Student Theses and Dissertations

Date of Award

2000

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Konarska Laboratory

Abstract

R N A polymerase II (pol II) has been implicated in the RNA-templated RNA synthesis during replication of some Viroids and Hepatitis Delta Virus (HDV). In this study HDV RNA-templated pol II transcription was examined in vitro, using HeLa cell nuclear extracts (NE). Under standard conditions for the DNAtemplated pol II transcription, a segment of the antigenomic (AG) HDV RNA encompassing the left-hand tip region of the HDV rod-like structure, serves as a template for efficient transcription. The transcription reaction is highly sensitive to a-amanitin in HeLa cell NE, but it is partially resistant to this toxin in NE from PMG cells that contain an a-amanitin resistant allele of the pol II largest subunit, strongly suggesting pol II involvement in the process. Direct RNase A, T1 and RNAse H digestion analyses demonstrate that the product of the HDV RNA dependent pol II transcription represents a chimeric molecule, in which the pol II transcript is covalently attached to the 5' half of the AG RNA template. Such a chimeric RNA product is generated by a cleavage of the template at a specific site followed by transcription that uses the new end as a primer. Pol II transcription of the RNA template proceedes with high fidelity but appears to pause after -40 transcribed nucleotides. A viral protein, delta antigen (HDAg), stimulates pol II elongation on the RNA template. The initiation of HDV RNA transcription that involves cleavage of the RNA template may be mechanistically similar to the endonucleolytic cleavage of the nascent transcript that, under certain conditions, occurs during the elongation phase of pol II transcription on a 'typical' DNA templates. Mutational and secondary structure mapping analyses of HDV RNA templates demonstrate that the secondary structure, rather than the primary sequence, specifies the functional pol II transcription templates. The secondary structure in the left-hand terminal hairpin of the HDV RNA also modulates HDV replication in vivo, in COS7 and HeLa cells. The correlation between the effects of secondary structure alterations on the efficiency of pol II transcription in vitro and HDV replication in vivo suggests that the observed RNA-templated pol II transcription in vitro reflects an important regulatory step of the HDV replication cycle. Based on these findings, a modified pol ll-mediated rolling-circle model for HDV replication is proposed. It involves initiation of transcription by cleavage of one RNA template, followed by pol II switching to a new circular template. The model also suggests that, both the initiation and the template switching steps are regulated by specific secondary structure elements in the terminal hairpin of HDV RNA.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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