Figure 1.
Schematic representations of the dual luciferase reporter constructs, and of transfection and infection assays.
A) Schematic outline of the firefly and Gaussia luciferase reporter constructs. The firefly and Gaussia 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 Gaussia luciferase reporter construct (GFsNP) additionally contains the 3′ terminal half of the firefly luciferase gene (indicated as Fs) behind the stop codon of the Gaussia 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 Gaussia luciferase expression levels can be measured consecutively using a dual luciferase assay system (Promega) 24 h post-transfection or post-infection.
Figure 2.
Competition between firefly and Gaussia luciferase reporter genome segments.
Plasmids encoding firefly (FNP) or Gaussia (GNP) luciferase reporter constructs were transfected alone (Single) or in combination (Co). Luciferase expression was induced by simultaneous co-transfection of polymerase and NP expression plasmids (transfection assay). A) Firefly luciferase activity after transfection of FNP or FNP together with GNP. B) Gaussia luciferase activity after transfection of GNP or GNP together with FNP. C) Normalized ratio of firefly to Gaussia luciferase activity (Fluc/Gluc) when FNP and GNP were transfected singly or in combination. D) Quantitative RT-PCR analysis of mRNA levels derived from FNP and GNP after single or co-transfection of these constructs. RNAs were extracted 24 h post-transfection and subjected to quantitative RT-PCR. 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 samples in which a single reporter construct was transfected. E) Normalized ratio of firefly to Gaussia luciferase activity (Fluc/Gluc) when reporter gene constructs FNP and GNP were transfected singly or in combination. The amounts of reporter gene constructs transfected are indicated. Significant differences in A–D are indicated (**; P<0,01).
Figure 3.
Competition for viral proteins.
Normalized ratio of firefly to Gaussia 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).
Figure 4.
The effect of gene length and segment UTR.
A) Plasmids encoding firefly (FNP) or Gaussia 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 Gaussia luciferase activity (Fluc/Gluc) after single or co-transfection of FNP and GNP or GFsNP (extended version). B) Normalized ratio of firefly to Gaussia luciferase activity (Fluc/Gluc) after single or co-transfection of FNP and different versions of the extended Gaussia 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 Gaussia luciferase reporter construct and the corresponding Gaussia luciferase expression levels after single transfection is shown.
Table 1.
vRNA segment lengths and UTR sequences of IAV-WSN used in the reporter constructs.
Figure 5.
Competition between reporter and natural influenza A virus genome segments.
Normalized luciferase activity of firefly (FNP) (A) or Gaussia (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).
Figure 6.
The effect of panhandle-stabilizing mutations in the UTR.
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 Gaussia luciferase activity (Fluc/Gluc) after single or co-transfection of FNP and different versions of the extended Gaussia reporter construct carrying either NP, NPph or NPphR UTRs (GFsNP, GFsNPph, and GFsNPphR, respectively). C) Normalized ratio of firefly to Gaussia 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 Gaussia reporter construct carrying either NP (GNP and GFsNP) or NPph (GNPph and GFsNPph) UTRs.
Figure 7.
Comparison of transfection and infection assay.
Luciferase activity of firefly (FNP or FNPph) or Gaussia (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.
Table 2.
Quantitative RT-PCR primers for mRNA of Gaussia or firefly luciferase reporter segments flanked by NP or NPph UTRs.