vRNA segment lengths and UTR sequences of IAV-WSN used in the reporter constructs.

a<p>The conserved regions in the 3′ and 5′ UTRs are underlined. The start and stop codons are italicized. The bold characters indicate the mutated nucleotides.</p

The effect of gene length and segment UTR.

<p>A) Plasmids encoding firefly (FNP) or <i>Gaussia</i> luciferase reporter constructs were transfected alone (Single; s) or in combination (Co; c). Luciferase expression was induced by simultaneous co-transfection of polymerase and NP expression plasmids (transfection assay). A) Normalized ratio of firefly to <i>Gaussia</i> luciferase activity (Fluc/Gluc) after single or co-transfection of FNP and GNP or GFsNP (extended version). B) Normalized ratio of firefly to <i>Gaussia</i> luciferase activity (Fluc/Gluc) after single or co-transfection of FNP and different versions of the extended <i>Gaussia</i> reporter construct carrying UTRs derived from the eight IAV-WSN genome segments. C) Correlation between fold inhibition of firefly luciferase expression upon co-transfection of a <i>Gaussia</i> luciferase reporter construct and the corresponding <i>Gaussia</i> luciferase expression levels after single transfection is shown.</p

Schematic representations of the dual luciferase reporter constructs, and of transfection and infection assays.

<p>A) Schematic outline of the firefly and <i>Gaussia</i> luciferase reporter constructs. The firefly and <i>Gaussia</i> luciferase genes, flanked by 3′ and 5′ UTR of the NP segment, were inserted in antisense orientation between a PolI promoter and a ribozyme sequence, resulting in FNP and GNP, respectively. The extended <i>Gaussia</i> luciferase reporter construct (GFsNP) additionally contains the 3′ terminal half of the firefly luciferase gene (indicated as Fs) behind the stop codon of the <i>Gaussia</i> gene. B) HEK 293T cells were transfected with one or both reporter constructs (single or co-transfection). Luciferase expression is induced by expression of viral RNA polymerases (PB1, PB2, PA) and NP either by simultaneous co-transfection of expression plasmids (transfection assay) or by infection with IAV at an MOI 1 at 24 h post-transfection (infection assay). The firefly and <i>Gaussia</i> luciferase expression levels can be measured consecutively using a dual luciferase assay system (Promega) 24 h post-transfection or post-infection.</p

Competition between reporter and natural influenza A virus genome segments.

<p>Normalized luciferase activity of firefly (FNP) (A) or <i>Gaussia</i> (GNP) (B) luciferase reporter constructs after co-transfection with empty plasmid (pUC18) or transcription plasmids encoding one of the eight IAV-WSN vRNA segments using the transfection assay. C) Correlation between fold-inhibition of firefly luciferase activity upon co-transfection of one of the eight IAV-WSN vRNA encoding plasmids and the length of the vRNA segments. Significant differences in A and B are indicated (*; P<0,05).</p

Competition for viral proteins.

<p>Normalized ratio of firefly to <i>Gaussia</i> luciferase activity (Fluc/Gluc) after single (Single) or co-transfection (Co) of FNP and GNP in the presence of increasing amounts of transfected plasmids encoding PB1, PB2, PA and NP (A), NP alone (B) or PB1, PB2 and PA (C).</p

Comparison of transfection and infection assay.

<p>Luciferase activity of firefly (FNP or FNPph) or <i>Gaussia</i> (GNP or GNPph) luciferase reporter constructs using the transfection (A) or infection assay (B). B) Cells were infected with IAV-WSN at an MOI of 1 TCID50 units per cells, which resulted in approximately 50% infected cells as determined by immunocytochemical analysis using NP-specific antibodies. C) Fold difference in luciferase expression levels between FNPph and FNP and between GNPph and GNP in either the transfection or infection assay. D) Quantitative RT-PCR analysis of mRNA levels derived from GNP or GNPph using the transfection (trans) or infection (inf) assay. For the transfection assay, cells were co-transfected with expression plasmids encoding PB1, PB2, PA and NP. For the infection assay, cells transfected with reporter plasmids were infected with IAV-WSN. The comparative Ct method was used to determine the relative mRNA levels using the housekeeping gene GAPDH as a reference. The mRNA levels were normalized relative to the mRNA expression level of the GNP reporter construct in the transfection assay.</p

The effect of panhandle-stabilizing mutations in the UTR.

<p>A) Schematic representation of the proposed conformational structure of IAV-WSN wild type NP UTR in corkscrew or panhandle conformation (left panel; refs 10 and 30) and the improved base-pairing by panhandle-stabilizing mutations in the 3′ (NPph) or 5′ (NPphR) UTR (right panel). B) Normalized ratio of firefly to <i>Gaussia</i> luciferase activity (Fluc/Gluc) after single or co-transfection of FNP and different versions of the extended <i>Gaussia</i> reporter construct carrying either NP, NPph or NPphR UTRs (GFsNP, GFsNPph, and GFsNPphR, respectively). C) Normalized ratio of firefly to <i>Gaussia</i> luciferase activity (Fluc/Gluc) after single or co-transfection of firefly luciferase constructs with NP or NPphs UTR (FNP and FNPph, respectively) and the short or extended <i>Gaussia</i> reporter construct carrying either NP (GNP and GFsNP) or NPph (GNPph and GFsNPph) UTRs.</p

Reactivity of recombinant F proteins with neutralizing antibodies.

<p>A) ELISA analysis of recombinant F proteins. Purified F proteins were coated on 96-well plates; when indicated samples were treated with TPCK trypsin (+ T) prior to coating. The reactivity of the recombinant proteins with different neutralizing MAbs was analyzed by applying 2-fold serial dilutions of Palivizumab (starting with 0.375 µg/ml), AM22 (starting with 3.5 µg/ml) or D25 (starting with 5 µg/ml). Binding of the antibodies was detected using HRP-conjugated secondary antibodies. Fwt-GCN and Flys-GCN contain a C-terminal LysM domain and ST3 tag. Fwt and Flys contain a C-terminal ST3 tag. B) Neutralization of RSV by MAbs. The amount needed of each MAb to achieve 50% neutralization of virus infectivity is graphed. The error bars indicate the standard deviations (* P<0.05 in Student’s t test).</p

Binding of recombinant F proteins to BLPs.

<p>A) Binding efficacy of the various F proteins (Fwt, Fwt-GCN, Flys, and Flys-GCN; all carrying a C-terminal LysM domain, but no ST3 tag) to BLPs was determined by incubation of an equal amount (28 µg) of the F proteins with 1 mg of BLPs using standard conditions. The amount of F protein bound to the BLPs was determined by comparative SDS-PAGE analysis in which Colloidal Blue staining of F proteins was compared with that of BSA standards. B) To analyze the binding pattern of F to BLPs, BLPs carrying Flys-GCN with a LysM domain (BLP-F) were generated using standard conditions followed by incubation with Palivizumab and FITC-labeled goat-anti-human secondary antibodies. As a control “empty” BLPs were used. The preparations were analyzed using bright field and fluorescence microscopy. C) BLPs displaying Fwt-GCN or Flys-GCN were analyzed using bright field microscopy.</p

Schematic representation of the different recombinant soluble RSV F protein constructs.

<p>RSV F proteins lacking the transmembrane domain (TM) and cytoplasmic tail (CT) were genetically fused to a CD5 signal peptide (CD5) and to a carboxy-terminal tag (tag). When indicated a GCN4 trimerization motif (GCN) was introduced between the F protein and the tag. The tag either consisted of a triple Strep-tagII, a LysM peptidoglycan binding domain, or of a combination of the two (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071072#pone.0071072.s001" target="_blank">Fig. S1</a>). The F2 and F1 subunits of F are indicated, as well as the p27 peptide (P27) that is released after furin cleavage. Protease cleavage sites are indicated by black arrows. Grey arrows indicate mutated furin cleavage sites. The approximate location of the fusion peptide (FP), heptad repeat A (HRA) and B (HRB) is also shown.</p