Defining picornaviral replication complexes by molecular virology and state-of-the art imaging - Novel strategies for disease control

背景：小核糖核酸病毒是许多严重疾病的罪魁祸首，包括脊髓灰质炎、口蹄疫（FMDV）和一些新出现的疾病，如人类手足口病。迫切需要开发新的治疗策略来解决小核糖核酸病毒感染的持续问题。基因组复制是一个关键的治疗目标，通常被隔离在“复制复合体”中，被认为可以提高效率并保护生命周期中这一脆弱的部分。然而，对于小核糖核酸病毒，这些定义不明确。目标：该项目是一个更大的研究组合的一部分，旨在提高我们在基础水平上对重要分子过程的理解，并开发新的小核糖核酸病毒疗法，由BBSRC， MRC和世卫组织/盖茨基金会和合作者（英国和海外）的研究资助。新颖性：该项目将重点关注FMDV，该病毒复制的许多方面尚不清楚。据我们所知，其他地方没有进行类似的工作。及时性：我们最近建立了一个互补系统来研究FMDV基因组之间的串扰[Herod et al ., 2015,2016,2017]。这些研究强调了多重共感染在病毒复制中的重要性。我们将在这里利用这个系统，见下文。实验方法：我们的工作采用复制子-微型基因组，允许以安全的无病毒方式研究复制，这对基因组复制机制的研究非常方便。利用互补性（连同内部可用的最先进的成像和结构技术），我们可以解剖复制复合体的RNA和蛋白质成分的功能。i.e.1。鉴定FMDV基本非结构蛋白和前体在复制中的新作用，重点研究了利用诱变和复制抑制剂的聚合酶（3D）和复制引物（VPg）。以时间解决的方式调查复制地点（复制工厂）的发展和结构，例如通过PALM/STORM和EM。

Background:Picornaviruses are responsible for a number of serious diseases, including polio, foot-and-mouth disease (FMDV) and some emerging diseases, e.g. human hand foot and mouth disease. There is an urgent need to develop new therapeutic strategies to address the continuing issue of picornavirus infection. Genome replication, a key therapeutic target, is usually sequestered within 'replicationcomplexes', thought to improve efficiency and protect this vulnerable part of the lifecycle. However, for picornaviruses, these are poorly defined. Objectives:The project is part of a larger research portfolio designed to improve our understanding of important molecular processes at a fundamental level and to develop new picornavirus therapies, supported by research grants from BBSRC, MRC and WHO/Gates foundation and collaborators (UK and overseas).Novelty:This project will focus on FMDV, many aspects of the replication of this virus are poorly understood. To our knowledge, no similar work is being undertaken elsewhere.Timeliness:We have recently established a reciprocal complementation system to study the cross talk between FMDV genomes [Herod et al, 2015,2016,2017]. These studies have highlighted the importance of multiple co-infection in viral replication. We will exploit this system here, see below.Experimental approach:Our work employs replicons - mini-genomes that allow the study of replication in a safe virus-free way which is highly convenient for the study of the mechanics of genome replication. Using reciprocal complementation (together with state-of the art imaging and structural techniques available in-house) we can dissect the functions of both the RNA and protein components of replication complex. i.e.1. Identifying novel roles of the essential FMDV non-structural proteins and precursors in replication, focusing on the polymerase (3D) and primer of replication (VPg) using mutagenesis and replication inhibitors.2. Investigating the development and structure of sites of replication (replication factories) in a time-resolved manner e.g. by PALM/STORM and EM.