Effect of NS1 Alanine substitutions on DENV replication.

Abstract

<p>(<b>A</b>) <i>Schematic representation of the DENV reporter virus genome and NS1 protein domains</i>. The full-length luciferase reporter DENV genome (DVR2A) is shown at the top, with the 5’ and 3’ NTRs depicted with their putative secondary structures. Polyprotein cleavage products are separated by vertical lines and labeled as specified in the introduction. A <i>Renilla luciferase</i> coding sequence was inserted in-between the capsid cyclization sequence (CAE) and the <i>Tosea asigna</i> virus 2A cleavage site that ensures proper processing after polyprotein synthesis. NS1 protein domains shown below are indicated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005277#ppat.1005277.g001" target="_blank">Fig 1</a>. Glycosylation sites are shown with green hexagons, while the <i>β-roll</i>, <i>Wing</i> and <i>β-ladder</i> domains are shown in blue, yellow and red, respectively. Two <i>connector</i> sub-domains within the <i>Wing</i> domain are shown in orange. (<b>B</b>) <i>Replication kinetics of DENV NS1 mutants</i>. VeroE6 cells were electroporated with <i>in-vitro</i> transcribed luciferase virus RNAs containing NS1 mutations specified at the bottom. Cells were lysed 4, 24, 48, 72, 96 and 120 h after transfection and luciferase activity in cell lysates was determined. Data were normalized to the 4h-value that reflects transfection efficiency. The background of the assay was determined with the active site NS5 polymerase mutant (GND). Colored lines on the top of each panel refer to the color-coded representation of NS1 protein domains as shown in (A). NS1 mutants severely impaired in viral RNA replication (normalized luciferase activity ≤0) are underlined. For ease of comparison, data obtained with WT and GND are repeated in the left of each panel. Mean values and standard deviations of three independent experiments are shown.</p