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Serologic testing shows that hepatitis C virus (HCV) may have a role in the pathogenesis of B-cell non-Hodgkin lymphomas (B-cell NHLs). We tried to demonstrate HCV RNA sequences in paraffin-embedded tissue from B-cell NHLs by reverse-transcription double polymerase chain reaction (RT-PCR) and Southern blotting. We studied 31 consecutive cases of B-cell NHLs; lymph nodes from 32 patients with diseases other than B-cell NHL were negative controls. Positive-strand HCV RNA was tested with primers for the 5' untranslated region. Replicative negative strand HCV RNA was tested with strand-specific RT-PCR for the 5' untranslated region. Immunohistochemical staining for HCV was done using an antibody to HCV core protein. Positive-strand HCV RNA was detected in 8 patients with B-cell NHL; negative-strand HCV RNA was detected in 6 of these cases, indicating viral replication. All control cases were negative for HCV RNA. Immunohistochemistry showed no staining of lymphoma cells for HCV core proteins in any case. HCV and B-cell NHLs may be associated. RT-PCR on paraffin-embedded lymphoma tissue is an alternative method of testing for HCV. The value of immunohistochemistry could not be ascertained. The exact role of HCV in the pathogenesis of B-cell NHL needs to be studied further.

The mechanisms of hepatocyte death and the events that lead to a high rate of chronic liver disease in patients infected with hepatitis C virus are not known. We established a HCV replication system in HepG2 cell culture and utilized this model to address the effect of HCV proteins on HepG2 cell growth and viability. After transfection of HepG2 cells with full-length RNA, a truncated RNA, or an antisense RNA, cell proliferation and cell viability were analyzed by thymidine uptake and the trypan blue exclusion method, respectively. Full-length RNA transfected HepG2 cells showed a decrease in cell proliferation and viability compared to cells transfected with HCV truncated RNA and antisense RNA control. A subset of cells expressing HCV proteins underwent apoptosis as documented by morphological studies, ultrastructural analysis, cell cycle analysis by flow cytometry, terminal transferase enzyme mediated end labeling of DNA, and DNA laddering. This study suggests that expression of HCV proteins can lead to cell death by apoptosis, which may be an important event in the pathogenesis of chronic hepatitis C virus infection in humans.

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It was demonstrated previously that HepG2 cells produce negative strand RNA and virus-like particles after transfection with RNA transcribed from a full-length hepatitis C virus (HCV) cDNA clone [Dash et al. (1997) American Journal of Pathology, 151:363-373]. To determine in vivo infectivity of these in vitro synthesized viral particles, a chimpanzee was inoculated intravenously with HCV derived from HepG2 cells. The infected chimpanzee was examined serially for elevation of liver enzymes, for the presence of HCV RNA in the serum by reverse transcription nested polymerase chain reaction (RT-PCR), anti-HCV antibodies in the serum, and inflammation in the liver. The chimpanzee developed elevated levels of liver enzymes after the second week, but the levels fluctuated over a 10-week period. HCV RNA was detected in the serum of the chimpanzee at the second, seventh and ninth weeks after inoculation, and remained positive up to 25 weeks. Liver biopsies at Weeks 18 and 19 revealed of mild inflammation. Nucleotide sequence analysis of HCV recovered from the infected chimpanzee at the second and ninth weeks showed 100% sequence homology with the clone used for transfection studies. Serum anti-HCV antibodies were not detected by EIA during the 25 weeks follow-up period. These results suggest that intravenous administration of the virus-like particles derived from RNA-transfected HepG2 cells are infectious, and therefore, the pMO9.6-T7 clone is an infectious clone. These results provide new information that in vitro synthesized HCV particles produced from full-length HCV clone can cause infection in a chimpanzee. This study will facilitate the use of innovative approaches to the study of assembly of HCV particles and mechanisms of virus infectivity in cell culture.

The 5' and 3' untranslated regions (UTR) of the hepatitis C virus (HCV) genome contain stem-loop structures, which are important in viral gene expression and replication. In this study, the functional roles of the predicted stem-loop structures of HCV 5' UTR and 3' UTR in viral gene expression were examined using a chimeric clone of full-length HCV genomic cDNA clone and the gene for green fluorescent protein (GFP). High level expression of the HCV-GFP chimera in Huh-7 cells was accomplished by using a replication defective adenovirus that expresses T7 RNA polymerase and transcription plasmid containing full-length HCV-GFP chimera under the control of a T7 promoter. The HCV-GFP clone, with deletion of stem-loop I, expressed proteins in transfected Huh-7 cells at comparable levels to the wild type HCV clone. Other mutations of the 5' UTR, which either deleted or altered the base pairing of stem-loops II to IV, completely abolished the expression of HCV-GFP chimera. In contrast, deletion of 3' UTR sequences had no effect on HCV protein expression. These findings suggest that the stem-loop structures II to IV of HCV 5' UTR are necessary for protein expression, but that stem loop I is dispensable for protein translation. The stem-loop structures of 3' UTR of HCV genome appear to have no direct role in viral gene expression.

Hepatitis C virus (HCV) is a hepatotropic virus that belongs to the family Flaviviridae. Chronic HCV infection can result in fibrosis, cirrhosis, and hepatocellular carcinoma (1). It is estimated that approximately 2% of the world population (ϳ170 million people) is infected with HCV, making it a major world health concern (2-6). The HCV genome consists of a positive-strand RNA molecule of approximately 9,600 nucleotides encoding both structural and nonstructural proteins. Nonstructural proteins 3 (NS3) and 5B (NS5B) have serine protease and RNA-dependent RNA polymerase functions, respectively, which are essential for proteolytic processing of the nonstructural-protein region of the HCV polyprotein and for viral RNA replication.Because the HCV polymerase and protease are necessary for the viral replication cycle, both have been attractive targets for anti-HCV drug development (7). Several inhibitors of the NS3 · 4A protease (e.g., Incivek In addition to being essential for proteolytic cleavage of the viral polyprotein, the HCV NS3 · 4A protease has also been shown to suppress the innate immune response by cleaving beta interferon (IFN-␤) stimulator 1 (IPS-1) (13) and Toll/interleukin 1 (IL-1) receptor domain-containing adapter inducing IFN-␤ (TRIF) (14) in cultured cells. In response to viral replication intermediates, such as viral RNA and proteins, IPS-1 and TRIF adaptor protein act as signaling intermediates in retinoic acidinducible gene (Rig-I) and/or Toll-like receptor 3 (TLR3) pathways (15-17). Activation of these pathways induces IRF3 activation and subsequent transcriptional activation of type I