Project Summary/Abstract Hepatitis delta virus (HDV) is a human pathogen that causes the most serious forms of acute and chronic liver disease. There is no effective licensed therapy for this virus, which has a unique replication cycle that is not well understood. The HDV genome is a circular single stranded RNA that uses host RNA polymerase II for replication. In addition to the genome, two antigenomesense RNAs are produced in infected cells: the circular antigenome, which serves as a template for genome synthesis, and an mRNA, which encodes a nucleocapsid- like protein called the hepatitis delta antigen. The genome and antigenome are synthesized by a rolling circle mechanism in which the polymerase initiates, then transits the circular RNA template numerous times. Newly made RNAs are cleaved by ribozymes into unit-length monomers that are ligated into circles by a host RNA ligase. These processes determine the amount of viral RNA that accumulates in infected cells, which in turn affect virus spread, persistence and host responses, but their mechanisms and dynamics are not well understood. In recent work, in which we identified the position at which synthesis of genome RNA initiates, we developed tools to track these processes by following the 5’ ends of RNAs generated during rolling circle replication – one 5’ end is the initiate site, the other is the 5’ end created by ribozyme cleavage. The genome 5’ end itself is unusual in that it starts with a G that is either untemplated or templated in an unusual way, such as by a U rather than C. Our methods provide 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 dynamics of HDV RNA synthesis in infected cells and, in turn, to determine the HDV sequences and structures that affect initiation and elongation. There are two aims. In Aim 1 we will identify the sequence and structural determinants that determine where and how efficiently HDV genome RNA and mRNA synthesis initiates. We will analyze different HDV isolates and genotypes, including a recently identified ”HDV-like” virus. In Aim 2 we will characterize the dynamics of HDV genome and antigenome RNA synthesis during the course of HDV replication in cells. We will thus determine the relative contributions of initiation and elongation HDV RNA levels and identify how changes in these contributions affect changes in HDV RNA levels during replication in cells.