Figure 3: A subset of Bat NP-specific amino acids of CH4 causes severe attenuation and irregular packaging of genome segments.

(a) Alignment of SC35M NP and NP mutant proteins harbouring 14 (NP14), 10 (NP10) or 7 (NP7) Bat NP-specific amino acids of CH4. Upper panel shows the sequence logo of the consensus sequence of NP of available IAV strains (n=27,675 strains). (b) Positions of Bat NP-specific amino acids in the modelled crystal structure of SC35M NP. The program PyMOL was used to assign the indicated positions. Bat-specific amino acids are marked in the colour code used in a. Note that amino acid 239 is not surface exposed. (c) Relative SC35M polymerase activities in the presence of the mutant NP proteins determined by polymerase reconstitution assays. Bat-specific amino acids and nucleotides are indicated. 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 recombinant virus rNP7. At the indicated time post infection (p.i.), virus titres were determined by plaque assay. (e) 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 rNP7. Values obtained for SC35M were set to 1. *P<0.05. (f) Ratio of incorporated NP and M1 proteins in viral particles between SC35M and rNP7. Protein levels were determined by Western blot analysis of virus stocks with equal infectivity (PFU). *P<0.05; ***P<0.001. (g) Relative ratio of genome segments identified in viral particles preparations of SC35M and rNP7. RNA was prepared from virus stocks with identical infectivity (PFU) and subjected to quantitative RT-PCR. Levels of viral genome transcripts obtained with wt SC35M were set to 1. *P<0.05; **P<0.01; ***P<0.001. (h) Relative ratio of the viral transcript level in wt SC35M- or rNP7-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 RNA. Normalized values obtained in cells infected with wt 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 three independent experiments.