Hepatitis delta virus (HDV) is a significant human pathogen that causes serious acute and chronic liver disease. There is no effective licensed therapy for this virus, which has a unique replication cycle that is not fully understood. HDV is a negative strand RNA virus that produces three RNAs in infected cells: the genome, which is packaged, the antigenome, which serves as a template for genome synthesis, and an mRNA. The genome and antigenome accumulate to high levels in infected cells – over 100,000 copies per cell. These RNAs are synthesized by host RNA polymerase in a highly unusual process that involves an RNA template rather than DNA. The mechanisms affecting or controlling the different amounts of these RNAs, including rates of initiation and extent of elongation have not been studied. Even basic information about the synthesis of the genome and antigenome RNAs is lacking. For example, the nature the 5’ ends of these RNAs, including the transcription start site and its chemical composition (e.g. capped, triphosphate) and how many units of these RNAs are transcribed during engagement of the polymerase with the circular RNA template are not known. Analysis of these processes has been challenging because of the ribozyme activity that processes the transcripts produced by rolling circle replication and because of limitations in transfection methods that have been used to initiate and study HDV replication. We have recently established a method for initiating HDV RNA replication following transfection of in vitro-synthesized circular HDV RNAs, including ones lacking ribozyme activity. This method provides new approaches to analyzing the early stages of HDV RNA synthesis in ways that have not been possible. This proposal is directed at exploiting these new approaches to determine the contributions of initiation and elongation to the amounts of the HDV RNAs that appear in infected cells and, in turn, to determine the HDV sequences and structures that affect initiation and elongation. There are three aims. Aim 1 will identify and characterize the initiation sites of the HDV genome and antigenome. This aim will also provide a definitive answer to the relationships between the synthesis of the mRNA and the antigenome, both of which occur on the genome template. Aim 2 will characterize the genome and antigenome elongation processes, answering key questions about how elongation contributes to the amounts of these RNAs that accumulate in cells. Aim 3 will place the information gained in the first two aims in context by comparing the contributions of initiation and elongation to variations in HDV RNA levels among different HDV clones, genotypes and mutations