Specific HDV RNA-templated transcription by pol II in vitro

RNA polymerase II is implicated in the RNA-templated RNA synthesis during r eplication of viroids and Hepatitis Delta Virus (HDV); however, neither the RNA template nor protein factor requirements for this process are well def ined. We have developed an in vitro transcription system based on HeLa cell nuclear extract (NE), in which a segment of antigenomic RNA corresponding to the left-hand tip region of the HDV rod-like structure serves as a templ ate for efficient and highly specific RNA synthesis. Accumulation of the un ique RNA product is highly sensitive to alpha-amanitin in HeLa NE and only partially sensitive to this drug in NE from PMG cells that contain an allel e of the alpha-amanitin-resistant subunit of pol II, strongly suggesting po l II involvement in this reaction, Detailed analysis of the RNA product rev ealed that it represents a chimeric molecule composed of a newly synthesize d transcript covalently attached to the 5' half of the RNA template. Select ion of the start site for transcription is remarkably specific and depends on the secondary structure of the RNA template, rather than on its primary sequence. Some features of this reaction resemble the RNA cleavage-extensio n process observed for pol II-arrested complexes in vitro. A possible invol vement of the described reaction in HDV replication is discussed.