The details are described in Materials and Methods and in the text. The mutations were placed into the HDV genome in the context of pDL542, and then the construct containing the mutation was transfected into AgS-293 cells. In these settings, the HDV genome (with or without the mutation) doesn’t make AgS, but it is replication-competent, and will support RNA-directed RNA replication, if functional AgS is provided in-trans. In this case, wt AgS was provided from the integrated fragment of HDV DNA in the presence of tetracycline. The described settings allowed us to analyze the influence of an individual mutation on the ability of HDV genome to support RNA-directed RNA synthesis separately from the effect that AgS bearing the mutation might have on HDV G RNA replication.