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A newly discovered protein-coding ORF in enteroviruses: from mechanism to application

肠道病毒中新发现的蛋白质编码ORF：从机制到应用

基本信息

批准号：
MR/T000376/1
负责人：
Andrew Firth
金额：
$ 97.92万
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=MR%2FT000376%2F1
关键词：
newly discovered protein coding ORF

项目摘要

Enteroviruses are common and important human pathogens. While many infections are mild, some strains can cause severe and even fatal disease with symptoms ranging through fever, hand foot and mouth disease, myocarditis, viral meningitis, encephalitis, acute hemorrhagic conjunctivitis, and acute flaccid paralysis. Although poliovirus (the enterovirus that causes poliomyelitis) has been eradicated from much of the globe it remains a threat, and other emerging enteroviruses can also cause severe polio-like symptoms. Currently, vaccines are only routinely available for poliovirus (approved globally) and EV-A71 (approved in China).Enteroviruses are also some of the best studied viruses and serve as a useful model system for understanding the biology of all "positive-sense" RNA viruses which can be found in every virology textbook. The enterovirus genome is ~7400 nucleotides in length and, ever since poliovirus was first sequenced in 1981, was believed to encode only 11 proteins, including enzymatic proteins that replicate the viral genome and structural proteins that package progeny genomes into protein capsids for transmission. These 11 proteins are translated from the genome (which functions directly as a messenger RNA) concatenated end-to-end as a single giant "polyprotein", which is then cut up by one of the viral proteins (a proteolytic enzyme) into the 11 virus proteins.However, we recently showed that this picture was incomplete: in fact one more protein is cryptically encoded within the genome of many enteroviruses. We call the additional protein UP (Upstream Protein) since it is encoded upstream of the polyprotein. Using an organoid system ("mini gut" 3d cell cultures grown in the lab), we went on to show that UP plays an important role specifically in gut epithelial cells where it is involved in releasing newly formed virus particles from membranous compartments. This is exciting because the gut is the site where enteroviruses first replicate on infecting a new host before, potentially, invading other cell types and organs. In contrast, the closely related rhinoviruses replicate in the upper respiratory tract and - perhaps not surprisingly - they ubiquitously lack the UP protein. We published this research in Nov 2018 in Nature Microbiology.However this previous work just scratched the surface of understanding the biological function of the novel UP protein. Many very important questions remained unanswered, such as why do some enteroviruses appear to lack the UP protein, how does UP differ between different enteroviruses, what controls UP expression, with which membranes does UP associate, and how exactly does UP function to facilitate release of virus from membranous compartments. Through five work packages, we will now perform a detailed functional analysis of the UP protein:1) Bioinformatic analysis - correlation of UP with virus provenance and clinical data2) Assessing and deciphering the regulation of UP expression3) Biochemical and functional characterization of the UP protein4) Biological functionality of UP in human intestinal organoids5) Physiological role of UP and potential as a vaccine candidateThis research is exciting for several reasons. The discovery of a new protein opens up a whole new avenue for enterovirus research that will give new insights into virus biology and pathogenesis. We are ideally poised to exploit this new research direction. Understanding the function of UP may also help explain why some enteroviruses can infect the gut whereas others infect the upper respiratory tract. These findings may aid rapid prediction of the tropism of newly emerging enteroviruses. More importantly, UP knockout viruses could be ideal candidates for attenuated virus vaccines since they grow well in cell culture (to give good vaccine yield) but are attenuated in the gut; further, this strategy could be quickly applied to newly emerging enteroviruses.

肠道病毒是人类常见的重要病原体。虽然许多感染是温和的，但一些菌株可引起严重甚至致命的疾病，症状包括发烧、手足口病、心肌炎、病毒性脑膜炎、脑炎、急性出血性结膜炎和急性弛缓性麻痹。虽然脊髓灰质炎病毒（导致脊髓灰质炎的肠道病毒）已经从地球仪的大部分地区根除，但它仍然是一种威胁，其他新出现的肠道病毒也可能导致严重的脊髓灰质炎样症状。目前，疫苗只能常规用于脊髓灰质炎病毒（全球批准）和EV-A71（中国批准）。肠道病毒也是研究最好的病毒之一，并作为了解所有“正义”RNA病毒生物学的有用模型系统，这些病毒可以在每一本病毒学教科书中找到。肠道病毒基因组长度约为7400个核苷酸，并且自从脊髓灰质炎病毒在1981年首次测序以来，据信仅编码11种蛋白质，包括复制病毒基因组的酶蛋白和将子代基因组包装到蛋白衣壳中用于传播的结构蛋白。这11种蛋白质从基因组（直接作为信使RNA）翻译出来，首尾相连，形成一个巨大的“多蛋白”，然后被其中一种病毒蛋白（蛋白水解酶）切割成11种病毒蛋白。然而，我们最近发现这幅图是不完整的：事实上，在许多肠道病毒的基因组中还有一种蛋白质被神秘地编码。我们称这种额外的蛋白质为UP（上游蛋白质），因为它是在多聚蛋白的上游编码的。使用类器官系统（在实验室中生长的“迷你肠道”3d细胞培养物），我们继续表明UP在肠道上皮细胞中起着重要作用，它参与从膜隔室释放新形成的病毒颗粒。这是令人兴奋的，因为肠道是肠道病毒在感染新宿主后首先复制的场所，然后可能入侵其他细胞类型和器官。相比之下，密切相关的鼻病毒在上呼吸道复制，并且-也许并不奇怪-它们普遍缺乏UP蛋白。我们于2018年11月在Nature Microbiology上发表了这项研究。然而，之前的工作只是触及了了解新型UP蛋白生物学功能的表面。许多非常重要的问题仍然没有答案，例如为什么有些肠道病毒似乎缺乏UP蛋白，不同肠道病毒之间UP有何不同，是什么控制UP表达，UP与哪些膜相关，以及UP如何确切地发挥作用以促进病毒从膜室释放。通过五个工作包，我们现在将对UP蛋白进行详细的功能分析：1）生物信息学分析-UP与病毒来源和临床数据的相关性2）评估和破译UP表达的调节3）UP蛋白的生化和功能表征4）UP在人类肠道器官中的生物功能5）UP的生理作用和作为候选疫苗的潜力这项研究令人兴奋，原因有几个。一种新蛋白的发现为肠道病毒研究开辟了一条全新的途径，将为病毒生物学和发病机制提供新的见解。我们已经做好了充分准备来开拓这一新的研究方向。了解UP的功能也可能有助于解释为什么一些肠道病毒可以感染肠道，而另一些则感染上呼吸道。这些发现可能有助于快速预测新出现的肠道病毒的嗜性。更重要的是，UP敲除病毒可能是减毒病毒疫苗的理想候选者，因为它们在细胞培养物中生长良好（以获得良好的疫苗产量），但在肠道中减毒;此外，这种策略可以快速应用于新出现的肠道病毒。