FLU DFIM

Funding Application 1.0 (DFIM)

Proteins Associated with the Viral Polymerase and vRNP Complexes Question: do any of the proteins proposed to interact with the viral polymerase complex or viral RNP (by NP) have functional consequences to viral replication? Is there a difference in interactions/effects on the activity of avian/human v-pol and replication? VPOL activity exhibits strain-specific variances, and is a determinant of pathogenesis (refs). Interactome maps for these may be important for understanding highly pathogenic and avian influenza variants.

Goal summary: I.	Identify from among proteomics data host factors that modulate influenza viral polymerase activity by functional genomics. II. Map mechanisms of modulation on the viral life cycle (validation of proteomics and functional genomics), and strain-specific differences. III. Model modulation by a dynamic funcitonal interactome map (DFIM) incorporating additional phylogenetic, structural, genomic, or experimental data. IV. Develop a medium-throughput assay for analyzing the viral polymerase activity/DPIM of emerging highly pathogenic influenza strains. V.	Map vpol activity/DPIM to pathogenesis data from human patients and animal model experiments, modeling viral polymerase pathogencity factors.

Dynamic Funcitonal Interactome Map

DFIM - up to 4D (space, time, abundance, activity) network of a viral-host protein interactions in a series of subprocesses in viral infection, replication, assembly and release. Proteomics data (discovery and functional) can provide an estimate of the 4D parameters, by subcellular localization (space), replication sequence at timepoints and with appropriate drugs/mutants (time), estimates of protein content by MS/biochemical data (abundance), and functional studies on interactions, post-translational processing, and effects of mutation/overexpression on viral replication (activity).

Viral polymerase subprocesses may be studied individually: I.	vPOL assembly in the nucleus II. vPOL transcriptional activity by minigenome reporter assays III. vPOL fidelity by a minigenome mutant recovery assay

Amenable because biochemical purification protocols exist for subcellular fractions where each of these events may occur; and known marker proteins (for biochemistry, imaging, and interaction controls) exist in each subprocess. Thus, transfected/infected cells can be fractionated and the interacting and non-interacting proteomes mapped for each subcellular compartment, encompassing a subprocess. Most viruses take advantage of subcellular compartmentalization and cytoskeletal structures for separating distinct subprocesses (events) in their life cycles. Different proteomics technologies must be applied to generate a 4D DFIM, however. These need to be developed and field-tested as the project progresses. Interactions: the co-IP and mechanistic studies coming from the limited proteomics experiments performed so far will serve to highlight the potential of this approach, develop proficiency at (1) and (3) above, and develop methods for generating the dynamic proteome interaction map. Quantitative Biology: q-proteomics/interactomics, q-peptidomics, q-gex, q-metabolomics and integration into a systems biology model of dynamics. Test model by deletion/overexpression/d.n., knockdown, metabolic/drug alteration, to get operating parameters for system through the course of viral infection. q-Proteomics: various methods exist .. both in situ and mostly ex situ .. including 2DdiGE with bicolor dye labels; ICAT or isotope growth-labeling coupled with parallel/shotgun MC/MS-MS. Integration into Systems Virology

The dynamic proteome map can be coupled with: 1.	deletion analyses of cellular proteins involved in influenza infection 2.	advanced, real-time and cryo-imaging of virion morphogenesis 3.	mechanistic studies of viral proteins and cell response including immunity 4.	systems biology: transcriptome, metabolome, translation, processing systems 5.	development of rapid protein interaction mapping for flu variants