以前,建立了一个系统,其中来自小鼠肝炎病毒(MHV)的基因间区域插入到MHV缺陷干扰(DI)RNA中,导致辅助病毒感染的细胞中亚基因组DIRNA的转录。在本研究中,构建了DIcDNA,该cDNA在位于基因6和7之间的0.3kb长的基因间区域的中间包含一个UCUAAAC共有序列。从这个DIcDNA克隆,构建21个突变型DIRNA,使得七个共有序列核苷酸中的每一个单独地改变为三个替代碱基。这些突变体用于定义MHV转录共有序列UCUAAAC的完整性变化如何影响mRNA转录。除了具有序列UGUAAAC和UCGAAAC的两个突变体外,所有突变体都支持有效的亚基因组DIRNA转录。这表明MHV转录调节足够灵活以识别改变的共有序列。接下来,这些和其他突变体用于检查亚基因组DIRNA上的前导体融合位点。序列分析表明,分析的所有亚基因组DIRNA都包含两个五核苷酸序列;第一个序列似乎是由前导序列贡献的,和前导体融合很可能发生在第二序列的第一或第二核苷酸。这一观察结果与提出的冠状病毒转录模型(S.C.贝克和M.C.赖,EMBOJ.9:4173-4179,1990),其中指出核苷酸错配可以通过RNA聚合酶校对活性来校正。
Previously, a system in which an intergenic region from mouse hepatitis virus (MHV) inserted into an MHV defective interfering (DI) RNA led to transcription of a subgenomic DI RNA in helper virus-infected cells was established. In the present study, a DI cDNA containing one UCUAAAC
consensus sequence in the middle of the 0.3-kb-long intergenic region located between genes 6 and 7 was constructed. From this DI cDNA clone, 21 mutant DI RNAs were constructed so that each of the seven
consensus sequence nucleotides was changed individually to the three alternative bases. These mutants were used to define how changes in the integrity of MHV transcription
consensus sequence UCUAAAC affected mRNA transcription. Except for two mutants with the sequences UGUAAAC and UCGAAAC, all of the mutants supported efficient subgenomic DI RNA transcription. This indicated that MHV transcription regulation was sufficiently flexible to recognize altered
consensus sequences. Next, these and other mutants were used to examine the leader-body fusion site on the subgenomic DI RNAs. Sequence analysis demonstrated that all subgenomic DI RNAs analyzed contained two pentanucleotide sequences; the first sequence seemed to be contributed by the leader, and the leader-body fusion most likely took place at either the first or the second nucleotide of the second sequence. This observation was not consistent with the proposed coronavirus transcription model (S. C. Baker and M. M. C. Lai, EMBO J. 9:4173-4179, 1990) which states that nucleotide mismatch can be corrected by RNA polymerase proofreading activity.