Figure 1: Limited compatibility of SC35M/bat NP chimeras to rescue and package viral genomes of SC35M.

(a) SC35M polymerase activity in the presence of Bat NP. HEK293T cells were transiently transfected with expression plasmids coding for PB2, PB1, PA of SC35M, the indicated NP proteins, a minigenome encoding the firefly luciferase and a Renilla luciferase expression plasmid to normalize for variations in transfection efficiency. In the negative control (Neg.) PB1 was omitted. Western blot analysis was performed to determine the expression levels of NP. (b) Cartoon depicting NP segments of A/SC35M (SC35M NP), HL17NL10 (Bat NP) or a NP segment (SC35M₂₅₀-NP_ORF-Bat) harbouring the non-coding regions of SC35M NP, 5′ and 3′ coding sequences of SC35M NP and the complete ORF of Bat NP. +successful rescue; −no rescue. (c) Relative SC35M polymerase activities in the presence of the mutant NP proteins (CH1–CH5). Mean and s.d. of three independent experiments are indicated in parenthesis. SC35M rescue experiments were performed with NP segments encoding the indicated mutant proteins. +successful rescue; −no rescue. (d) MDCKII cells were infected at an MOI of 0.001 with wt SC35M or rCH2. At the indicated time points post infection (p.i.), virus titres were determined by plaque assay. (e) Relative ratio of the viral transcript level in wt SC35M- or rCH2-infected cells. Steady state levels of viral transcripts (mRNA, cRNA and vRNA) and 5S ribosomal RNA (5S rRNA) were determined by primer extension analysis using total RNA from MDCKII cells infected at an MOI of 5 with wt SC35M or rCH2 for 6 h. Signal intensities were normalized to the signal intensities obtained with 5S rRNA. Normalized values obtained in wt SC35M-infected cells were set to 1 (all non-significant). (f) Viral infectivity of SC35M and rCH2 (PFU) using identical HA titre. **P<0.01. (g) Relative ratio of the number of viral particles (counted by electron microscopy) divided by the number of infectious particles (determined by plaque assay) between wt SC35M and rCH2. Values obtained for SC35M were set to 1. *P<0.05. (h) Ratio of incorporated NP protein in viral particles between SC35M and rCH2. Protein levels were determined by Western blot analysis of virus stocks with equal infectivity (PFU). **P<0.01. (i) Relative ratio of genome segments in viral particles preparations of SC35M and rCH2. RNA was prepared from virus stocks with equal PFU and subjected to quantitative RT-PCR. Levels of viral genome transcripts obtained with SC35M were set to 1. *P<0.05; **P<0.01; ***P<0.001. Student’s t test was used for two-group comparisons. Error bars indicate the mean and s.d. of at least three independent experiments.