A new influenza virus virulence determinant: The NS1 protein four C-terminal residues modulate pathogenicity

Jackson et al. 10.1073/pnas.0800482105.

Supporting Information

Files in this Data Supplement:

SI Figure 5
SI Figure 6
SI Figure 7
SI Figure 8

SI Figure 5

Fig. 5. The distribution of NS1 in virus-infected cells. MDCK cells were infected with wt or rNS1 viruses at an MOI of 1 and incubated for 6 h at 37°C. Cells were fixed and stained with a rabbit anti-NS1 polyclonal serum, followed by a FITC conjugated secondary antibody. Cell nuclei were stained with DAPI. Images were captured at 63´ magnification.

SI Figure 6

Fig. 6. Mouse experiments. Five-week-old BALB/c mice (four per group) were inoculated intranasally with wt or rNS1 influenza viruses at a range of serial 10-fold dilutions. (A) The body weight of mice infected with 10³ pfu was measured up to 14 days p.i. Mice were euthanized after the loss of 30% of their initial body weight. (B) The data expressed as the percentage survival of mice infected with 10³ pfu.

SI Figure 7

Fig. 7. NDV-GFP IFN reporter assay in influenza virus-infected A549 cells. A549 cells were either infected with wt or rNS1 influenza virus at an MOI of 5 or treated with 150 units/ml IFN-b and incubated at 37°C for 6 h, followed by infection with NDV-GFP for a further 18 h. Cells were fixed and GFP expression was imaged at 63´ magnification. Immunofluorescence using anti-Udorn antisera was performed to ensure all cells were infected with influenza virus (data not shown).

SI Figure 8

Fig. 8. Quantification of IFN-a mRNA levels in influenza virus-infected cells. MDCK cells were infected with rWSN wt, rNS1, rUd-NS1-rna, or rUd wt influenza viruses, parainfluenza virus 5 (PIV5), PIV5 VdelC (a mutant virus that induces IFN synthesis), or Sendai virus at an MOI of 5 and incubated at 37°C for 24 h. Cells were lysed, and total cellular RNA was extracted by using the RNEasy Kit (Qiagen). mRNAs were reverse transcribed by using an oligo(dT) primer and Super Reverse Transcriptase (Molecular Genetic Resources). Canine IFN-a cDNA was amplified by quantitative PCR (qPCR) using SYBR Green (Bio-Rad) and canine IFN-a specific primers (sequences available on request). cDNA amplification was performed by using an iQ5 Real-Time PCR Detection System (Bio-Rad) and quantified by iQ5 Optical System Software (version 2.0) (Bio-Rad). rUd wt = A/Udorn/72 virus, rUd-NS1-rna = a recombinant A/Udorn/72 virus encoding an NS1 protein containing an R38K mutation such that it lacks a functional RNA binding domain. The results represent the average of three independent experiments.