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

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

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 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

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

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

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

Primers.

a<p>The location of primers is relative to the full genome sequence of the PEDV CV777 strain (GenBank accession No. AF353511).</p>b<p>Endonuclease restriction sites used for cloning are indicated in bold.References.</p

Coronavirus genome organization and targeted RNA recombination scheme.

<p>(A) Genomic organization of PEDV. (B) Targeted RNA recombination scheme to make the interspecies chimeric virus mPEDV (Stage 1) or recombinant PEDV derivatives e.g. lacking the ORF3 gene as shown here (Stage 2). The ectodomain-encoding region of the MHV S gene is shown as a light-grey box in the mPEDV genome. Synthetic RNAs transcribed from the transfer vectors (Fig. 2A) were electroporated into PEDV (Stage 1) or mPEDV (Stage 2) infected cells, respectively. A single recombination event (indicated by a curved line) anywhere within the 3′ region of ORF1b present in the donor RNA and viral genome generates a recombinant genome. Selection of recombinant progeny viruses against parental viruses was done on the basis of the acquired ability to form plaques in murine cell monolayers (Stage 1) or on the basis of the ability to infect VERO cells and the concomitantly lost ability to infect murine cells (Stage 2).</p

Characterization of a PEDV recombinant virus lacking ORF3.

<p>(A) Genetic analysis of PEDV-ΔORF3. RT-PCR was performed covering the S-ORF3-E-M region (primers 4538/4977) using RNA templates isolated from wtPEDV, r-wtPEDV and PEDV-ΔORF3, and analyzed by gel electrophoresis. The expected sizes of the RT-PCR products (numbered 1 to 3) are indicated in the genome maps. For primer sequences, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069997#pone-0069997-t001" target="_blank">Table 1</a>. (B) Multi-step growth kinetics of r-wtPEDV and PEDV-ΔORF3. VERO cells were infected with each recombinant PEDV (MOI = 0.01), washed after three hours and viral infectivity in the culture media was determined at different times p.i. by a quantal assay on VERO cells from which TCID₅₀ values were calculated.</p