Identification of the functional regions required for hepatitis D virus replication and transcription by linker-scanning mutagenesis of viral genome.

To define the important cis-elements in hepatitis delta virus (HDV) RNA, the viral genome was mutated by a linker-scanning mutagenesis strategy that maintained the native rod-like structure of HDV RNA. Mutant HDV cDNAs or their corresponding RNA transcripts were transfected into a Huh-7-derived cell line which continuously expressed small hepatitis delta antigen to study the viral replication and transcription. Here we report the following findings. (i) Although most of the mutant RNAs could self-process to generate the 1.7-kb genomic RNA and all their stabilities were similar, positions which surround the genomic ribozyme domain were found to be important for the self-processing of the dimeric RNA. (ii) The replication of viral RNA was greatly diminished in many mutants, suggesting that multiple regions in HDV RNA were required for replication. (iii) In certain mutants, replication of the HDV antigenomic RNA was selectively abolished but that of the genomic RNA was not. Therefore, this was the first report to show that the cis-elements needed for the replication of genomic or antigenomic HDV RNA could be different. (iv) A continuous region (nt 1625 to nt 431), spanning the HDAg mRNA initiation site and containing the in vitro identified RNA promoter, was found to be important for mRNA production in vivo. (v) The HDV RNA replication and transcription was previously proposed to be governed by a single "double-acting promoter." However, two mutants which were deficient in mRNA synthesis still retained active viral RNA replication. It suggested that the HDV replication could initiate from sites other than this single promoter. This study therefore provided an insight into the cis-elements required for HDV RNA replication and transcription and further contributed to our understanding of HDV life cycle.