PURIFICATION OF HEPATITIS C VIRUS PARTICLES AND ANALYSIS OF ITS STRUCTURAL PROTEINS

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The largest obstacle to the viral characterization of HCV was the inability to sustain sufficient replication in the culture. HCV existed in two entities in the circulation, one of which had a high density (1.15-1.21g/cm^3) and was associated with gamma-globulins ; the other had a low density (1.06-1.12g/cm^3), appeared to be free virions and paralleled the infectivity titer. A buoyant density of HCV recovered from the circulation was estimated to be 1.09-1.11g/cm^3. Electron microscopy of plasma from infected individuals visualized viral particles with a diameter of 55-60nm. By peeling off the envelope with detergent such as Nonidet P-40 and Tween 80, particles were obtained, which had a diameter of 33 nm (peaked at 1.24-1.25g/cm^3) and an abilty to bind with monoclonal antibodies raised against oligopeptides mimicking the HCV core protein and IgG fractions from sera of persistently infected individuals. Specificity was determined by using rabbit polyclonal antibodies raised against isolate-specific E2/hypervariable region oligopeptides or mouse monoclonal antibodies directed to HCVcore which had been coupled with colloidal gold particles. This direct immunogold electron microscopic study confirmed that HCV virions are 55 to 60 nm diameter spherical particles and have 33nm inner core.Density heterogeneities of HCV in the circulation due to the binding of low density lipoproteins and immunoglobulins as well as low virus titers hampered the large-scale preparation of free-virions from HCV RNA-positive plasmas, but we could observe tens of HCV particles per grids in average. Accumulated knowledge and techniques were also useful for the physicochemical analysis of a recently identified human hepatitis virus, GB virus C/hepatitis G virus.