Insertion of a New Transcriptional Unit into the Genome of Mouse Hepatitis Virus

The subgenomic mRNAs of the coronavirus mouse hepatitis virus (MHV) are composed of a leader sequence, identical to the 5′ 70 nucleotides of the genome, joined at distant downstream sites to a stretch of sequence that is identical to the 3′ end of the genome. The points of fusion occur at intergenic sequences (IGSs), loci on the genome that contain a tract of sequence homologous to the 3′ end of the leader RNA. We have constructed a mutant of MHV-A59 containing an extra IGS inserted into the genome immediately downstream of the 3′-most gene, that encoding the nucleocapsid (N) protein. We show that in cells infected with the mutant, there is synthesis of an additional leader-containing subgenomic RNA of the predicted size. Our study demonstrates that (i) an IGS can be a sufficient cis-acting element to dictate MHV transcription, (ii) the relative efficiency of an IGS must be influenced by factors other than the nucleotides immediately adjacent to the 5′AAUCUAAAC3′ core consensus sequence or its position relative to the 3′ end of the genome, (iii) a downstream IGS can exert a polar attenuating effect on upstream IGSs, and (iv) unknown factors prevent the insertion of large exogenous elements between the N gene and the 3′ untranslated region of MHV. These results confirm and extend conclusions previously derived from the analysis of defective interfering RNAs.

冠状病毒鼠肝炎病毒（mouse hepatitis virus, MHV）的亚基因组mRNA（subgenomic mRNA）由一段与病毒基因组5′端70个核苷酸序列完全一致的前导序列（leader sequence）组成，该前导序列通过远端下游的拼接位点，与一段和病毒基因组3′端序列完全一致的区域相连。这类融合位点均位于基因间序列（intergenic sequences, IGSs）上，这类基因组位点包含一段与前导RNA的3′端同源的序列片段。我们构建了MHV-A59的突变毒株，在其基因组3′端最外侧的基因——即编码核衣壳蛋白（nucleocapsid (N) protein）的基因——的紧邻下游区域，插入了一段额外的IGS。实验结果显示，在该突变毒株感染的细胞中，会合成一条长度符合预期、额外携带前导序列的亚基因组RNA。本研究证实了如下结论：（i）IGS可作为足够的顺式作用元件（cis-acting element），调控MHV的转录过程；（ii）IGS的相对效率，除了受其5′端核心保守序列5′AAUCUAAAC3′相邻的核苷酸，或是其相对于基因组3′端的位置影响外，还会受到其他因素的调控；（iii）下游的IGS可对上游的IGS产生极性减弱效应；（iv）目前尚不明确的因素会阻碍在MHV的N基因与3′非翻译区（3′ untranslated region, 3′UTR）之间插入大型外源性元件。上述研究结果验证并拓展了此前通过缺陷干扰RNA（defective interfering RNAs）分析所得到的结论。