Department of Pediatrics and Microbiology & Immunology, Vanderbilt University Medical Center, Nashville, TN, USA.
RNA Biol. 2011 Mar-Apr;8(2):270-9. doi: 10.4161/rna.8.2.15013. Epub 2011 Mar 1.
In order to survive and propagate, RNA viruses must achieve a balance between the capacity for adaptation to new environmental conditions or host cells with the need to maintain an intact and replication competent genome. Several virus families in the order Nidovirales, such as the coronaviruses (CoVs) must achieve these objectives with the largest and most complex replicating RNA genomes known, up to 32 kb of positive-sense RNA. The CoVs encode sixteen nonstructural proteins (nsp 1-16) with known or predicted RNA synthesis and modification activities, and it has been proposed that they are also responsible for the evolution of large genomes. The CoVs, including murine hepatitis virus (MHV) and SARS-CoV, encode a 3'-to-5' exoribonuclease activity (ExoN) in nsp14. Genetic inactivation of ExoN activity in engineered SARS-CoV and MHV genomes by alanine substitution at conserved DE-D-D active site residues results in viable mutants that demonstrate 15- to 20-fold increases in mutation rates, up to 18 times greater than those tolerated for fidelity mutants of other RNA viruses. Thus nsp14-ExoN is essential for replication fidelity, and likely serves either as a direct mediator or regulator of a more complex RNA proofreading machine, a process previously unprecedented in RNA virus biology. Elucidation of the mechanisms of nsp14-mediated proofreading will have major implications for our understanding of the evolution of RNA viruses, and also will provide a robust model to investigate the balance between fidelity, diversity and pathogenesis. The discovery of a protein distinct from a viral RdRp that regulates replication fidelity also raises the possibility that RNA genome replication fidelity may be adaptable to differing replication environments and selective pressures, rather than being a fixed determinant.
为了生存和繁殖，RNA 病毒必须在适应新的环境条件或宿主细胞的能力与维持完整和具有复制能力的基因组的需求之间取得平衡。套式病毒目（order Nidovirales）中的几个病毒科，如冠状病毒（CoVs），必须利用已知的最大和最复杂的复制 RNA 基因组来实现这些目标，该基因组的正链 RNA 长达 32kb。CoVs 编码 16 种非结构蛋白（nsp1-16），具有已知或预测的 RNA 合成和修饰活性，并且有人提出它们也负责大基因组的进化。CoVs，包括鼠肝炎病毒（MHV）和 SARS-CoV，在 nsp14 中编码 3'到 5'外切核酸酶活性（ExoN）。通过在保守的 DE-D-D 活性位点残基上将丙氨酸取代，使工程 SARS-CoV 和 MHV 基因组中的 ExoN 活性失活，导致产生可行的突变体，其突变率增加 15-20 倍，比其他 RNA 病毒的保真度突变体的容忍度高 18 倍。因此，nsp14-ExoN 对于复制保真度是必需的，并且可能是更复杂的 RNA 校对机器的直接介导物或调节剂，这在 RNA 病毒生物学中以前是前所未有的。阐明 nsp14 介导的校对的机制将对我们理解 RNA 病毒的进化具有重大意义，并且还将为研究保真度、多样性和发病机制之间的平衡提供一个强大的模型。发现一种与 RdRp 不同的调节复制保真度的蛋白质也提出了这样一种可能性，即 RNA 基因组复制保真度可能适应不同的复制环境和选择压力，而不是作为一个固定的决定因素。
