Abraham Teresa M, Loeb Daniel D
McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
J Virol. 2007 Nov;81(21):11577-84. doi: 10.1128/JVI.01414-07. Epub 2007 Aug 15.
Previous analysis of hepatitis B virus (HBV) indicated base pairing between two cis-acting sequences, the 5' half of the upper stem of epsilon and phi, contributes to the synthesis of minus-strand DNA. Our goal was to identify other cis-acting sequences on the pregenomic RNA (pgRNA) involved in the synthesis of minus-strand DNA. We found that large portions of the pgRNA could be deleted or substituted without an appreciable decrease in the level of minus-strand DNA synthesized, indicating that most of the pgRNA is dispensable and that a specific size of the pgRNA is not required for this process. Our results indicated that the cis-acting sequences for the synthesis of minus-strand DNA are present near the 5' and 3' ends of the pgRNA. In addition, we found that the first-strand template switch could be directed to a new location when a 72-nucleotide (nt) fragment, which contained the cis-acting sequences present near the 3' end of the pgRNA, was introduced at that location. Within this 72-nt region, we uncovered two new cis-acting sequences, which flank the acceptor site. We show that one of these sequences, named omega and located 3' of the acceptor site, base pairs with phi to contribute to the synthesis of minus-strand DNA. Thus, base pairing between three cis-acting elements (5' half of the upper stem of epsilon, phi, and omega) are necessary for the synthesis of HBV minus-strand DNA. We propose that this topology of pgRNA facilitates first-strand template switch and/or the initiation of synthesis of minus-strand DNA.
先前对乙型肝炎病毒（HBV）的分析表明，两个顺式作用序列（即ε和φ上茎的5' 半部分）之间的碱基配对有助于负链DNA的合成。我们的目标是鉴定前基因组RNA（pgRNA）上参与负链DNA合成的其他顺式作用序列。我们发现，pgRNA的大部分可以被删除或替换，而不会显著降低合成的负链DNA水平，这表明大部分pgRNA是可有可无的，并且该过程不需要特定大小的pgRNA。我们的结果表明，负链DNA合成的顺式作用序列存在于pgRNA的5' 和3' 末端附近。此外，我们发现，当一个包含pgRNA 3' 末端附近存在的顺式作用序列的72个核苷酸（nt）片段被引入该位置时，第一链模板转换可以被引导到一个新位置。在这个72 nt区域内，我们发现了两个新的顺式作用序列，它们位于受体位点两侧。我们表明，这些序列之一，名为ω，位于受体位点的3' 端，与φ碱基配对，有助于负链DNA的合成。因此，三个顺式作用元件（ε上茎的5' 半部分、φ和ω）之间的碱基配对对于HBV负链DNA的合成是必要的。我们提出，pgRNA的这种拓扑结构促进了第一链模板转换和/或负链DNA合成的起始。
