Hepatitis delta virus (HDV) contains a viroid-like circular RNA that is presumed to replicate via a rolling circle replication mechanism mediated by cellular RNA polymerases. However, the exact mechanism of rolling circle replication for HDV RNA and viroids is not clear. Using our recently described cDNA-free transfection system (L. E. Modahl and M. M. Lai, J. Virol. 72:5449-5456, 1998), we have succeeded in detecting HDV RNA replication by metabolic labeling with [ 32 P]orthophosphate in vivo and obtained direct evidence that HDV RNA replication generates high-molecular-weight multimeric species of HDV RNA, which are processed into monomeric and dimeric forms. Thus, these multimeric RNAs are the true intermediates of HDV RNA replication. We also found that HDV RNA synthesis is highly temperature sensitive, occurring most efficiently at 37 to 40°C and becoming virtually undetectable at temperatures below 30°C. Moreover, genomic HDV RNA synthesis was found to occur at a rate roughly 30-fold higher than that of antigenomic RNA synthesis. Finally, in lysolecithin-permeabilized cells, the synthesis of full-length antigenomic HDV RNA was completely resistant to high concentrations (100 g/ml) of ␣-amanitin. In contrast, synthesis of genomic HDV RNA was totally inhibited by ␣-amanitin at concentrations as low as 2.5 g/ml. Thus, these results suggest that genomic and antigenomic HDV RNA syntheses are performed by two different host cell enzymes. This observation, combined with our previous finding that hepatitis delta antigen mRNA synthesis is likely performed by RNA polymerase II, suggests that the different HDV RNA species are synthesized by different cellular transcriptional machineries.

Cellular DNA-dependent RNA polymerase II (pol II) has been postulated to carry out RNA-dependent RNA replication and transcription of hepatitis delta virus (HDV) RNA, generating a full-length (1.7-kb) RNA genome and a subgenomic-length (0.8-kb) mRNA. However, the supporting evidence for this hypothesis was ambiguous because the previous experiments relied on DNA-templated transcription to initiate HDV RNA synthesis. Furthermore, there is no evidence that the same cellular enzyme is involved in the synthesis of both RNA species. In this study, we used a novel HDV RNA-based transfection approach, devoid of any artificial HDV cDNA intermediates, to determine the enzymatic and metabolic requirements for the synthesis of these two RNA species. We showed that HDV subgenomic mRNA transcription was inhibited by a low concentration of ␣-amanitin (<3 g/ml) and could be partially restored by an ␣-amanitin-resistant mutant pol II; however, surprisingly, the synthesis of the full-length (1.7-kb) antigenomic RNA was not affected by ␣-amanitin to a concentration higher than 25 g/ml. By several other criteria, such as the differing requirement for the de novo-synthesized hepatitis delta antigen and temperature dependence, we further showed that the metabolic requirements of subgenomic HDV mRNA synthesis are different from those for the synthesis of genomic-length HDV RNA and cellular pol II transcripts. The synthesis of the two HDV RNA species could also be uncoupled under several different conditions. These findings provide strong evidence that pol II, or proteins derived from pol II transcripts, is involved in mRNA transcription from the HDV RNA template. In contrast, the synthesis of the 1.7-kb HDV antigenomic RNA appears not to be dependent on pol II. These results reveal that there are distinct molecular mechanisms for the synthesis of these two RNA species.Hepatitis delta virus (HDV) is a subviral particle containing a circular RNA genome of 1.7 kb which resembles plant viroid RNAs and contains ribozyme activities (27). HDV RNA can replicate itself in cultured cells, requiring only a virus-encoded protein, the hepatitis delta antigen (HDAg) (13, 26). HDAg, however, does not possess an RNA polymerase activity. Thus, it has always been assumed that HDV utilizes host cell RNA polymerases to replicate its RNA genome, in a mechanism similar to that of viroid RNA replication (40). However, the dependence of HDV RNA replication on a viral protein (HDAg) distinguishes HDV RNA synthesis from the synthesis of plant viroid RNA, which does not encode any protein. Furthermore, unlike plant cells (41), animal cells are not known to have RNA-dependent RNA polymerases. These issues raised interesting questions concerning which cellular polymerases are responsible for HDV RNA-dependent RNA synthesis and how they are converted from DNA-to RNAtemplated polymerases.The common belief that HDV RNA synthesis is carried out by cellular RNA polymerase II (pol II) came from early in vitro transcription studies using nuclear extracts of HDV-replica...

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The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.Hepatitis C virus (HCV) is now recognized as the major cause of non-A, non-B hepatitis. It has been estimated that about 170 million people worldwide are infected with HCV, of whom 70 to 80% will develop chronic liver disease, leading to cirrhosis in 10 to 20% and liver cancer (hepatocellular carcinoma) in 1 to 5% of chronically infected individuals (6). The linear, single-stranded, positive-sense HCV RNA genome of ca. 9.5 kb contains a single open reading frame (ORF) encoding a polyprotein which is cleaved into the individual mature viral proteins by host-and virus-specific proteinases. Three structural proteins have been identified, the core protein and two envelope proteins, E1 and E2.It has been reported that cellular and humoral immune responses play a pivotal role in the host defense mechanism against HCV (8, 36). Most HCV carriers have circulating antibodies to the virus envelope proteins and to a region located in the extreme amino terminus of E2, hypervariable region 1 (HVR1), which has been reported to contain neutralizing Bcell epitopes and a T-cell epitope (16,17,44). HVR1 probably represents the major site of HCV genetic drift, with amino acid substitutions leading to escape from recognition by existing anti-HVR1 antibodies. Due to the variability within the HVR1 region, it has been proposed that these mutations are responsible for the persistence of HCV infection through neutralizing antibody escape mutants (25,46,52). Qualitative antibody changes accompany HVR1 epi...