Macnaughton T B, Wang Y J, Lai M M
Howard Hughes Medical Institute, University of Southern California School of Medicine, Los Angeles 90033-1054.
J Virol. 1993 Apr;67(4):2228-34. doi: 10.1128/JVI.67.4.2228-2234.1993.
Hepatitis delta virus (HDV) contains a circular RNA genome of 1.7 kb. HDV RNA replication is thought to proceed via a rolling-circle mechanism that is dependent on autocatalytic cleavage and ligation reactions. However, it has never been established that these ribozyme activities are indeed involved in HDV RNA replication. To investigate the possible biological significance of HDV RNA self-cleavage, we constructed several HDV dimer cDNAs containing single-base substitutions of the 3' nucleotide of the genomic and the antigenomic self-cleavage sites. These mutations were known to affect self-cleavage in vitro to various extents. The effects of these mutations on HDV RNA replication were examined in hepatic and nonhepatic cell lines. The results showed that all of the mutants which had lost the in vitro self-cleavage activity could not replicate. The only mutant which retained full cleavage activity replicated as efficiently as the wild-type RNA. Thus, this study established that self-cleavage activity is required for HDV RNA replication in cells. Interestingly, the level of HDV RNA detected in cells transfected with this replication-competent mutant and that detected in cells transfected with the wild-type construct were similar in COS-7 cells but vastly different in HepG2 and Huh-7 cells, suggesting that HDV RNA self-cleavage activity may be modulated by cell-specific factors. We also compared the effects of mutations when the primary transcripts of these constructs were of either genomic or antigenomic sense. In constructs which synthesize primary transcripts of genomic sense, all of the antigenomic self-cleavage mutants produced as much hepatitis delta antigen (HDAg) as did the wild-type construct, even in the absence of detectable HDV RNA replication, whereas the genomic self-cleavage mutants produced very little HDAg. These and other data suggest that (i) the primary HDV RNA transcripts of both genomic and antigenomic polarities must first be processed to serve as a template for HDV RNA transcription, (ii) efficient cleavage at the antigenomic self-cleavage site is not required for HDAg expression, and (iii) HDV RNA replication most likely occurs by a double-rolling-circle mechanism.
丁型肝炎病毒（HDV）含有一个1.7kb的环状RNA基因组。HDV RNA复制被认为是通过一种依赖于自催化切割和连接反应的滚环机制进行的。然而，这些核酶活性是否确实参与HDV RNA复制从未得到证实。为了研究HDV RNA自我切割的可能生物学意义，我们构建了几个HDV二聚体cDNA，它们在基因组和反基因组自我切割位点的3'核苷酸处含有单碱基取代。已知这些突变在体外会不同程度地影响自我切割。在肝细胞系和非肝细胞系中检测了这些突变对HDV RNA复制的影响。结果表明，所有失去体外自我切割活性的突变体都不能复制。唯一保留完全切割活性的突变体复制效率与野生型RNA相同。因此，本研究证实了自我切割活性是HDV RNA在细胞中复制所必需的。有趣的是，用这种具有复制能力的突变体转染的细胞中检测到的HDV RNA水平与用野生型构建体转染的细胞中检测到的水平在COS-7细胞中相似，但在HepG2和Huh-7细胞中差异很大，这表明HDV RNA自我切割活性可能受细胞特异性因子调节。我们还比较了这些构建体的初级转录本为基因组或反基因组意义时突变的影响。在合成基因组意义初级转录本的构建体中，所有反基因组自我切割突变体产生的丁型肝炎抗原（HDAg）与野生型构建体一样多，即使在没有可检测到的HDV RNA复制的情况下也是如此，而基因组自我切割突变体产生的HDAg很少。这些以及其他数据表明：（i）基因组和反基因组极性的初级HDV RNA转录本都必须首先进行加工才能作为HDV RNA转录的模板；（ii）HDAg表达不需要在反基因组自我切割位点进行有效切割；（iii）HDV RNA复制很可能通过双滚环机制发生。
