Affiliation:
1. McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706
Abstract
ABSTRACT
Synthesis of minus-strand DNA of human hepatitis B virus (HBV) can be divided into three phases: initiation of DNA synthesis, the template switch, and elongation of minus-strand DNA. Although much is known about minus-strand DNA synthesis, the mechanism(s) by which this occurs has not been completely elucidated. Through a deletion analysis, we have identified a
cis
-acting element involved in minus-strand DNA synthesis that lies within a 27-nucleotide region between DR2 and the 3′ copy of DR1. A subset of this region (termed Φ) has been hypothesized to base pair with the 5′ half of ε (H. Tang and A. McLachlan, Virology,
303:
199-210, 2002). To test the proposed model, we used a genetic approach in which multiple sets of variants that disrupted and then restored putative base pairing between the 5′ half of ε and Φ were analyzed. Primer extension analysis, using two primers simultaneously, was performed to measure encapsidated pregenomic RNA (pgRNA) and minus-strand DNA synthesized in cell culture. The efficiency of minus-strand DNA synthesis was defined as the amount of minus-strand DNA synthesized per encapsidation event. Our results indicate that base pairing between Φ and the 5′ half of ε contributes to efficient minus-strand DNA synthesis. Additional results are consistent with the idea that the primary sequence of Φ and/or ε also contributes to function. How base pairing between Φ and ε contributes to minus-strand DNA synthesis is not known, but a simple speculation is that Φ base pairs with the 5′ half of ε to juxtapose the donor and acceptor sites to facilitate the first-strand template switch.
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology
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