Journal
VIROLOGY
Volume 379, Issue 1, Pages 30-37Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.virol.2008.06.021
Keywords
spliced RNA; pregenomic RNA; CMV-IE promoter; minus-strand DNA synthesis; plus-strand DNA synthesis
Categories
Funding
- National Institutes of Health [P01 CA22443, T32 CA009135 (EBL)]
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Hepatitis B virus synthesizes multiple spliced RNAs that can be reverse transcribed into viral DNA. We thoroughly characterized the contribution of spliced RNAs to DNA synthesis in transfected cultures of Huh7 and HepG2 cells. We found that LIP to 50% of DNA within intracellular capsids is derived from five spliced RNAs. Expressing HBV P protein and pgRNA from separate plasmids and the use of the CMV-IE promoter contributes to these high levels of encapsidated DNA derived from spliced RNA. A spliced RNA called Sp1 was the predominant species expressed in both cell lines. All spliced RNAs Support the synthesis minus-strand DNA and duplex linear DNA. Only one of the spliced RNAs, Sp14, Supported the synthesis of relaxed circular DNA because splicing removed an important cis-acting sequence (hM) in the other four RNAs. Additionally, we created a variant that was deficient in the synthesis of spliced RNA and supported DNA synthesis at wildtype levels. Our results reinforce and extend the idea that a significant fraction of HBV DNA synthesized under common experimental conditions is derived from spliced RNA. It is important that their presence be considered when analyzing HBV DNA replication in transfected cell cultures. (C) 2008 Elsevier Inc. All rights reserved.
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