RNA-dependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerases

Cellular DNA-dependent RNA polymerase II (pol II) has been postulated to ca rry out RNA-dependent RNA replication and transcription of hepatitis delta virus (HDV) RNA, generating a full-length (1.7-kb) RNA genome and a subgeno mic-length (0.8-kb) mRNA. However, the supporting evidence for this hypothe sis was ambiguous because the previous experiments relied on DNA-templated transcription to initiate HDV RNA synthesis. Furthermore. there is no evide nce 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 enzymat ic and metabolic requirements for the synthesis of these two RNA species. W e showed that HDV subgenomic mRNA transcription was inhibited by a low conc entration of alpha-amanitin (<3 mu g/ml) and could be partially restored by an alpha-amanitin-resistant mutant pol II; however, surprisingly, the synt hesis of the full-length (1.7-kb) antigenomic RNA was not affected by alpha -amanitin to a concentration higher than 25 mu g/ml. By several other crite ria, such as the differing requirement for the de novo-synthesized hepatiti s delta antigen and temperature dependence, we further showed that the meta bolic requirements of subgenomic HDV mRNA synthesis are different from thos e for the synthesis of genomic-length HDV RNA and cellular pol II transcrip ts. The synthesis of the two HDV RNA species could also be uncoupled under several different conditions. These findings provide strong evidence that p ol II, or proteins derived from pol LI transcripts, is involved in mRNA tra nscription 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 result s reveal that there are distinct molecular mechanisms for the synthesis of these two RNA species.