We propose to expand the scope of R01 CA57973-16 """"""""Structure and function of the HCV RNA replicase"""""""", which currently funds our studies of hepatitis C virus (HCV) replication. HCV infects over 130 million people worldwide, with often devastating consequences including liver cirrhosis and hepatocellular carcinoma. We are currently investigating the structures and functions of individual viral proteins that make up the HCV RNA replication complex, we are also attempting to catalog interactions between viral proteins that contribute to the architecture of the replicase, and ultimately to reconstitute replication in vitro. In the one month since the renewal of R01 CA57973-16 funding, we have made excellent progress towards the aims of the parent grant, including preparing a manuscript describing the mechanism of NS3 helicase activity (Gu M and Rice CM, in preparation). We are now proposing to extend the aims of the parent grant to include the identification and characterization of functional host factor interactions with the HCV replicase. This new line of investigation will complement our currently funded studies of genetic, biochemical, and structural interactions between viral proteins, and accelerate progress towards a more complete understanding of the central multiprotein complex catalyzing HCV replication. We will heavily bias our studies towards the identification of functional interactions by performing pull-down assays of individual viral proteins from native complexes within an infected cell. To do this we will discover permissive sites for tag insertion within each HCV gene, we will optimize conditions to pull-down the viral protein and associated factors, and we will independently validate and perform mechanistic studies on each interaction. The requested funding will be used to jump-start this project by acquiring significant reagent sets and by supporting one currently unfunded postdoctoral associate, partially funding one Ph.D. student, and hiring an additional technician. The proposed expenditures will thereby further the aims of the Recovery Act while dramatically increasing our understanding of the HCV replicase complex.

Public Health Relevance

Hepatitis C virus (HCV) is a liver-tropic human virus that currently infects approximately 130 million people worldwide. We are studying the mechanisms by which HCV replicates its RNA genome and produces new infectious particles, with the hopes of identifying interactions that can be exploited for the development of anti-viral drugs. Currently funded studies are investigating the interactions between viral proteins that are important for replication complex function;the new studies proposed here expand our research to explore the importance of cellular factors in HCV propagation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA057973-16S1
Application #
7811290
Study Section
Special Emphasis Panel (ZRG1-IDM-C (95))
Program Officer
Read-Connole, Elizabeth Lee
Project Start
2009-09-30
Project End
2011-09-29
Budget Start
2009-09-30
Budget End
2011-09-29
Support Year
16
Fiscal Year
2009
Total Cost
$561,080
Indirect Cost
Name
Rockefeller University
Department
Microbiology/Immun/Virology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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Billerbeck, Eva; Wolfisberg, Raphael; Fahnøe, Ulrik et al. (2017) Mouse models of acute and chronic hepacivirus infection. Science 357:204-208
Moreno, Elena; Gallego, Isabel; Gregori, Josep et al. (2017) Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment. J Virol 91:
Luna, Joseph M; Michailidis, Eleftherios; Rice, Charles M (2016) Mopping up miRNA: An integrated HBV transcript disrupts liver homeostasis by sequestering miR-122. J Hepatol 64:257-259
Scheel, Troels K H; Luna, Joseph M; Liniger, Matthias et al. (2016) A Broad RNA Virus Survey Reveals Both miRNA Dependence and Functional Sequestration. Cell Host Microbe 19:409-23
Ramanan, Vyas; Trehan, Kartik; Ong, Mei-Lyn et al. (2016) Viral genome imaging of hepatitis C virus to probe heterogeneous viral infection and responses to antiviral therapies. Virology 494:236-47
Gu, Meigang; Rice, Charles M (2016) The Spring ?-Helix Coordinates Multiple Modes of HCV (Hepatitis C Virus) NS3 Helicase Action. J Biol Chem 291:14499-509
Swanson, Michael D; Boudreaux, Daniel M; Salmon, Loïc et al. (2015) Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral activity. Cell 163:746-58
Saeed, Mohsan; Andreo, Ursula; Chung, Hyo-Young et al. (2015) SEC14L2 enables pan-genotype HCV replication in cell culture. Nature 524:471-5
Luna, Joseph M; Scheel, Troels K H; Danino, Tal et al. (2015) Hepatitis C virus RNA functionally sequesters miR-122. Cell 160:1099-110

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