Luytjes W, Gerritsma H, Spaan W J
Department of Virology, Leiden University, The Netherlands.
Virology. 1996 Feb 1;216(1):174-83. doi: 10.1006/viro.1996.0044.
We have analyzed the replication of deletion mutants of defective interfering (DI) RNAs derived from the coronavirus mouse hepatitis virus (MHV)-A59 in the presence of MHV-A59. Using two parental DI RNAs, MIDI and MIDI delta H, a twin set of deletion mutants was generated with progressively shorter stretches of 5' sequence colinear with the genomic RNA. All deletion mutants contained in-frame ORFs. We show that in transfected cells and after one passage the DI RNAs were detectable and that their accumulation was positively correlated with the length of 5' sequence they contained. However, accumulation of two twin mutants, delta 2, in which sequences from nucleotide position 467 were fused to those from position 801, was undetectable. In passage 4 cells, but not in transfected or in passage 1 cells, recombination with genomic RNA led to the appearance of the parental DI RNAs. The accumulation of these parental RNAs was inversely correlated with the length of 5' sequence on the deletion mutants and was highest in the delta 2 samples. In sharp contrast to the data reported for MHV-JHM-derived DI RNAs, we show that MHV-A59-derived mutant RNAs do not require an internal sequence domain for replication. The data suggest that coronavirus replication involves an RNA superstructure at the 5' end of the genome or one comprising both ends of the genomic RNA. We also conclude from the recombination data that in-frame mutants with impaired replication signals are more fit than out-frame mutants with intact replication signals.
我们分析了源自冠状病毒小鼠肝炎病毒（MHV）-A59的缺陷干扰（DI）RNA缺失突变体在MHV-A59存在下的复制情况。利用两个亲本DI RNA，即MIDI和MIDI delta H，构建了一组缺失突变体，其与基因组RNA共线性的5'序列片段逐渐缩短。所有缺失突变体均含有读码框内的开放阅读框（ORF）。我们发现，在转染细胞中以及传代一次后，DI RNA均可检测到，且它们的积累与所含5'序列的长度呈正相关。然而，两个双突变体delta 2（其中核苷酸位置467处的序列与位置801处的序列融合）的积累无法检测到。在传代4的细胞中，但在转染细胞或传代1的细胞中未出现这种情况，与基因组RNA的重组导致亲本DI RNA的出现。这些亲本RNA的积累与缺失突变体上5'序列的长度呈负相关，在delta 2样本中积累最高。与报道的源自MHV-JHM的DI RNA的数据形成鲜明对比的是，我们发现源自MHV-A59的突变RNA复制不需要内部序列结构域。数据表明，冠状病毒复制涉及基因组5'端的RNA超结构或包含基因组RNA两端的超结构。我们还从重组数据得出结论，复制信号受损的读码框内突变体比复制信号完整的读码框外突变体更具适应性。
