PROJECT 1 (PI Megan Shaw, MSSM):
The underlying hypothesis of this application is that critical molecular features of host-pathogen interactions and responses dictate the pathogenic outcome of viral infection. Thus, a comprehensive understanding of viral-host interactions, innate responses to viral infection, and viral evasion strategies is pivotal for predictive modeling of viral pathogenesis. Here, we will provide a comprehensive overview of the genetic, chemical, and biochemical networks that play a role in controlling influenza virus infection by investigating host-virus interactions in an ex vivo setting using primary human cells.
The impact of influenza virus replication on the host will be studied using next generation sequencingtechnologies to interrogate cellular RNA populations to define transcriptome-level changes (RNA-seq),conduct genome-wide survey of promoters engaged by RNA polymerase (GRO-seq), as well as evaluate epigenetic alterations in the chromosomal landscape (CHiP-seq). Furthermore, global alterations in intracellular and extracellular metabolite levels, protein abundance, as well as post-translational modifications induced upon viral infection will be measured. Combining these approaches with genome-wide
functional genomic screening and high-throughput protein interactome analysis will enable the generation of high-resolution networks that accurately depict the hierarchies of interactions between influenza virus and the host. By conducting these analyses simultaneously with three viruses that drive varying pathogenic outcomes, computational modeling of these data will enable us to identify critical nodes of the viral-host network that are predictive of viral pathogenesis. The in vivo and clinical impact of these nodes will be evaluated in Projects 2 and 3, respectively.
PROJECT 2 (PI Adolfo Garcia-Sastre, MSSM):
The underlying hypothesis of our consortium is that host genes and networks involved in viral replication and in early host responses modulate viral pathogenesis and therefore represent targets for therapeutic intervention. In Project 2, we propose an OMICS approach to identify key early genes/networks involved in influenza virus pathogenesis. This will be achieved by modeling global host responses during influenza virus infection in a mouse model in collaboration with the Modeling Core E and the OMICs Cores B, C and D (Genomics, Proteomics and Metabolomics). In Aim 1, we will investigate the early global host response associated with lethal, severe and moderate influenza A virus infection in a mouse model. We will be using three clinically relevant strains of influenza A virus that differ in their virulence, allowing for comparisons of the host responses and interactions associated with different ranges of disease severity. In Aim 2, we will
investigate host proteins interacting with influenza virus proteins during viral infection in mouse lungs, in collaboration with the Proteomics Core C, using the same influenza A virus strains as in Aim 1. The models constructed in collaboration with the Modeling Core E by the integration of the data generated in vivo with those ex vivo in Project 1 will predict key genes and networks likely to be involved in virus replication and host responses. In Aim 3, the model-identified networks will be validated by conducting perturbations including: use of specific virus mutants that disrupt key host-virus interactions, use of virus with specific mutations involved in host tropism and pathogenesis, use of pharmacological inhibitors, and use of mouse k.o. or of antisense targeting of key host genes in the mouse model. For antisense targeting in vivo we will
be using a validated and innovative technology based on lung delivery of peptide-conjugated morpholino antisense oligomers (PPMO), pioneered by our collaborators Hong Mouton and David Stein. In all these experiments, viruses will be generated in collaboration with the Virus Core (Core G). In Aim 4, the results of these perturbations, approximately 40 per year, on the model networks will be analyzed in a medium
throughput or targeted–OMICS approach, and the data will be incorporated into the model in collaboration with the Modeling Core E for model refinement. Targeted host genes in our Project 2 will also be studied for variants and impact in human macrophage function by Project 3. We hypothesize that our integrated approach will result in the identification of novel host targets for therapeutic intervention during influenza
PROJECT 3 (PI Steven Wolinsky, Northwestern):
Influenza A virus depends on the host cell machinery for its replication. Recognition of the virus by the host triggers a complex signaling cascade that results in the expression of numerous interferon (IFN)-stimulated genes that respond to the virus and interfere with its life cycle. As an ancient antiviral defense mechanism, the innate immune response is a collection of functionally distinct subsystems that have evolved to counter
infection by viruses. The influenza A virus, however, can bring about measures that subvert many components of the host innate immune response to infection. Differences in the genes relevant to the virus life cycle or immunity, from genetic variations or epigenetic factors, can contribute to host resilience. Many aspects of the virus-host interaction have not been described fully. Here, we will conduct a cohort study to
determine whether mutation of genes encoding intermediates in signaling pathways and networks host factors identified by our ’omics’ approach for which a plausible biological mechanism of action is found influences the virus life cycle. We will discover rare and disruptive variants in human genes required during virus replication or for host cell modifiers of infection relevant to the virus life cycle or immunity by targeted capture and high-throughput resequencing of the selected genes across individuals. We will map and
quantify expression quantitative trait loci by high-throughput sequencing of cDNA libraries and produce genome-wide maps of chromatin accessibility to link genetic variation to changes in gene regulation and molecular phenotype. We will elucidate assess the impact of the predicted function-altering changes in host factors required for virus replication and innate immune defense to understand the mechanisms by which
they affect the steps in the influenza A virus life cycle. We will follow the extant men over a subsequent season of influenza prospectively to associate changes in molecular phenotypes with changes in molecular and cellular processes and disease-related phenotype, With this approach, we anticipate finding novel cellular proteins required during virus replication, new host cell modifiers of infection, and their functional
importance in restriction of infection that will provide valuable insights into the biological basis of disease.
ADMINISTRATIVE CORE A (Adolfo Garcia-Sastre, MSSM):
The Administrative Core (Core A) will provide a management plan to coordinate the whole FLUOMICS consortium by 1) the establishment of an organizational structure centered around an Executive Committee responsible for monitoring overall program progress, implementing a Pilot Research Program, and making decisions on staffing plans, allocation of resources, scientific core usage and other policies; 2) the coordination of conference calls and of annual meetings between the PIs, selected key personnel, the Steering Committee and NIAID; 3) assisting the members of this consortium in reagents and data sharing, manuscript preparations, public release of data and submission of annual progress reports to the NIH; and 4) providing training opportunities in Systems Biology for the infection disease scientist. The Core Director has expertise in the coordination of program projects and big consortiums, as he is the Director of one of the NIAID Centers of Excellence of Influenza Research and Surveillance. This FLUOMICS proposal complements and does not overlap with the Center of Excellence, as the influenza center does not include the use of a systems biology approach in its research agenda. The Core Co-Director is in charge of the training program to be implemented in years 2 to 5 and he has past experience in organizing training for students in systems biology approaches.
GENOMICS CORE B (Chris Benner, Salk Institute):
Innate signaling pathways can regulate influenza virus replication, and there are viral countermeasures, but there remain critical gaps in our knowledge about how these responses impact viral disease pathogenesis. The overarching goal of this highly integrated program is to systematically address these questions using a systems-based approach to reveal new therapeutic approaches. The goal of the Genomics Core (Core B) is to provide a central resource that will facilitate high throughput sequencing of the transcriptome (mRNA-seq, GRO-Seq, and Nanostring gene expression analysis) and the epigenome (ChIP-Seq) in influenza virus infected cells. The core will also provide data analysis and integration to uncover the virus-host transcriptional response network. This information will be used by other program investigators in the Modeling Core to define host molecular networks and pathways that impact influenza virus infection and to
define rare and disruptive gene polymorphisms that influence influenza virus disease pathogenesis (Project 3). Cellular networks, pathways, and genes that are implicated in the virus-host transcriptinal response network will be targeted by RNAi-knockdown to determine how perturbations in the system impact the transcriptional response network to infection by wild-type and specific mutant influenza A viruses. These studies are critical for the overall goal of the program aimed at uncovering the global effects of influenza virus on cellular functions and on the system of key antiviral responses and virus countermeasures.
PROTEOMICS CORE C (Nevan Krogan, UCSF):
In this study, we aim to functionally interrogate host-pathogen relationships in human influenza viruses. The Proteomics Core (Core C) will employ a systematic affinity tag/purification-mass spectrometry approach to identify the viral-host protein complexes. The data generated using these initial, unbiased approaches will fuel more targeted, hypothesis-driven research in the subsequent projects. In tandem with this work and with more targeted downstream work, we will be closely monitoring for links to host factors involved in quality control processes, including chaperone function, protein ubiquitination, and protein degradation, which will link this work to the collaborations with Drs. Garcia-Sastre, Chanda and Benner.
METABOLOMICS CORE D (Ed Dennis, UCSD):
The goal of the Metabolomics Core (Core D) is to provide a central resource that will facilitate high throughput characterization of the metabolome in influenza virus infected cells and tissues. We will validate relevant metabolites and localize metabolites within infected tissues samples. The core will also design strategies to uncover host metabolic pathways that are impacted by, and that influence influenza virus infection and disease pathogenesis.
MODELING CORE E (Nevan Krogan, UCSF):
The overall goal of the Modeling Core (Core E) is to drive the integration of global –OMICS data to identify virus-host networks that control the innate immune response and influence pathogenicity. This will be accomplished through two main objectives a) to design and provide tools to analyze -OMICS data and b) to serve as an engine for integrating –OMICS data into network models of pathogenicity that are subject to further
refinement in an iterative fashion. This Core will employ existing bioinformatics and systems biology approaches as well as develop novel approaches to identify cellular proteins and networks which influence influenza virus replication and contribute to virulence in vivo. The modeling core will be the engine for translating –OMICS data into biological insight and has a central role in the successful completion of this program. Co-directors Bandyopadhyay and Krogan have a strong history of innovation and collaboration with each other and others on this proposal and are well suited to direct the modeling efforts. Predictions that are based upon our models will be tested in primary cell culture and in animal model systems by employing targeted –OMICS technologies as well as in vivo experimentation and analysis of clinical phenotypes.
DATA MANAGEMENT and RESOURCE DISSEMINATION CORE F (Sumit Chanda, Sanford-Burnham Institute):
Building a predictive model for influenza pathogenesis will require reiterative cycles of data generation and computational analysis. Given the role of large-scale datasets in this effort, effective data management and resource dissemination is critical for not only the success of the program, but for the broader scientific community to fully realize and exploit resources generated by this Center. Towards that end, the Data Management and Resource Dissemination Core (Core E) will act as a central repository for all data and resources generated by the Center, and ensure that these materials are readily accessible by not only other scientists in the program, but also the broader scientific community. The Core will adapt practices, protocols, approaches, and software that have been previously and successfully utilized to integrate and disseminate large-scale datasets within the context of a large program project. Internally, we will focus on project tracking, data consolidation and integration, quality control, and managing a centralized database that is accessible and user-friendly. In addition, the core will work to integrate publically available data, and make these integrated large-scale datasets, and associated resources, available to the scientific community to enable the exploration of novel hypothesis based on the information generated by the program.
VIRUS CORE G (Randy Albrecht, MSSM):
The Virus Core (Core G) is an essential resource for the needs of projects 1 and 2, which will benefit from a centralized Virus Core by: i) the established expertise with reverse genetics techniques which will facilitate rescue of recombinant influenza viruses that are described below and itemized in Table 1B, ii) the maintenance of influenza virus stocks that have been sequence-confirmed and assessed for quality by hemagglutination and plaque assays, and iii) reduced inter-experimental variation by consistent use of specific virus stock preparations. The Department of Microbiology, Mount Sinai School of Medicine, NY, NY is a pioneer in the application of reverse genetics and the development of recombinant viruses. The well-equipped facilities, established procedures, and properly trained personnel provide a cost-effective Virus
Core that will result in efficient production of wild-type and recombinant influenza virus stocks that will be essential for projects 1 and 2. Specifically, Dr. Megan Shaw (Co-PI of project 1) and Dr. Adolfo Garcia-Sastre (Co-PI of project 2) will directly benefit by their close proximity to and direct communication with the Virus Core. Specific functions of the Virus Core are to i) maintain working stocks of wild-type influenza viruses for use by projects 1 and 2, and ii) generate recombinant influenza viruses for use by projects 1 and 2.