The physical state of the negative strand of hepatitis C virus RNA in serum of patients with chronic hepatitis C.
Negative strands of the hepatitis C virus (HCV) genome (a positive-stranded RNA virus) have been found in a nuclease-resistant form in the serum of patients with HCV infections. We determined whether a complete negative-strand copy is present in the serum, whether the negative strand is particle-associated, and finally, whether it is virion-associated and encapsidated like the positive (genomic) strand. Isopyknic sucrose and cesium chloride density ultracentrifugation followed by a strand-specific reverse transcription-polymerase chain reaction on the collected 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/cm3) in sucrose. After treatment of the plasma with detergent (0.1% Nonidet P-40) to remove the viral envelope and centrifugation on cesium chloride gradients, the positive strands shifted to a density of 1.35 g/cm3, and the negative strands were not detected. By using antibodies specific 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 resuspended in either PBS or PBS with 0.1% Nonidet P-40 to expose the core. These pellets were then incubated in antibody-coated microtiter wells. RNA extracted from the bound and unbound fractions was tested for HCV RNA. The anti-core antibody was able to bind positive strands but not negative strands only in detergent-treated samples. In the nondetergent-treated pellets, the anti-E1 and -E2 bound the positive strand, but only anti-E1 bound the negative strands. These findings indicate that while both strands of HCV RNA can be detected in serum, the positive strand is encapsidated within the enveloped core, and the negative strand appears to be in a membrane particle associated with the viral envelope protein E1 but does not appear to be within the HCV core of circulating virions.
Shindo
M
M
Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Di Bisceglie
A M
AM
Akatsuka
T
T
Fong
T L
TL
Scaglione
L
L
Donets
M
M
Hoofnagle
J H
JH
Feinstone
S M
SM
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D016174:Hepacivirus / Q000737:chemistry*; D006526:Hepatitis C / Q000097:blood / Q000382:microbiology*; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000737:chemistry*
D017931:DNA Primers; D012367:RNA, Viral