Zheng H, Fu T B, Lazinski D, Taylor J
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
J Virol. 1992 Aug;66(8):4693-7. doi: 10.1128/JVI.66.8.4693-4697.1992.
It has been shown previously that during replication of the genome of human hepatitis delta virus (HDV), a specific nucleotide change occurs to eliminate the termination codon for the small delta antigen (G. Luo, M. Chao, S.-Y. Hsieh, C. Sureau, K. Nishikura, and J. Taylor, J. Virol. 64:1021-1027, 1990). This change creates an extension in the length of the open reading frame for the delta antigen from 195 to 214 amino acids. These two proteins, the small and large delta antigens, have important and distinct roles in the life cycle of HDV. To further investigate the mechanism of this specific nucleotide alteration, we developed a sensitive assay involving the polymerase chain reaction to monitor changes on HDV RNA sequences as they occurred in transfected cells. We found that the substrate for the sequence change was the viral genomic RNA rather than the antigenomic RNA. This sequence change occurred independently of genome replication or the presence of the delta antigen. Less than full-length genomic RNA could act as a substrate, but only if it also contained a corresponding RNA sequences from the other side of the rodlike structure, which is characteristic of HDV. We were also able to reproduce the HDV base change in vitro, by addition of purified viral RNA to nuclear extracts of cells from a variety of species.
先前的研究表明，在人类丁型肝炎病毒（HDV）基因组复制过程中，会发生特定的核苷酸变化，从而消除小δ抗原的终止密码子（G. Luo、M. Chao、S.-Y. Hsieh、C. Sureau、K. Nishikura和J. Taylor，《病毒学杂志》64:1021 - 1027，1990年）。这种变化使δ抗原开放阅读框的长度从195个氨基酸延伸至214个氨基酸。这两种蛋白质，即小δ抗原和大δ抗原，在HDV的生命周期中具有重要且不同的作用。为了进一步研究这种特定核苷酸改变的机制，我们开发了一种灵敏的检测方法，该方法涉及聚合酶链反应，用于监测HDV RNA序列在转染细胞中发生的变化。我们发现，序列变化的底物是病毒基因组RNA，而非反基因组RNA。这种序列变化的发生独立于基因组复制或δ抗原的存在。小于全长的基因组RNA可以作为底物，但前提是它还包含来自杆状结构另一侧的相应RNA序列，这是HDV的特征。我们还能够通过向来自多种物种的细胞的核提取物中添加纯化的病毒RNA，在体外重现HDV的碱基变化。
