RNA Recombination in Coronaviruses

冠状病毒中的 RNA 重组

• 批准号: 10934125
• 负责人: Andrew Laurence Routh
• 金额: $ 39.8万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10934125
• 关键词: RNA; Recombination; Coronaviruses

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.

RNA 病毒极其多样化且进化迅速。它们的 RNA 依赖性 RNA 聚合酶容易发生突变，缺乏校对能力（冠状病毒除外），并且经常在同源和非同源模板之间进行重组。为了快速适应新环境、逃避免疫反应和回避抗病毒治疗，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，因此是至关重要的。随着 SARS-CoV-2 的持续传播，通过组合生成亚基因组信使 RNA、结构变异 (SV) 和缺陷 RNA (D-RNA)，RNA 重组是 CoV 复制和进化的重要特性。重组已被认为是推动新型变异体出现的主要因素，我们最近证明，SARS-CoV-2 变异体已产生突变，可改善受体结合、破坏先天免疫反应或逃避适应性免疫。 SARS-CoV-2 的重组能力比其他 CoV（例如 MERS 和 MHV）高 10 倍（Gribble 等人，2021，PLoS Path），使用我们实验室开发的“Tiled-ClickSeq”（Jaworski 等人）。 al, 2021, eLife），我们发现了相同的趋势我们还证明，SARS-CoV-2 的刺突蛋白中富含 U 的基序两侧的微缺失在细胞培养过程中会自发出现，并改变病毒的发病机制（Johnson 等人，2021 年，《自然》）。 SARS-CoV-2 的新变体（例如 Alpha 变体）中的微插入缺失也具有富含 U 的基序，总而言之，这一证据表明富含 U 的区域定义了 RNA 重组。然而，虽然重组率明显很高，但 SARS-CoV-2 具有强大的传播瓶颈，限制了少数变异体（例如 Alpha 和 Omicron）的传播。并且可能是在慢性感染（例如免疫功能低下）患者的宿主适应过程中出现的，因此，在正确的生理环境中表征 RNA 重组对于了解哪些 RNA 至关重要。重组物种能够出现，以及如何选择它们。在本提案中，我们将描述 RNA 重组的分子机制，并描述生理方面和感染位点如何决定是否选择新变异并从而确定出现的因素。此外，这些研究将揭示推动 RNA 病毒进化的基本原理。描述病毒出现的基本原理和可能会影响我们对一系列病毒病原体的理解。