An innovative and straightforward approach to construct and manipulate viral infectious clones

项目摘要/摘要。 像SARS-CoV-2(冠状病毒科)和登革病毒(DENV；黄病毒科)这样的RNA病毒导致 全球范围内的重大疾病。引起关注的SARS-CoV-2变种的兴起突显了理解 疾病严重性、传播潜力和逃避免疫能力背后的病毒基因决定因素， 这需要分子工具来制造病毒突变。然而，目前的方法是构建和 操纵病毒感染性克隆依赖于活的宿主(细菌或酵母)，这可能会导致不必要的突变 病毒基因组中的缺失，特别是像冠状病毒和黄病毒这样的较大病毒。由于这些原因 问题，很少有实验室能成功地完成这项工作。由于宿主造成了这些不想要的突变，一种策略 这消除了对活着的东道主的需求，这是最好的前进道路。 我们的长期目标是创造简单易用的工具，促进基础病毒学研究，旨在 减少疾病负担，符合美国国立卫生研究院的使命。该项目旨在开发和优化一个范例-- 用于构建和操作病毒感染性克隆的变化但直接的无宿主方法， 促进寻求确定疾病、传播和其他表型的病毒遗传决定因素的研究。 这种方法也可用于构建重组疫苗病毒和诊断工具，如Report 病毒。我们将使用一种令人兴奋的技术--复制循环反应(RCR)--它可以重建大肠杆菌 试管中的DNA复制机器。RCR可以高效地扩增高达1Mb的单个DNA分子 以简单易用的格式实现保真度。在这里，我们建议开发基于RCR的系统来构建新的 感染性克隆(目标1)和操纵现有克隆(目标2)。Aim 1将使用化学合成的DNA来 为快速传播的SARS-CoV-2 Omicron变异株和DENV Puo-218株建立感染性克隆 经批准的登革热疫苗成分登革热。这一目标的影响将是一个创新的系统 产生新的感染性克隆。AIM 2将使用基于RCR的系统来进行突变和删除 SARS-CoV-2 Omicron变种在现有的感染性克隆中；然后我们将研究这些变种的影响 在原代人类细胞中进行病毒复制。这个目标的影响将是一种直接的方法来产生 病毒突变体，促进机制研究，以了解致病的病毒遗传决定因素， 传播和免疫逃避。这些研究还将确定在Spike外影响病毒的突变 复制。我们预计这些研究将对分子病毒学产生持续的影响，使更多 构建和操纵感染性克隆的实验室。我们预计这里产生的技术将广泛应用于 也适用于其他正义RNA病毒和负义RNA、DNA病毒。最后，我们 将公开和不受限制地分享这里生成的所有方法和工具。

Project Summary / Abstract. RNA viruses like SARS-CoV-2 (family Coronaviridae) and dengue virus (DENV; family Flaviviridae) cause significant disease globally. The rise of SARS-CoV-2 variants of concern has highlighted the need to understand the viral genetic determinants underlying disease severity, transmission potential, and ability to evade immunity, which requires molecular tools to make viral mutants. However, current approaches to constructing and manipulating viral infectious clones rely on a living host (bacteria or yeast), which can lead to unwanted mutations and deletions in the viral genome, especially for larger viruses like coronaviruses and flaviviruses. Due to these issues, few labs can successfully perform this work. Since the host causes these unwanted mutations, a strategy that removes the need for a living host represents the best way forward. Our long-term goal is to create simple-to-use tools that facilitate fundamental virology research aimed at reducing disease burden, in line with NIH’s mission. This project aims to develop and optimize a paradigm- shifting but straightforward host-free approach for constructing and manipulating viral infectious clones, facilitating studies seeking to identify viral genetic determinants of disease, transmission, and other phenotypes. This approach can also be used to construct recombinant vaccine viruses and diagnostic tools like reporter viruses. We will use an exciting technique—replication cycle reaction (RCR)—which reconstitutes the E. coli DNA replication machinery in a tube. RCR can efficiently amplify a single DNA molecule of up to 1 Mb with high fidelity in a simple-to-use format. Here, we propose developing this RCR-based system to construct new infectious clones (Aim 1) and manipulate existing ones (Aim 2). Aim 1 will use chemically synthesized DNA to create infectious clones for the rapidly spreading SARS-CoV-2 Omicron variant and DENV strain Puo-218, a component of the approved DENV vaccine, Dengvaxia. The impact of this aim will be an innovative system to generate new infectious clones. Aim 2 will use the RCR-based system to make mutations and deletions in the SARS-CoV-2 Omicron variant in an existing infectious clone; we will then study the impact these mutations have on viral replication in primary human cells. The impact of this aim will be a straightforward method to generate viral mutants, facilitating mechanistic studies to understand the viral genetic determinants of pathogenesis, transmission, and immune evasion. These studies will also identify mutations outside Spike that impact viral replication. We expect these studies will have a sustained impact on molecular virology by empowering more labs to construct and manipulate infectious clones. We anticipate the techniques generated here to be broadly applicable to other positive-sense RNA viruses and negative-sense RNA and DNA viruses as well. Finally, we will share all the methods and tools generated here openly and without restriction.