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

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)

Research Focus 1 – Workflow

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 high pathogenicity (possibly higher than PR8)

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.

Identification of key transcriptional regulators 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. Orange = higher in PR8 infection than x31 Green = lower in PR8 infection than x31

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.

Profile proteins in influenza infected lungs MRM (peptide atlas)

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

Lipidomics: Levels of Mediators in a Time Course Experiment 0 8d 13d BAL CD45 AEC d

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.

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. Sub-aim C: in vitro Infection Model (mTECs) Insert Plate well Media Apical Basolateral

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.

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

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

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

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

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

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