C-terminally mCherry-tagged NS1 supports DENV RNA replication and virus production and co-localizes with envelope and capsid upon ΔNS1^TCP infection.

Abstract

<p>(<b>A</b>) <i>Sub-cellular distribution of NS1</i>^{<i>mCherry</i>}. Two days post-cell seeding, CTRL, NS1^WT- or NS1^mCherry-expressing cells were fixed, permeabilized and NS1 was detected with a rabbit polyclonal NS1-specific primary and AlexaFluor-488-conjugated secondary antibody. Cells were analyzed by confocal microscopy as described in materials and methods. The upper panel depicts a schematic representation of the NS1^mCherry fusion protein. (<b>B</b>) <i>Replication competence of ΔNS1</i>^<i>TCP</i><i>in VeroE6_NS1</i>^{<i>mCherry</i>}<i>cells</i>. VeroE6 cells expressing no NS1 (CTRL) or NS1^WT or NS1^mCherry were infected with 1 MOI of ΔNS1^TCP and luciferase activity was measured in the lysates 24, 48 and 72 h post-infection. Released infectivity was assessed by inoculating NS1^WT-expressing cells with culture media collected 72 h p.i. and measuring luciferase activity 48 h later. The dashed lines indicate the limit of detection of the assay. (<b>C-D</b>) <i>Co-localization and 3D reconstruction of NS1 with the structural proteins capsid and envelope</i>. (C) VeroE6 cells specified on the left, were mock-infected (Non-Infected) or infected with 1 MOI of ΔNS1^TCP. Two-days later, cells were fixed, permeabilized and stained with E- and capsid-specific antibodies. Cells were analyzed by confocal microscopy. Scale bars represent 10 μm. (D) Deconvolved fluorescence image of VeroE6_NS1^mCherry cells infected with ΔNS1^TCP particles. VeroE6 cells stably expressing C-terminally tagged NS1^mCherry were infected with ΔNS1^TCP as described above. Two-days later, cells were fixed, permeabilized and stained with E- and capsid-specific antibodies. <i>Left panel</i>: representative image of an NS1^mCherry- ΔNS1^TCP-infected cell 48 h p.i. (scale bar represents 2 μm). Boxed areas are enlarged in the numbered panels on the right (scale bar represent 300 nm). Overview and enlargements show 3D reconstructed images created with the Imaris software as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005277#sec010" target="_blank">Materials and Methods</a>.</p