de Groot R J, van der Most R G, Spaan W J
Department of Virology, Institute of Medical Microbiology, Faculty of Medicine, Leiden University, The Netherlands.
J Virol. 1992 Oct;66(10):5898-905. doi: 10.1128/JVI.66.10.5898-5905.1992.
The genome of the defective interfering (DI) mouse hepatitis virus DI-a carries a large open reading frame (ORF) consisting of ORF1a, ORF1b, and nucleocapsid sequences. To test whether this fusion ORF is important for DI virus replication, we constructed derivatives of the DI-a genome in which the reading frame was truncated by a nonsense codon or a frameshift mutation. In vitro-transcribed DI RNAs were transfected into mouse hepatitis virus-infected cells followed by undiluted passage of the resulting virus-DI virus stocks. The following observations were made. (i) Truncation of the fusion ORF was not lethal but led to reduced accumulation of DI RNA. (ii) When pairs of nearly identical in-frame and out-of-frame DI RNAs were directly compared by cotransfection, DI viruses containing in-frame genomic RNAs prevailed within three successive passage even when the out-of-frame RNAs were transfected in 10-fold molar excess. (iii) When DI viruses containing out-of-frame genomic RNAs were passaged, mutants emerged and were selected for that had restored the reading frame. We conclude that translation of the fusion ORF is indeed required for efficient propagation of DI-a and its derivatives.
缺陷干扰（DI）小鼠肝炎病毒DI-a的基因组携带一个由ORF1a、ORF1b和核衣壳序列组成的大开放阅读框（ORF）。为了测试这种融合ORF对DI病毒复制是否重要，我们构建了DI-a基因组的衍生物，其中阅读框被无义密码子或移码突变截断。将体外转录的DI RNA转染到感染小鼠肝炎病毒的细胞中，然后对产生的病毒-DI病毒株进行原液传代。得到以下观察结果。（i）融合ORF的截断并非致死性的，但导致DI RNA积累减少。（ii）当通过共转染直接比较几乎相同的框内和框外DI RNA对时，即使框外RNA以10倍摩尔过量转染，含有框内基因组RNA的DI病毒在连续三代传代中仍占优势。（iii）当含有框外基因组RNA的DI病毒传代时，出现了突变体并被选择出来，其阅读框得以恢复。我们得出结论，融合ORF的翻译确实是DI-a及其衍生物高效增殖所必需的。
