A forward genetic screen identifies pseudoreversions in NS4A and NS4B compensating replication-inactivating mutations in NS1.

(A) Homology model of the NS1 dimer based on the DENV NS1 structure (PDB 4O6B) with missing residues modelled according to the ZIKV NS1 structure (PDB 5K6K). The model was built using the MOE 2015 software package and molecular graphics were prepared with UCSF Chimera [66]. Wing, β-ladder and β-roll domains are shown in blue, turquoise and orange, respectively. The connector subdomains in Wing domain are shown in dark blue. The membrane is indicated with a horizontal line. (B) Left panel: schematic of the experimental approach used to select for pseudoreversions rescuing replication of NS1 mutants. Point mutations in NS1 were inserted into a selectable subgenomic DENV replicon (sgDVH2A) encoding a hygromycin phosphotransferase gene (HygroR) downstream of by the cyclisation sequence residing in the capsid-coding region (CS) and upstream of the 2A protease of Thosea asigna virus (2AP) and the signal sequence of NS1 corresponding to the last 24 amino acid residues of E (ET). VeroE6 cells were electroporated with selectable replicon RNAs containing given mutations and cultured in the presence of hygromycin B. After three to four weeks, single cell clones were propagated; total RNA was extracted and amplified for sequence analysis. Right panel: number of hygromycin B-resistant cell colonies obtained after selection for each primary mutation in NS1 (indicated at the bottom) with the color of the bar corresponding to the color of the NS1 domain into which the respective mutation was introduced. (C) Schematic summarizing the localization of second site mutations in the NS4A-2K-NS4B polyprotein that is drawn according to its hypothetical membrane topology [7,36]. The circle color of primary mutations in NS1 (lower left) corresponds to pseudoreversions identified for each respective mutant.