Large-scale compatibility assessments between ACE2 proteins and diverse sarbecovirus spikes

项目总结 从动物蓄水池向人类的病毒溢出会摧毁公共卫生系统，使 世界经济，当前的SARS-CoV-2大流行证明了这一点。野生动物栖息地的持续破坏， 人口的扩张和常规的全球旅行继续增加了另一场病毒大流行 在接下来的几十年内再次发生。贝塔冠状病毒是一个极其多样化的病毒家族。 在亚洲、欧洲和非洲观察到的，已被证明能够在人类中传播人畜共患病 在过去20年中在全球范围内引发了多次疫情。我们仍然缺乏基本的 对决定分子亲和性的分子和遗传因素的理解 哪些贝塔冠状病毒最有可能在未来传染给人类。 类SARSβ冠状病毒利用ACE2作为细胞进入受体的能力是一个主要因素 确定冠状病毒在物种间或生物体组织内的嗜性程度。而当 SARS-CoV-2已经被深入研究，对这种病毒的大多数其他成员几乎一无所知 一家人。传统的研究一次只能测试少数几种情况，不能完全抽样大范围的情况 相关的实验条件，特别是对数百种未定性的贝塔冠状病毒。大号- 需要规模、最小偏差、基于单元格的进入分析来模拟这些因素如何汇聚到一起来决定 感染的概率。 我们将配对细胞工程和合成生物学的新方法，使DNA测序成为可能 多重遗传分析进行一系列大规模感染分析，揭示决定因素 对贝塔冠状病毒进入的易感性。这些大规模的实验将揭示ACE2如何测序和 细胞表面密度影响病毒进入的效率。通过测试受体结合结构域的文库 从蝙蝠冠状病毒的生态监测中鉴定出的序列，我们将识别哪些病毒拥有 与人类ACE2有足够的亲和力，以加强跨物种传播，并创建一个描述所有 这些病毒进化它们的序列以参与ACE2的不同方式。通过对 SPEKE和ACE2蛋白序列、表达水平和细胞进入效率的关系，我们将 识别SARS-CoV-2和其他类似SARS的蝙蝠冠状病毒的潜在动物宿主，并预测哪些 病毒与人类血管紧张素转换酶2有足够的结合，可能引发下一次大流行。

PROJECT SUMMARY Viral spillover from animal reservoirs into humans can decimate public health systems and cripple the world economy, as evident with the current SARS-CoV-2 pandemic. Continued wildlife habitat destruction, human expansion, and routine global travel keep increasing the likelihood that another viral pandemic will occur again within the next few decades. Beta coronaviruses are an incredibly diverse family of viruses observed across Asia, Europe, and Africa, that have proven capable of zoonotic spillover into humans as they have caused multiple worldwide outbreaks over the last two decades. We still lack the fundamental understanding of the molecular and genetic factors that dictate the molecular compatibilities that determine which beta coronaviruses are most likely to jump into humans in the future. The ability of SARS-like beta coronaviruses to utilize ACE2 as a receptor for cell entry is a major factor determining the extent of coronavirus tropism across species or within the tissues of an organism. While SARS-CoV-2 has been heavily studied, almost nothing is known about most other members of this virus family. Traditional studies can only test a handful of conditions at a time, incompletely sampling the vast range of relevant experimental conditions, particularly for the hundreds of uncharacterized beta coronaviruses. Large- scale, minimally-biased, cell-based entry assays are needed to model how these factors converge to dictate the probability of infection. We will pair new methods in cell engineering and synthetic biology with DNA-sequencing enabled multiplex genetic assays to perform a series of large-scale infection assays revealing the factors determining susceptibility to beta coronavirus entry. These large-scale experiments will reveal how ACE2 sequence and cell surface density impact the efficiency of virus entry. By testing a library of receptor binding domain sequences identified from ecological surveillance of bat coronaviruses, we will identify which viruses possess sufficient affinity to human ACE2 to potentiate cross-species transmission, and create a catalog describing all of the different ways these viruses have evolved their sequences to engage ACE2. By modeling the relationship between spike and ACE2 protein sequence, expression level, and efficiency of cell entry, we will identify potential animal reservoirs for SARS-CoV-2 and other SARS-like bat coronaviruses, and predict which viruses have sufficient binding with human ACE2 to potentially spark the next pandemic.