Biochemical and structural characterization of the SARS-CoV-2 non-structural protein 16 (Nsp16), a cap ribose 2’O-methyltransferase

SARS-Cov-2 Nsp 16属于病毒2 '-O-甲基转移酶（2' OMT）的保守家族，其特征仍然很差。SARS-CoV和MERS Nsp 16 2 'OMT显示对于干扰素抗性和病毒发病机理是必需的{Menachery，2014 #14}。然而，冠状病毒2 'OMT的一般机制以及它们在加帽RNA之外的完整RNA靶谱在很大程度上仍然难以捉摸。在SARS-Cov Nsp 16的情况下，更长的加帽RNA底物被更有效地甲基化，这表明特定的序列要求或/和远离m7 G帽的残基的甲基化。 因此，我们的共同项目的总体目标是详细表征SARS-CoV-2 Nsp 16 2 'OMT在调节基因表达中的分子机制、结构和功能，并潜在地开发Nsp 16抑制剂。Nsp 16及其辅助蛋白Nsp 10与m7 GpppA-RNA复合的结构最近才被报道{Krafcikova，2020 #1; Lin，2020 #3; Rosas-Lemus，2020 #4; Viswanathan，2020 #17}。然而，SARS-CoV-2对较长RNA底物的特异性识别以及Nsp 16和Nsp 10的关键残基仍不清楚。使用重组Nsp 16和Nsp 10蛋白，我们的初步实验表明，病毒的RNA加工机制是重组的RNA序列，这是修改。体外SELEX实验旨在鉴定未加帽的RNA底物。我们将调查Nsp 16/Nsp 10在复杂的不同的加帽和uncapped RNA片段，使用生化实验和NMR，并将试图提供复杂的结构细节，从X-射线，冷冻EM和NMR的复杂。Landthaler和Popowicz/Sattler实验室正在协同合作，利用他们在蛋白质表达、RNA修饰酶和RNA结合蛋白的结构和生化表征以及RNA相互作用蛋白的高通量分析方面的专业知识。意义：Nsp 16/Nsp 10复合物对于病毒mRNA转录物的加帽以有效翻译和逃避免疫监视至关重要。这项工作将首次全面分析SARS-CoV-2 Nsp 16的化学、结构和RNA底物特异性，以及病毒RNA的2 'O-甲基化状态。作为该项目的一部分开发的测定和策略可以容易地适应在细胞质中复制的其他病毒的2 'OMT，包括埃博拉病毒、麻疹病毒、辛德毕斯病毒、寨卡病毒、登革热病毒和牛痘病毒，以提供新的抗病毒疗法的途径。

SARS-Cov-2 Nsp16 belongs to a conserved family of viral 2’-O-methyltransferases (2’OMTs) that are still poorly characterized. The SARS-CoV and MERS Nsp16 2’OMTs were shown to be necessary for interferon resistance and viral pathogenesis {Menachery, 2014 #14}. However, the general mechanisms of coronavirus 2’OMTs as well as their full RNA target spectrum beyond capped RNA remain largely elusive. In case of SARS-Cov Nsp16, longer capped RNA substrates were methylated more efficiently suggesting specific sequence requirements or/and methylation of residues distant from the m7G-cap. It is therefore the general aim of our joined project to characterize the molecular mechanism, structure and function of the SARS-CoV-2 Nsp16 2’OMT in modulating gene expression in detail and to potentially develop Nsp16 inhibitors. Structures of Nsp16 and its accessory protein Nsp10 in complex with a m7GpppA-RNA have only recently been reported {Krafcikova, 2020 #1; Lin, 2020 #3; Rosas-Lemus, 2020 #4; Viswanathan, 2020 #17}. However, the specific recognition of longer RNA substrates by SARS-CoV-2 and critical residues of Nsp16 and Nsp10 remain unclear. Using recombinant Nsp16 and Nsp10 proteins, our initial experiments indicate that the viral RNA-processing mechanism is re-organized depending on the RNA sequence that is modified. In vitro SELEX experiments are aimed to identify non-capped RNA substrates. We will investigate Nsp16/Nsp10 in a complex with diverse capped and uncapped RNA fragments using biochemical experiments and NMR and will attempt to provide structural details of the complex from X-ray, cryo-EM and NMR of the complex. The Landthaler and Popowicz/Sattler labs are synergizing here to use their expertise in protein expression, structural and biochemical characterization of RNA-modifying enzymes and RNA binding proteins and high-throughput analysis of RNA-interacting proteins. SIGNIFICANCE: The Nsp16/Nsp10 complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance. The proposed work will provide the first comprehensive analysis of the chemistry, structure and RNA substrate specificity of SARS-CoV-2 Nsp16 and the 2’O-methylation status of viral RNA. Assays and strategies developed as part of this project can readily be adapted to 2’OMTs of other viruses that replicate in the cytoplasm including Ebola viruses, Measles virus, Sindbis virus, Zika virus, Dengue virus and Vaccinia virus to provide a path for novel antiviral therapies.