1. Develop a molecular network model of the interaction between influenza virus and the innate immune system. A Systems Approach to Infectious Disease Research: Influenza

2. Influenza Contract Research Collaboration Exp. Prep & Pathophysiology – St. Jude (Doherty, Thomas, Webby) Genomics & Proteomics – ISB (Aderem) UW (Rubens); Lipidomics – UCSD (Dennis, Quehenberger); Vanderbilt (Brown) Computation – ISB (Shmulevich/Aderem)

3. Research Focus 1 – Workflow

4. Viral Models • PR8 (A/Puerto Rico/34) • Mouse adapted, reverse genetics virus with high pathogenicity • x31 (HA/NA from A/Aichi/2/68) • Mouse adapted, reverse genetics virus on PR8 backbone with low pathogenicity • rg/VN1203 (HA/NA from A/Vietnam/1203/04) • “Clipped” HA from high path H5N1, reverse genetics virus on PR8 backbone with highpathogenicity (possibly higher than PR8)

5. Research Focus 1 –Specific Aims Sub-aim A To determine the regulatory networks controlling the innate immune response to infection with influenza strains of varying pathogenicity, and to determine their contribution to resolving infection and inducing pathology.

6. Identification of key transcriptional regulators Orange = higher in PR8 infection than x31 Green = lower in PR8 infection than x31 Extended transcriptional regulatory network showing differentially expressed transcription factor genes and associations with downstream interferon-responsive genes. A directed edge indicates that (1) the target gene cis-regulatory region contains one or more high-quality matches for the TF binding site motif recognized by the source gene’s product (TF), and (2) the colors of the source and target are consistent with known function (activation and/or repression) of the source TF. A TF gene was included in the diagram only if its binding site motif is enriched within cis-regulatory regions of genes whose expression levels were altered by the pathogenicity of the virus.

7. Research Focus 1 –Specific Aims Sub-aim B To characterize the spectrum of proteins and bioactive lipid mediators induced in the lung in response to infection with influenza strains of varying virulence, and to identify those that correlate with enhanced pathogenicity.

8. Profile proteins in influenza infected lungs • MRM (peptide atlas)

9. Sub-aim B2 –Lipid profiling • Profile eicosanoids in the lung following influenza infection • Identify novel lipids that are produced in the lung during influenza infection Begins in Year 2

10. Lipidomics: Levels of Mediators in a Time Course Experiment 0 8d 13d BAL CD45 AEC 0 6 9 11 13 19d

11. Research Focus 1 –Specific Aims Sub-aim C To map the regulatory networks induced in epithelial cells by influenza strains of varying pathogenicity, and establish the mechanisms that create highly pathogenic phenotypes.

12. Insert Apical Plate well Media Basolateral Sub-aim C: in vitro Infection Model (mTECs) Mouse tracheal epithelial cell (mTEC) culture is used as a model for epithelial cells lining the airways. • Polarized cells growing on a membrane with the apical side exposed to air. • Cells form a monolayer sealed by tight junctions. • Cells are fully differentiated, form cilia and closely resemble epithelial cells in vivo.

13. Research Focus 1 –Specific Aims Sub-aim D To identify the critical determinants of pathogenicity by generating mutant viruses containing individual genetic elements of highly virulent H5N1, and assess their impact on the networks defined in Sub-aims A-C.

14. Sub-aim D: Identify Critical Determinants of Pathogenicity “PR8:5” “PR8:D92E” “PR8” (Five amino-acid deletion in NS1) (Point mutation in NS1) Both of these mutations have been shown to increase virulence.

15. Research Focus 2- Specific Aims Subaim A: Characterize the host proteins targeted by influenza proteins that are associated with virulence.

16. Research Focus 2- Specific Aims Subaim B: Characterize the unique lipid envelope components of pathogenic strains of influenza.

17. Research Focus 2 – Viral Lipidomics PR8 (H1N1) rgVN1203 (H5N1) x31 (H3N2) Measure lipid envelope composition from various strains infecting mTECs using mass-spectrometry approaches.

18. Research Focus 3 - Specific Aims Sub-aim A: Determine the S. aureus transcriptional regulatory networks induced during superinfection following influenza infection. Sub-aim B: Determine the innate immune regulatory networks induced during superinfection with S. aureus.

19. Public Web Portal

20. Internal Contract Collaboration Site