RNA Recombination in Coronaviruses

冠状病毒中的 RNA 重组

• 批准号: 10585621
• 负责人: Andrew Laurence Routh
• 金额: $ 17.13万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10585621
• 关键词: 2019-nCoV; Air; Antiviral Therapy; Automobile Driving; COVID-19; COVID-19 patient; Cell Culture Techniques; Cell Differentiation process; Cells; Chronic; Coronavirus; Databases; Disease; Disease Outcome; Environment; Epidemic; Etiology; Event; Evolution; Generations; Genetic Recombination; Human; IFNAR1 gene; Immune; Immune Evasion; Immune response; Immunocompromised Host; Infection; Innate Immune Response; Learning; Life Cycle Stages; Liquid substance; Livestock; Lung; Measures; Messenger RNA; Microscopy; Middle East Respiratory Syndrome; Minority; Modeling; Molecular; Mus; Mutate; Mutation; Nature; Open Reading Frames; Patients; Phylogenetic Analysis; Physiological; Play; Polymerase; Property; Proteins; RNA; RNA Viruses; RNA-Directed RNA Polymerase; Replication Initiation; Reporter Genes; Reproducibility; Role; SARS-CoV-2 infection; SARS-CoV-2 positive; SARS-CoV-2 spike protein; SARS-CoV-2 variant; Sampling; Serial Passage; Site; Source; System; Testing; Time; Tissues; Variant; Viral; Viral Genome; Viral Pathogenesis; Virulence; Virus; adaptive immunity; cell type; clinical research site; current pandemic; endonuclease; fitness; future outbreak; improved; insertion/deletion mutation; microdeletion; mouse model; novel; novel coronavirus; pandemic disease; pathogenic virus; prevent; protein expression; receptor binding; recombinant RNA; reverse genetics; transmission process; trend; vaccine failure; variants of concern; viral RNA; viral fitness

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 病毒异常多样且进化迅速。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。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 复制和进化的基本特性。随着 SARS-CoV-2 的持续传播，重组已被视为推动新变种出现的主要因素。 SARS-CoV-2 变体已产生突变，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 的重组能力是其他 CoV（例如 MERS 和 MHV）的 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 的区域附近。使用我们实验室开发的“Tiled-ClickSeq”（Jaworski 等人，2021，eLife），我们在 COVID19 患者样本中发现了相同的趋势。我们还证明，SARS-CoV-2 刺突蛋白中富含 U 基序两侧的微缺失在细胞培养物传代过程中自发出现，并改变病毒发病机制（Johnson 等人，2021 年，《自然》）。值得注意的是，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，这些证据表明，富含 U 的区域定义了 RNA 重组热点，从而导致了新型冠状病毒变体的出现。然而，尽管重组率明显很高，但 SARS-CoV-2 具有强大的传播瓶颈，限制了少数变种的传播。 Alpha 和 Omicron 等新变体包含多个重组事件，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 重组物种能够出现以及如何选择它们至关重要。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出现。描述产生新出现的菌株和变种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，这些研究将揭示驱动 RNA 病毒进化的基本原则。当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。