Kim Y N, Lai M M, Makino S
Department of Microbiology, University of Texas, Austin 78712.
Virology. 1993 May;194(1):244-53. doi: 10.1006/viro.1993.1255.
All of the coronavirus defective interfering (DI) RNAs analyzed thus far contain an open reading frame (ORF) from which DI RNA-specific protein(s) are translated, although the function of the DI-specific protein and the significance of the ORF are not known. A complete cDNA clone of a mouse hepatitis virus (MHV) DI RNA, NE-1, containing a single nucleotide deletion in the 5' region of the ORF was obtained and analyzed. Due to this single nucleotide deletion, a DI-specific protein of 7.5-kDa was made from NE-1, in contrast to the 88-kDa protein made from the wild-type DI RNA. NE-1 RNA was efficiently replicated after transfection into MHV-infected cells. However, after one passage of NE-1 RNA-containing virus, the 88-kDa wild-type protein was synthesized, indicating that the large ORF was restored during NE-1 DI RNA replication. Sequence analysis of NE-1 DI RNA from infected cells demonstrated that in approximately half of the DI RNA population, the ORF was restored by RNA recombination between NE-1 DI RNA and helper virus genomic sequence. The sequences of other DI RNAs contained an additional nontemplated A at the five-A sequence nine nucleotides upstream of the deletion site, resulting in a stretch of six consecutive As. In these "edited"-type DI RNAs, the original nucleotide deletion was maintained and no RNA recombination was observed. This "editing" produced an ORF of the same size as the wild-type DI RNA. We conclude that the DI RNA with a large ORF has a selective advantage. There was no significant difference in replication efficiency among these RNAs when they replicated alone. However, cotransfection of two DI RNA species and time course experiments suggested that homologous interference and other mechanism(s) during the early stage of virus multiplication are responsible for the accumulation of DI RNAs containing the large ORF.
迄今为止分析的所有冠状病毒缺陷干扰（DI）RNA都包含一个开放阅读框（ORF），可从中翻译出DI RNA特异性蛋白，尽管DI特异性蛋白的功能以及该ORF的意义尚不清楚。获得并分析了小鼠肝炎病毒（MHV）DI RNA NE-1的完整cDNA克隆，该克隆在ORF的5'区域有一个单核苷酸缺失。由于这个单核苷酸缺失，NE-1产生了一种7.5 kDa的DI特异性蛋白，与之形成对比的是，野生型DI RNA产生的是88 kDa的蛋白。将NE-1 RNA转染到感染MHV的细胞后能高效复制。然而，含NE-1 RNA的病毒传代一次后，就合成了88 kDa的野生型蛋白，这表明在NE-1 DI RNA复制过程中，大的ORF得以恢复。对感染细胞中NE-1 DI RNA的序列分析表明，在大约一半的DI RNA群体中，ORF是通过NE-1 DI RNA与辅助病毒基因组序列之间的RNA重组而恢复的。其他DI RNA的序列在缺失位点上游9个核苷酸处的五聚腺苷序列中额外含有一个非模板化的A，导致出现一段连续6个A的序列。在这些“编辑”型DI RNA中，原始的核苷酸缺失得以保留，未观察到RNA重组。这种“编辑”产生了一个与野生型DI RNA大小相同的ORF。我们得出结论，具有大ORF的DI RNA具有选择性优势。这些RNA单独复制时，复制效率没有显著差异。然而，两种DI RNA的共转染以及时间进程实验表明，病毒增殖早期的同源干扰和其他机制导致了含大ORF的DI RNA的积累。
