The prenylated form of the human 2'-5'-oligoadenylate synthetase 1 (OAS1) protein has been shown to potently inhibit the replication of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. However, the OAS1 orthologue in the horseshoe bats (superfamily Rhinolophoidea), the reservoir host of SARS-related coronaviruses (SARSr-CoVs), has lost the prenylation signal required for this antiviral activity. Herein, we used an ancestral state reconstruction approach to predict and reconstitute in vitro, the most likely OAS1 protein sequence expressed by the Rhinolophoidea common ancestor prior to its prenylation loss (RhinoCA OAS1). We exogenously expressed the ancient bat protein in vitro to show that, unlike its non-prenylated horseshoe bat descendants, RhinoCA OAS1 successfully blocks SARS-CoV-2 replication. Using protein structure predictions in combination with evolutionary hypothesis testing methods, we highlight sites under unique diversifying selection specific to OAS1's evolution in the Rhinolophoidea. These sites are located near the RNA-binding region and the C-terminal end of the protein where the prenylation signal would have been. Our results confirm that OAS1 prenylation loss at the base of the Rhinolophoidea clade ablated the ability of OAS1 to restrict SARSr-CoV replication and that subsequent evolution of the gene in these bats likely favoured an alternative function. These findings can advance our understanding of the tightly linked association between SARSr-CoVs and horseshoe bats.

中文翻译：

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现代马蹄蝠祖先的 2'-5'-寡腺苷酸合成酶 1 (OAS1) 的复活可阻断 SARS-CoV-2 的复制。

人类 2'-5'-寡腺苷酸合成酶 1 (OAS1) 蛋白的异戊二烯化形式已被证明可有效抑制严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 的复制，该病毒是 2019 年冠状病毒病的罪魁祸首（2019冠状病毒病大流行。然而，马蹄蝠（Rhinolophidea 超科）中的 OAS1 直系同源物（SARS 相关冠状病毒（SARSr-CoV）的储存宿主）已经失去了这种抗病毒活性所需的异戊二烯化信号。在此，我们使用祖先状态重建方法在体外预测和重建Rhinolophoidea共同祖先在其异戊二烯化丢失之前表达的最有可能的OAS1蛋白序列（RhinoCA OAS1）。我们在体外外源表达了古老的蝙蝠蛋白，结果表明，与其非异戊二烯化马蹄蝠后代不同，RhinoCA OAS1 成功阻断了 SARS-CoV-2 的复制。利用蛋白质结构预测与进化假设检验方法相结合，我们突出显示了犀牛总科中 OAS1 进化特有的独特多样化选择下的位点。这些位点位于 RNA 结合区和蛋白质 C 末端附近，异戊二烯化信号就在此处。我们的结果证实，Rhinolophidea 分支基部的 OAS1 异戊二烯化丧失消除了 OAS1 限制 SARSr-CoV 复制的能力，并且这些蝙蝠中该基因的后续进化可能有利于替代功能。这些发现可以增进我们对 SARSr-CoV 与马蹄蝠之间紧密联系的理解。