THE PHYSICAL STATE OF THE NEGATIVE STRAND OF HEPATITIS-C VIRUS-RNA INSERUM OF PATIENTS WITH CHRONIC HEPATITIS-C

Negative strands of the hepatitis C virus (HCV) genome (a positive-str anded RNA virus) have been found in a nuclease-resistant form in the s erum of patients with HCV infections. We determined whether a complete negative-strand copy is present in the serum, whether the negative st rand is particle-associated, and finally, whether it is virion-associa ted and encapsidated like the positive (genomic) strand. Isopyknic suc rose and cesium chloride density ultracentrifugation followed by a str and-specific reverse transcription-polymerase chain reaction on the co llected fractions was performed to determine whether both positive and negative strands were associated with similar particles. Both strands comigrated to approximately the same density (1.11-1.16 g/cm(3)) in s ucrose. After treatment of the plasma with detergent (0.1% Nonidet P-4 0) to remove the viral envelope and centrifugation on cesium chloride gradients, the positive strands shifted to a density of 1.35 g/cm(3), and the negative strands were not detected. By using antibodies specif ic for the HCV core or envelope glycoproteins E1 or E2 coated onto the wells of a microtiter plate, it was possible to specifically bind HCV or viral cores to the solid phase. Pelleted virus particles were resu spended in either PBS or PBS with 0.1% Nonidet P-40 to expose the core . These pellets were then incubated in antibody-coated microtiter well s. RNA extracted from the bound and unbound fractions was tested for H CV RNA. The anti-core antibody was able to bind positive strands but n ot negative strands only in detergent-treated samples. In the nondeter gent-treated pellets, the anti-E1 and -E2 bound the positive strand, b ut only anti-E1 bound the negative strands. These findings indicate th at while both strands of HCV RNA can be detected in serum, the positiv e strand is encapsidated within the enveloped core, and the negative s trand appears to be in a membrane particle associated with the viral e nvelope protein E1 but does not appear to be within the HCV core of ci rculating virions.