NS1 secretion is dispensable for the production of infectious DENV particles.

Abstract

<p>(<b>A</b>) <i>Schematic representation of the delta-NS1 DENV genome and helper cell lines employed</i>. A deletion comprising 97 codons in NS1 (Δ156–253) was introduced into the full-length DVR2A genome (DVR2A^ΔNS1). VeroE6 helper cells (right panel) containing the stably integrated pWPI expression vector without insert (CTRL), or encoding full-length <i>wild-type</i> NS1 (WT), or NS1 C-terminally fused with an HA epitope (HA) or NS1 C-terminally fused with a KDEL ER retention motif (KDEL), were generated by transduction with lentiviral vectors as described in materials and methods. (<b>B</b>) <i>Intra- and extra-cellular NS1 protein levels of VeroE6 helper cell lines</i>. Cell lysates or clarified supernatants were analyzed by immunoblotting, using the antibodies indicated on the right. The positions of molecular weight markers (kDa) are shown on the left. (<b>C-D</b>) <i>Replication efficiency and released infectivity of the ΔNS1 genome trans-complemented with NS1 variants</i>. (C) <i>In-vitro</i> RNA transcripts of DVR2A^ΔNS1 were electroporated into VeroE6 helper cell lines expressing NS1 variants specified in the top and replication efficiency was determined in cell lysates at 4, 24, 48 and 72 h after transfection. (<b>D</b>) Clarified supernatants containing infectious particles were harvested 72h.p.t. and used to infect each respective naïve VeroE6_helper cells. Luciferase activity in the lysates was measured 48h later (right panel). The background of the assay was determined by infecting näive VeroE6 cells lacking exogenous NS1 (CTRL) and therefore not supporting DVR2A^ΔNS1 replication. The dashed line indicates the limit of detection of the assay.</p