The mechanistic basis for the ZAP antiviral system targeting viral and cellular RNAs

ZAP是一种在大多数脊椎动物中发现的抗病毒蛋白，它通过与病毒RNA结合来限制多种病毒，以抑制其翻译成病毒蛋白，并针对其进行降解。对ZAP如何抑制病毒的充分了解可用于开发新的治疗应用。在病毒RNA中引入ZAP结合位点可以增加ZAP介导的抑制作用，从而创造新的减毒活病毒疫苗。另一个应用是将ZAP结合位点引入病毒中，这些病毒选择性地在肿瘤中复制，以杀死癌细胞并触发免疫反应。几种类型的癌症，包括肝癌、结肠癌和膀胱癌，ZAP的表达比邻近的非癌症组织低，这与疾病进展和生存率较差有关。ZAP限制性溶瘤病毒可能是治疗这些类型癌症的好方法，因为它们会比周围健康组织更有效地在癌细胞中复制。然而，为了利用这些可能性，我们需要更好地了解ZAP是如何抑制病毒复制的。首先，目前尚不清楚ZAP如何选择与哪些病毒RNA结合。因此，我们将表征ZAP是如何与目标RNA特异性结合的，并利用这一理解将最佳的ZAP结合位点引入病毒RNA，以增加它们对ZAP介导的抑制的敏感性。其次，虽然ZAP与许多细胞蛋白相互作用，但只有几种已知的蛋白质能调节其抗病毒活性。为了识别促进或抑制其活性的蛋白质，我们将使用基于其相互作用伙伴的全面列表的基因筛查。第三，目前还不清楚ZAP在细胞中的哪里起作用来抑制病毒基因的表达。我们将使用尖端显微镜技术来可视化ZAP是如何抑制病毒的。虽然ZAP抑制了广泛的病毒，但为了这项资助的目的，我们将使用三种医学上相关的病毒来表征ZAP抗病毒途径。我们将分析ZAP如何抑制艾滋病毒-1，它的全球传播导致了艾滋病大流行，并提供了一个简单的系统来分析ZAP的功能；SARS-CoV-2，导致新冠肺炎，需要哪些新的疗法；以及甲型流感病毒，它导致季节性呼吸道疾病和周期性的严重疾病大流行，需要制定新的疫苗接种策略。总体而言，了解ZAP是如何抑制病毒复制的将使我们能够潜在地开发这些新的疗法。

ZAP is an antiviral protein found in most vertebrates that restricts a diverse range of viruses by binding viral RNA to inhibit its translation into viral proteins and target it for degradation. A full understanding of how ZAP inhibits viruses can be used to develop new therapeutic applications. Introducing ZAP binding sites in viral RNA could increase ZAP-mediated inhibition to create new live attenuated virus vaccines. Another application is to introduce ZAP binding sites into viruses which selectively replicate in tumours to kill the cancer cells and trigger immune responses. Several types of cancer, including liver, colon and bladder, have lower ZAP expression than adjacent non-cancer tissue and this is associated with poor disease progression and survival. ZAP-restricted oncolytic viruses may be a good treatment for these types of cancer since they will replicate in the cancer cells more efficiently than the surrounding healthy tissue.However, to exploit these possibilities, we need a better understanding of how ZAP inhibits viral replication. First, it is unclear how ZAP selects which viral RNAs to bind to. Therefore, we will characterise how ZAP specifically binds target RNA and use this understanding to introduce optimal ZAP binding sites into viral RNA to increase their sensitivity to ZAP-mediated inhibition. Second, while ZAP interacts with many cellular proteins, only a few proteins are known to regulate its antiviral activity. To identify proteins that promote or inhibit its activity, we will use genetic screens based on a comprehensive list of its interacting partners. Third, it is unclear where in the cell ZAP acts to inhibit virus gene expression. We will use cutting edge microscopy techniques to visualise how ZAP inhibits a virus.While ZAP inhibits a broad range of viruses, for the purposes of this grant we will use three medically relevant viruses to characterise the ZAP antiviral pathway. We will analyse how ZAP inhibits HIV-1, whose worldwide spread has caused the AIDS pandemic and represents a straightforward system to analyse ZAP function; SARS-CoV-2, which causes COVID-19 and for which novel therapeutics are needed; and influenza A virus, which causes seasonal respiratory disease and periodic pandemics with severe disease and requires new vaccination strategies to be developed. Overall, understanding how ZAP inhibits viral replication will allow us to potentially develop these new therapies.