RNA Elements Directing Translation of the Duck Hepatitis B Virus Polymerase via Ribosomal Shunting
RNA Elements Directing Translation of the Duck Hepatitis B Virus Polymerase via Ribosomal Shunting
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DOI：
10.1128/jvi.00101-11
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发表时间：
2011-07-01
影响因子：
5.4
通讯作者：
Tavis, John E.
中科院分区：
文献类型：
--
作者：
Cao, Feng;Tavis, John E.
鸭乙型肝炎病毒 (DHBV) 逆转录酶 (P) 从双顺反子 mRNA（称为前基因组 RNA）的下游位置，通过特征不明的核糖体分流进行翻译。在这里，绘制了分流过程中不连续核糖体转移的位置，并鉴定了对分流重要的RNA元件，作为剖析分流机制的前奏。将突变引入DHBV基因组，将基因组表达载体转染到支持逆转录的细胞中，P翻译效率定义为P/mRNA的比率。提出了五项观察结果。首先，核糖体脱离了包含称为ε的RNA茎环的序列，ε是病毒复制的关键，但已知的ε元件并不是分流所必需的。其次，在 mRNA 上至少发现了两个核糖体着陆位点。第三，epsilon上游的所有序列、帽和P AUG之间的大多数序列以及P编码区内的序列对于分流来说是可有可无的。第四，mRNA上参与逆转录的元件或根据其他病毒中记录的机制预测参与分流的元件，包括出发位点附近的短开放阅读框，对于分流并不是必需的。最后，我们发现 mRNA 5' 部分的两个 RNA 元件有助于分流。这些观察结果与通过未表征的 RNA 二级结构起作用的信号引导的分流最为一致。总之，这些数据表明 DHBV 采用了一种新颖的分流机制或一种特征机制的主要变异。
The duck hepatitis B virus (DHBV) reverse transcriptase (P) is translated from the downstream position on a bicistronic mRNA, called the pregenomic RNA, through a poorly characterized ribosomal shunt. Here, the positions of the discontinuous ribosomal transfer during shunting were mapped, and RNA elements important for shunting were identified as a prelude to dissecting the shunting mechanism. Mutations were introduced into the DHBV genome, genomic expression vectors were transfected into cells which support reverse transcription, and P translation efficiency was defined as the ratio of P/mRNA. Five observations were made. First, ribosomes departed from sequences that comprise the RNA stem-loop called epsilon that is key to viral replication, but the known elements of epsilon were not needed for shunting. Second, at least two landing sites for ribosomes were found on the mRNA. Third, all sequences upstream of epsilon, most sequences between the cap and the P AUG, and sequences within the P-coding region were dispensable for shunting. Fourth, elements on the mRNA involved in reverse transcription or predicted to be involved in shunting on the basis of mechanisms documented in other viruses, including short open reading frames near the departure site, were not essential for shunting. Finally, two RNA elements in the 5' portion of the mRNA were found to assist shunting. These observations are most consistent with shunting being directed by signals that act through an uncharacterized RNA secondary structure. Together, these data indicate that DHBV employs either a novel shunting mechanism or a major variation on one of the characterized mechanisms.