Hepatitis C virus (HCV) has not yet been visualized or cultured. Because of the availability of a high-titer plasma pool, i.e., the anti-c100-3 reactive pool which contained 3 x 105 PCR units of HCV RNA, we characterized HCV from this plasma so that the physical properties of the virus can be understood. We used our semi-quantitative PCR method (determining HCV RNA by limiting dilution analysis) to monitor the presence of HCV. An aliquot of the plasma pool was adjusted to pH 7.4 and the buoyant density of HCV was determined by density gradient ultracentrifugation (10 to 60% w/w of sucrose). Three HCV RNA peaks were obtained, corresponding to sucrose densities of 1.06, 1.14 and 1.21 g/cm3. However, when the same plasma sample was adjusted to pH 3.5 and incubated at 4 degrees C for 18 h, the buoyant density of HCV was found to be 1.05 g/cm3. The HCV RNA present in the lower density peak was inert to RNase A digestion and it appeared to be present as an intact virus. The nature of the HCV RNA present in the two higher density peaks is not known. However, the highest density peak may represent HCV RNA present in a nucleocapsid since when a plasma sample was treated with 0.4% NP40 (a detergent known to remove the HCV envelope protein and expose the nucleocapsid), the HCV RNA was found at a density of 1.25 g/cm3. We also determined the stability of HCV RNA (as present in plasma) at pH 3.5; at this pH, levels of HCV RNA remained relatively constant for at least 16 days. When the same plasma was chromatographed on protein G sepharose, which binds IgG, almost all of the HCV RNA was in the bound fraction. Thus, our preliminary data indicate that HCV may be associated with anti-HCV as an immune complex and that, at pH 3.5, the free, intact virus which has a low density is dissociated from immune complex(es). This work is still in progress.
