Hepatitis C virus (HCV) is the leading cause of post transfusional hepatitis worldwide and infects up to 15% of low risk populations. It is a major cause of chronic liver disease in virtually all parts of the world. Little is known about the HCV replication cycle and the mechanism by which it liver injury is unknown. It is not understood how HCV binds to and enters permissive cells nor how HCV can escape the host immune defenses and establish a chronic infection. To better understand some of these events we have focused our efforts on characterizing the early steps of HCV infection, primarily those involved in binding and entry of HCV into cells and determining which viral components and which cellular components play critical roles in these initial steps.
The specific aims of our proposal are: 1) To develop and optimize a cell culture system to quantify the early events of HCV infection, including cell binding and entry, and to utilize this assay to determine how certain target cell characteristics, such as species origin, target cell growth and metabolism and cell surface molecules affect entry. To optimize our current qualitative assay, we will utilize a competitive PCR detection system with an internal RNA standard to obtain more quantitive data on the amount of HCV entering cells. 2) To identify an characterize some basic virologic and immunologic features of HCV that enhance or restrict the binding and penetration of HCV into host cells. It is our observation that HCV isolates from different patients and from various stages of HCV infection have different abilities to bind and enter cells. We believe that differences in viral titer, genotype, extent of quasispecies diversity, and biophysical state will play a role in the ability of HCV to bind and gain entry into host cells. In addition, host humoral immunity to specific viral proteins is likely to modulate host cell entry. 3) To identify and characterize regions on the HCV virion surface that are involved in binding and cell entry using monoclonal antibody inhibition studies and recombinant protein competition studies. It is our hypotheses that HCV binding and/or entry can be blocked by monoclonal antibodies to HCV structural proteins E2 and, perhaps E1. We will use a library of monoclonal antibodies to different regions of C, E1 and E2 to map entry domains and attempt to confirm these studies by inhibiting HCV entry with recombinant structural proteins or peptides.
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