RNA Recombination in Coronaviruses

RNA病毒是非常多样化和快速进化的。它们的RNA依赖性RNA聚合酶易于突变，缺乏校对能力（冠状病毒是唯一的例外），并且经常在同源和非同源模板之间进行重组。这赋予了快速适应新环境、逃避免疫反应和回避抗病毒治疗的能力。在许多RNA病毒中，RNA重组与复制保真度共同调节，并且是纠正有害突变所必需的，因此是病毒适应性的关键决定因素。通过组合产生亚基因组信使RNA、结构变体（SV）和缺陷RNA（D-RNA），RNA重组是CoV复制和进化的基本特性。随着SARS-CoV-2的持续传播，重组已被强调为驱动新变体出现的主要因素。SARS-CoV-2变异体已经产生了突变，被认为可以改善受体结合，破坏先天免疫反应或逃避适应性免疫。我们最近证明SARS-CoV-2比其他CoV如MERS和MHV的重组发生性高10倍以上（Gribble等人，2021，PLoS Path）。有趣的是，产生SV和D-RNA的RNA重组事件主要发现于富含U的束附近。使用我们实验室开发的“Tiled-ClickSeq”（Jaworski et al，2021，eLife），我们在COVID 19患者样本中发现了相同的趋势。我们还证明了SARS-CoV-2的刺突蛋白中侧翼为富U基序的微缺失在细胞培养物中传代期间自发出现并改变病毒发病机制（约翰逊等人，2021，Nature）。值得注意的是，SARS-CoV-2的新变体（例如α变体）中的微插入缺失也侧接富含U的基序。总而言之，这些证据表明富含U的片段定义了RNA重组热点，这些热点导致了新型CoV变体的出现。然而，尽管重组率明显很高，但SARS-CoV-2具有强大的传播瓶颈，限制了少数变异株的传播。新的变体，如α和Omicron包含多个重组事件，可能在慢性感染（如免疫功能低下）患者的宿主内适应过程中出现。因此，在正确的生理环境中表征RNA重组对于理解哪些RNA重组物种能够出现以及这些物种是如何被选择的至关重要。在这项提案中，我们将描述RNA重组的分子机制，并描述生理方面和感染部位如何决定是否选择新的变体并因此出现。表征引起新出现的菌株和变体的因素对于我们理解历史和未来的疫情事件至关重要。此外，这些研究将为驱动RNA病毒进化的基本原理提供信息。目前的大流行呈现出一种独特的情况，可以描述病毒出现的基本原理，并可能影响我们对一系列病毒病原体的理解。

RNA viruses are exceptionally diverse and rapidly evolving. Their RNA-dependent RNA polymerases are prone to mutation, lack proof-reading capabilities (with the unique exception of coronaviruses) and frequently undergo recombination between both homologous and non-homologous templates. This confers the ability to rapidly adapt to new environments, evade immune responses and side-step anti-viral therapies. In many RNA viruses, RNA recombination is co-regulated with replication fidelity and is required to correct deleterious mutations and thus is a critical determinant of viral fitness. Through the combined generation subgenomic messenger RNAs, structural variants (SVs), and Defective RNAs (D-RNAs), RNA recombination is an essential property of CoV replication and evolution. With the continued spread of SARS-CoV-2, recombination has been highlighted as a major factor driving the emergence of novel variants. SARS-CoV-2 variants have developed mutations thought to improve receptor binding, disrupt innate immune responses, or evade adaptive immunity. We recently demonstrated that SARS-CoV-2 is >10-fold more recombinogenic than other CoVs such as MERS and MHV (Gribble et al, 2021, PLoS Path). Interestingly, RNA recombination events that give rise to SVs and D- RNAs were predominantly found adjacent to U-rich tracts. Using ‘Tiled-ClickSeq’, developed in our lab (Jaworski et al, 2021, eLife), we found the same trends in COVID19 patient samples. We also demonstrated that micro- deletions flanked by U-rich motifs in the Spike protein of SARS-CoV-2 spontaneously arise during passaging in cell culture and alter viral pathogenesis (Johnson et al, 2021, Nature). Notably, microindels in novel variants of SARS-CoV-2 (e.g. Alpha variant) are also flanked by U-rich motifs. Altogether, this evidence suggests U-rich tracts define RNA recombination hotspots which have given rise to the emergence of novel CoVs variants. However, while recombination rates are demonstrably high, SARS-CoV-2 has a strong transmission bottleneck restricting the dissemination of minority variants. Novel variants such as Alpha and Omicron contain multiple recombination events and may have arisen during intrahost adaption in a chronically infected (e.g. immunocompromised) patient. Therefore, characterizing RNA recombination in the correct physiological setting is crucial to understand which RNA recombinant species are able to emerge and how these are selected. In this proposal, we will characterize the molecular mechanisms of RNA recombination and characterize how the physiological aspects and site of infection determines whether a novel variant is selected for and thus emergence. Characterizing the factors that give rise to new emergent strains and variants will be critical in our understanding of both historical and future outbreaks events. Furthermore, these studies will inform on the basic and fundamental principles that drive RNA virus evolution. The current pandemic presents a unique situation in which to characterize the basic principles of virus emergence and may impact our understanding of a range of viral pathogens.