Characterization of selected NS1 mutants reveals specific defects in infectious particle production.

Abstract

<p><b>(A)</b><i>Replication kinetics of NS1 mutants in the context of a sub-genomic replicon</i>. Schematic representation of the sub-genomic reporter replicon (sgDVR2A) is shown at the top. It is derived from the DV2 full-length genome by insertion of a <i>Renilla luciferase</i> coding sequence in-between the capsid cyclization sequence (CAE) and the <i>Tosea asigna</i> virus 2A cleavage site. The last 24 amino acid residues of the envelope coding region (TM) at the N-terminus of NS1 ensure proper membrane topology of the polyprotein after synthesis. Selected NS1 mutations affecting virus production were inserted into sgDVR2A, and <i>in vitro</i> transcribed RNAs were electroporated into VeroE6 cells. Luciferase activity was measured in the lysates 4, 24, 48 and 72 h later. Values are expressed as fold of the 4h-value which reflects transfection efficiency. The background of the assay is determined with the active site NS5 polymerase mutant (GND). Columns represent mean and standard deviations of three independent experiments. (<b>B</b>) <i>Intra- and extra-cellular infectivity titers of NS1 mutants</i>. The structure of the full-length DENV genome used for this analysis is shown at the top (DV). NS1 mutations specified at the bottom were inserted into DV and in vitro transcripts derived therefrom were transfected into Huh7 cells and used for determination of intra- and extracellular infectivity. Seventy-two hours post electroporation, supernatants and cell lysates were subjected to 3 freeze and thaw cycles and titers were determined by TCID₅₀ assay using Huh7 cells and an envelope protein-specific MAb. Mean values and standard deviations of three independent experiments are shown. (<b>C</b>) Evidence that NS1 mutations affect, in part, release of infectious DENV particles. To determine the relative contribution of defects in virus assembly and virus release, data presented in (B) were used to calculate the ratio of intra- vs. extra-cellular infectivity.</p