Hepatitis C virus (HCV) is a major cause of acute and chronic liver disease, and contributes to the development of hepatocellular carcinoma. HCV-associated disease is the leading cause of liver transplantation in the United States. Currently available HCV therapies are expensive, poorly tolerated, and successfully control the virus in only a fraction of patients. To develop more effective antiviral strategies, a clear understanding of the viral life cycle is essential. The least understood aspect of this life cycle is virus assembly, which occurs by intracellular budding of virus particles within the secretory pathway. Formation of virus particles has an unusual dependence on the cellular VLDL assembly pathway, but the connection between these processes is unclear. Furthermore, the viral nonstructural (NS) proteins contribute important but poorly defined regulatory roles in virus assembly. And to date, intermediate stages in HCV particle assembly have not been rigorously defined. To address these issues, we are determining how the HCV NS2 and NS3-4A proteins contribute to virus assembly. In Preliminary Data, we: 1) developed a highly efficient system for growing HCV in cell culture;2) engineered infectious virus variants with useful reporter genes, affinity tags, and fluorescent markers;3) conducted a mutagenesis study on the viral NS2 gene, revealing functional interactions that are important for virus assembly;4) developed the biochemical tools necessary to study NS protein structure-function, and 5) developed methods to image fully functional, fluorescently-labeled HCV core protein in live cells. Based on our preliminary genetic, biochemical, and cell biological analysis of NS2, we hypothesize that NS2 coordinates distinct, early steps in virus assembly through interactions with the E1-E2 glycoprotein and NS3-4A enzyme complexes. We will test this hypothesis by completing three Specific Aims.
In Aim 1, we will define NS2 protein-protein interactions that control virus assembly by specifically capturing NS2 and its associated proteins from virus-producing cells. We will examine interactions between NS2 and other viral and cellular proteins;then, by using a panel of NS2 mutants that are defective in virus assembly, we will map their determinants.
In Aim 2, we will identify and characterize intermediate stages of virus assembly by examining a subset of our NS2 mutants with live- and fixed-cell microscopy and cellular fractionation. By integrating these analyses over a focused set of mutants and other relevant conditions, we will gain a more detailed understanding of the HCV assembly pathway.
In Aim 3, we will extend our combined genetic and biochemical approach to define NS3-4A determinants of virus assembly. When completed, these studies will contribute to a clearer understanding of the HCV assembly process and reveal essential protein-protein interactions that can serve as targets for antiviral design.

Public Health Relevance

Hepatitis C virus (HCV) is a highly successful human pathogen that causes persistent infection, chronic hepatitis, and hepatocellular carcinoma. Our studies characterize the virus particle assembly process, which will provide new avenues to target HCV and thereby prevent or treat disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI087925-04
Application #
8448734
Study Section
Virology - A Study Section (VIRA)
Program Officer
Koshy, Rajen
Project Start
2010-02-01
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2013
Total Cost
$385,036
Indirect Cost
$152,386
Name
Yale University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Kohlway, Andrew; Pirakitikulr, Nathan; Barrera, Francisco N et al. (2014) Hepatitis C virus RNA replication and virus particle assembly require specific dimerization of the NS4A protein transmembrane domain. J Virol 88:628-42
Lindenbach, Brett D (2013) Virion assembly and release. Curr Top Microbiol Immunol 369:199-218
Lindenbach, Brett D; Rice, Charles M (2013) The ins and outs of hepatitis C virus entry and assembly. Nat Rev Microbiol 11:688-700
Counihan, Natalie A; Rawlinson, Stephen M; Lindenbach, Brett D (2011) Trafficking of hepatitis C virus core protein during virus particle assembly. PLoS Pathog 7:e1002302
Phan, Tung; Kohlway, Andrew; Dimberu, Peniel et al. (2011) The acidic domain of hepatitis C virus NS4A contributes to RNA replication and virus particle assembly. J Virol 85:1193-204
Stapleford, Kenneth A; Lindenbach, Brett D (2011) Hepatitis C virus NS2 coordinates virus particle assembly through physical interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes. J Virol 85:1706-17
Lindenbach, Brett D (2010) New cell culture models of hepatitis C virus entry, replication, and virus production. Gastroenterology 139:1090-3