A novel vector for the study of hepatitis delta virus replication

In order to study HDV replication without the difficulties caused by the use of a multimeric construction and to obtain a selectable expression vector, a minimal amount of antigenomic HDV cDNA, sufficient to initiate RNA dependent replication was cloned into the plasmid pUTSV1. The first plasmid, pUT Delta 1.7, contained 1.7 genomes of KDV cDNA. After transfection of pUT Delta 1.7 into HuH7 cells, antigenomic HDV RNA was produced, processed and could enter into the replicative cycle of KDV. However, after transfection of an antigenomic ribozyme mutant (pUT Delta 1.7(AGR)) constructed on the same model, plasmid DNA dependent production of genomic HDV RNA was observed, especially in COS7 cells. It seems that a promoter within vector sequences downstream from the HDV insert may initiate counter-clockwise transcription of the plasmid. The presence of two genomic ribozymes in the insert permits the excision of a genome length genomic HDV RNA from this counter-clockwise transcript. In order to allow quantitative analysis of HDV replication, this problem was eliminated by removing the second genomic ribozyme from the insert to give the vector pUT Delta 1.7. This vector can be used conveniently for transfection experiments to explore HDV biology. (C) 1998 Elsevier Science B.V.