High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.
High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.
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通过RNA测序和核糖体分析对冠状病毒基因表达的高分辨率分析。
DOI：
10.1371/journal.ppat.1005473
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发表时间：
2016-02
期刊：
影响因子：
6.7
通讯作者：
Brierley I
中科院分区：
文献类型：
--
作者：
Irigoyen N;Firth AE;Jones JD;Chung BY;Siddell SG;Brierley I
冠状病毒科的成员具有所有RNA病毒中最大的基因组，通常在30个内切酶的区域中。一些冠状病毒，如严重急性呼吸综合征相关冠状病毒（SARS-CoV）和中东呼吸综合征相关冠状病毒（MERS-CoV），具有重要的医学意义，死亡率高，并且在SARS-CoV的情况下，具有重大的大流行潜力。其他冠状病毒，如猪流行性腹泻病毒和禽冠状病毒，是重要的家畜病原体。核糖体分析是一种利用翻译核糖体保护约30个核苷酸的mRNA免受核糖核酸酶消化的能力的技术。核糖体保护的mRNA片段被纯化，进行深度测序并映射回转录组以给出翻译的全局“快照”。平行RNA测序允许通过转录本丰度进行标准化。在这里，我们将核糖体分析应用于感染小鼠冠状病毒，小鼠肝炎病毒，A59株（MHV-A59），一种与SARS-CoV和MERS-CoV同属的模型冠状病毒的细胞。所获得的数据使我们能够精确地研究病毒转录和翻译的动力学。我们研究了正义和负义基因组和亚基因组病毒RNA产生的时间进程以及不同病毒ORF的相对翻译效率。没有发现病毒mRNA比宿主mRNA更有效地翻译;相反，由于高水平的病毒转录物，病毒翻译在稍后的时间点主导宿主翻译。分析数据的三联体定相允许精确测定翻译的阅读框，并揭示了已知病毒蛋白编码区上游或嵌入其中的几个翻译的短开放阅读框。核糖体暂停网站被确定在病毒复制酶多聚蛋白pp 1a ORF和实验研究。与预期相反，核糖体没有被发现暂停在核糖体移码位点。据我们所知，这是核糖体分析首次应用于RNA病毒。核糖体谱分析是一种以亚密码子分辨率监测活细胞翻译的强大技术。它特别适用于病毒学，能够识别在感染过程中被翻译的病毒mRNA，并为病毒基因表达、调控和宿主-病毒相互作用提供新的见解。在这项工作中，我们进行了第一次核糖体分析的RNA病毒，作为一个模型系统的鼠冠状病毒株MHV-A59，β冠状病毒在同一属的医学重要的SARS-CoV和MERS-CoV。在小鼠组织培养细胞中MHV复制过程中，对感染细胞时间点进行平行核糖体分析和RNA测序，并用于确定病毒基因表达动力学以及病毒和宿主mRNA的相对翻译效率。该方法的灵敏度和精确度使我们能够揭示冠状病毒翻译的几个意想不到的特征，深入了解核糖体移码、核糖体暂停以及利用短的、潜在的调控性上游开放阅读框架。我们还确定了与核糖体分析技术普遍相关的技术相关的一些挑战，并开发了生物信息学策略来解决这些问题。
Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus. Ribosome profiling is emerging as a powerful technique to monitor translation in living cells at sub-codon resolution. It has particular applicability to virology, with the capacity to identify viral mRNAs that are being translated during infection and to provide new insights into virus gene expression, regulation and host-virus interactions. In this work, we carried out the first ribosome profiling analysis of an RNA virus, using as a model system the murine coronavirus strain MHV-A59, a betacoronavirus in the same genus as the medically important SARS-CoV and MERS-CoV. Parallel ribosome profiling and RNA sequencing of infected-cell time points was performed during the course of MHV replication in mouse tissue culture cells and used to determine virus gene expression kinetics and the relative translational efficiencies of virus and host mRNAs. The sensitivity and precision of the approach permitted us to uncover several unanticipated features of coronavirus translation, giving insights into ribosomal frameshifting, ribosome pausing, and the utilisation of short, potentially regulatory, upstream open reading frames. We also identified some challenges associated with the technique that are of general relevance to the ribosome profiling technique and developed bioinformatic strategies to address these.
影响因子：
3.7
作者：
Shieh CK;Soe LH;Makino S;Chang MF;Stohlman SA;Lai MM
通讯作者：
Lai MM