheres in a ribbon representation of the DENV NS1 dimer structure (PDB accession no. 4O6B) (also see Data Set S1 in the supplemental material). One monomer is shown in gray, and the other monomer is colored according to domains as in panel A. Insertions at Lys-174, Thr-126, and Ser-204 were highly tolerated. As shown in the perpendicular view (right panel), the most tolerated mutations are relatively distant from the inner hydrophobic face that is pointed downward.