Calicivirus Research Group, Department of Virology, Faculty of Medicine, Imperial College London, Norfolk Place, London W2 1PG, United Kingdom.
J Virol. 2010 Mar;84(6):2859-70. doi: 10.1128/JVI.02053-09. Epub 2010 Jan 6.
Interactions of host cell factors with RNA sequences and structures in the genomes of positive-strand RNA viruses play various roles in the life cycles of these viruses. Our understanding of the functional RNA elements present in norovirus genomes to date has been limited largely to in vitro analysis. However, we recently used reverse genetics to identify evolutionarily conserved RNA structures and sequences required for norovirus replication. We have now undertaken a more detailed analysis of RNA structures present at the 3' extremity of the murine norovirus (MNV) genome. Biochemical data indicate the presence of three stable stem-loops, including two in the untranslated region, and a single-stranded polypyrimidine tract [p(Y)] of variable length between MNV isolates, within the terminal stem-loop structure. The well-characterized host cell pyrimidine binding proteins PTB and PCBP bound the 3'-untranslated region via an interaction with this variable sequence. Viruses lacking the p(Y) tract were viable both in cell culture and upon mouse infection, demonstrating that this interaction was not essential for virus replication. However, competition analysis with wild-type MNV in cell culture indicated that the loss of the p(Y) tract was associated with a fitness cost. Furthermore, a p(Y)-deleted mutant showed a reduction in virulence in the STAT1(-/-) mouse model, highlighting the role of RNA structures in norovirus pathogenesis. This work highlights how, like with other positive-strand RNA viruses, RNA structures present at the termini of the norovirus genome play important roles in virus replication and virulence.
正链 RNA 病毒基因组中宿主细胞因子与 RNA 序列和结构的相互作用在这些病毒的生命周期中发挥着各种作用。迄今为止，我们对诺如病毒基因组中存在的功能性 RNA 元件的理解在很大程度上局限于体外分析。然而，我们最近使用反向遗传学方法鉴定了诺如病毒复制所必需的进化上保守的 RNA 结构和序列。我们现在对鼠诺如病毒 (MNV) 基因组 3' 末端存在的 RNA 结构进行了更详细的分析。生化数据表明，在末端茎环结构内存在三个稳定的茎环，包括两个在非翻译区，以及一个在不同 MNV 分离株之间长度可变的单链嘧啶核苷酸序列 [p(Y)]。特征明确的宿主细胞嘧啶结合蛋白 PTB 和 PCBP 通过与该可变序列的相互作用结合 3'-非翻译区。缺乏 p(Y) 区的病毒在细胞培养和小鼠感染中均具有活力，表明该相互作用不是病毒复制所必需的。然而，在细胞培养中与野生型 MNV 的竞争分析表明，p(Y) 区的缺失与适应度成本相关。此外，p(Y) 缺失突变体在 STAT1(-/-) 小鼠模型中的毒力降低，突出了 RNA 结构在诺如病毒发病机制中的作用。这项工作强调了正链 RNA 病毒末端的 RNA 结构如何像其他正链 RNA 病毒一样在病毒复制和毒力中发挥重要作用。
