As influenza is an obligate intracellular pathogen, host cell factors play an essential role in detecting influenza virus, mounting an anti-viral response and modulating viral replication. While reverse genetic approaches have been used to study each of the viral gene products, only recent technological advances have enabled a systematic genetic approach to uncover host factors required in the life cycle of the virus. We have recently developed a genome-wide lentivirus-based RNAi library that can silence human genes in primary and immortalized cell types. We propose to use this RNAi library to identify host factors that are essential for influenza A replication and virus-induced interferon production and to test their value as therapeutic targets in an animal model of influenza infection. We will test the roles of ~10,000 human genes, including: the 'druggable' genome, factors that physically interact with viral proteins that we will identify in a systematic protein interaction screen, genes whose expression is induced or repressed by influenza, other candidate genes and an unbiased collection of genes. To identify essential host factors, A549 lung epithelial cells will be infected with lentiviruses that express shRNAs that individually target each of these human genes. Quantitative measurements of viral and interferon production will be performed to identify bioactive shRNAs that enhance or inhibit these processes. Specific host factors will be selected for further studies if >2 distinct shRNAs targeting a gene silence target gene expression and induce similar phenotypes in primary human lung epithelial cells. The role of selected host factors in each step of the viral life cycle will be determined using assays for viral entry, replication, budding and innate immune signaling. Finally, to assess the prophylactic and therapeutic utility of targeting host factors to counter influenza infection in animals, siRNAs that silence a small number of selected host factors will be administered into mouse lungs. Mice will be infected with influenza before or after siRNA delivery and assessed for viral titer, immune responses, pathology, weight loss and survival. Host factors found to be critical in influenza virus infection in vivo are expected to serve as a new class of targets for small molecule anti-influenza therapeutics that may be less prone to viral resistance. Targeting host factors may thus provide a universal treatment for influenza infection that would address the need for treatment of high-risk groups and preparation for future pandemics. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
6U01AI074575-04
Application #
7893386
Study Section
Special Emphasis Panel (ZAI1-CCH-M (M2))
Program Officer
Krafft, Amy
Project Start
2007-09-03
Project End
2012-08-31
Budget Start
2009-07-01
Budget End
2009-08-31
Support Year
4
Fiscal Year
2008
Total Cost
$273,121
Indirect Cost
Name
Broad Institute, Inc.
Department
Type
DUNS #
623544785
City
Cambridge
State
MA
Country
United States
Zip Code
02142
Giraud, Matthieu; Jmari, Nada; Du, Lina et al. (2014) An RNAi screen for Aire cofactors reveals a role for Hnrnpl in polymerase release and Aire-activated ectopic transcription. Proc Natl Acad Sci U S A 111:1491-6
Arazi, Arnon; Pendergraft 3rd, William F; Ribeiro, Ruy M et al. (2013) Human systems immunology: hypothesis-based modeling and unbiased data-driven approaches. Semin Immunol 25:193-200
Shapira, Sagi D; Hacohen, Nir (2011) Systems biology approaches to dissect mammalian innate immunity. Curr Opin Immunol 23:71-7
Amit, Ido; Regev, Aviv; Hacohen, Nir (2011) Strategies to discover regulatory circuits of the mammalian immune system. Nat Rev Immunol 11:873-80
Shapira, Sagi D; Gat-Viks, Irit; Shum, Bennett O V et al. (2009) A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection. Cell 139:1255-67