Hepatitis C Virus Infection and Lipoproteins

The study of hepatitis C virus (HCV) replication and the search for effective anti-HCV drugs have been significantly hampered by the lack of a reliable cell culture system for HCV infection and propagation. We have recently developed a stable HCV culture system that robustly produces and secretes infectious virus into the culture media. Initial examination of HCV RNA-containing particles revealed that there is a remarkable disparity between the abundance of HCV virion RNA (vRNA) and infectious titer. The infectious HCV titer was at least 1,000 times lower than HCV vRNA copy number, suggesting that the majority of HCV RNA-containing particles were not infectious. Further charac1terization of HCV particles by density gradient centrifugation demonstrated that the density of HCV RNA-containing particles covers a broad range varying from 1,06 to 1.20 g/ml. Intriguingly, only low density particles were infectious, whereas high density particles, which accounted for the majority of HCV vRNA detected in the culture media, were not infectious (Appendix 1). Additionally, the detection of apolipoproteins Band E in the low-density fractions correlates very well with HCV infectivity. Our central hypothesis is that human lipoproteins are important for HCV virion assembly and infectivity. This hypothesis represents a novel conGept that warrants further exploratory investigation and holds promise for the discovery of novel antiviral drugs against HCV replication and infection. The overall goal of this application is to determine the properties and biochemical composition of HCV RNA-containing particles and to define the role of lipoproteins in HCV assembly and infectivity. The specific aims are: 1. To determine the properties and biochemical composition of HCV RNA-containing particles and the structural detE~rminants of HCV infectivity by using cell biological, biochemical, immunological, and proteomic approaches. 1.1. To determine the naturE!and properties of HCVRNA-containing particles. 1.2. To determine the bioch,emical composition of HCVRNA-containing particles and the structural determinants of HCVinfectivity. 2. To define the role of lipoproteins in HCVvirion production and infection. 2.1. To determine the role of lipoproteins in HCV virion production by altering the levels of apolipoprotein expression in stable HCV-producing cells. 2.2. To determine the role of lipoproteins in HCV infection using blocking antibodies specific to apolipoproteins. 2.3. To determine the underlying mechanisms for the inhibition of HCV infection by human lipoproteins (HDL, LDl., and VLDL). Our long-term goal is to determine the underlying molecular mechanisms of HCV assembly and infection. The experiments described in this application are expected 1) to yield significant new information towards a complete understanding of the nature and biochemical composition of infectious HCV virions; 2) to provide novel targets for antiviral drug discovery; and 3) to identify specific and potent inhibitors that block HCV entry; and 4) to provide a foundation to determine the molecular basis underlying HCV assembly and infection.