Molecular mechanism of TRIM25 and ZAP mediated antiviral inhibition of arenavirus replication

这一联合项目跨越了Foster、Borkar和Dunham小组的病毒学和结构生物学研究计划，重点关注抑制病毒感染的细胞抗病毒机制以及控制这些机制的病毒与宿主之间的相互作用。福斯特小组专注于最大的出血热家族，这种病毒被称为阿雷纳病毒。这些人畜共患病病毒正在迅速扩大其基因多样性，导致流行地区的年度疫情增加，并在全球范围内导致零星的输入性疫情，包括英国最近的病例(1)。了解宿主抗病毒蛋白如何阻止ArenaVirus复制对于解决目前在病毒生命周期过程中的知识空白是至关重要的，这对开发有效的疫苗和治疗方法至关重要(1)。Foster实验室最近的蛋白质组学研究确定了ArenaVirus核蛋白NP和抗病毒E3连接酶三部分含蛋白25(TRIM25)之间的相互作用，TRIM25是先天免疫反应的关键组成部分，可以抑制多种致病病毒的复制，但ArenaVirus以前未知(2)。NP是病毒中表达最丰富的蛋白，是病毒感染过程中宿主免疫抑制的主要协调者，包裹病毒RNA，形成感染启动所需的病毒核糖核复合体(VRNPs)。利用ArenaVirus感染和CRISPR/Cas9敲除研究，我们已经证明TRIM25显著限制ArenaVirus的复制，TRIM25重新定位到含有NP的病毒复制位点，NP的过度表达足以诱导这种重新定位。TRIM25最近被发现是锌抗病毒蛋白ZAP的关键辅助因子，它针对含有CpG二核苷酸的病毒RNA，导致促进病毒RNA降解和/或抑制病毒RNA翻译[2]。我们已经证明了ZAP也有效地抑制了病毒的复制，因为TRIM25也是一种RNA结合蛋白，TRIM25和ZAP的活性是如何被它们的RNA相互作用共同调节的还有待确定。TRIM25的分子结构是通过其盘绕结构域形成的反平行二聚体，可通过其N-末端环域的二聚化进一步聚合，含有E3-泛素连接酶活性。ZAP和其他配体结合的C末端PRYSPRY结构域位于螺旋线圈(2)的两侧。我们假设TRIM25可能在螺旋vRNP复合体周围多聚体，驱动分解并暴露RNA位点，用于ZAP的招募和随后的RNA降解。因此，本项目旨在结合结构生物学(Borkar小组的专业知识)、生物化学和分子病毒学技术来表征TRIM25如何通过诱变和ArenaVirus RNA通过诱变和复制实验与ArenaVirus NP蛋白以及有或没有ZAP的ArenaVirus RNAs相互作用在复杂的和存在ArenaVirus RNA的情况下，通过电子显微镜和/或X射线结晶学显示TRIM25-NP和TRIM25-NP-RNA-ZAP复合体，结合改变RNA和辅因子结合的特定突变。鉴于这种抗病毒机制在不同的RNA和DNA病毒中的分子易操纵性，这项工作的发现将提供关键证据，可能改变我们对先天性免疫机制的理解，并影响治疗设计。

This joint project bridges across the virology and structural biology research programs in the Foster, Borkar and Dunham groups, focussing on the cellular antiviral mechanisms that that inhibit viral infection and the virus -host interactions that govern these. The Foster group focusses on the largest family of haemorrhagic fever causing viruses known as arenaviruses. These zoonotic viruses are rapidly expanding in their genetic diversity leading to increased annualoutbreaks in endemic regions and to sporadic imported outbreaks in globally, including recent cases in the UK (1). Understanding how host antiviral proteins block arenavirus replication is imperative to addressing current knowledge gaps on the viral lifecycle processes that are key for the development of effective vaccines and treatments (1).Recent proteomics studies in the Foster lab, identified the interaction between the arenavirus nucleoprotein, NP and the antiviral E3 ligase tripartite motif-containing protein 25 (TRIM25), a key component of the innate immune response that inhibits the replication of a diverse range of pathogenic viruses, but previously unknown for arenaviruses (2). NP is the most abundantly expressed arenavirus protein, is a major orchestrator of host immunosuppression during virusinfection and encapsidates the viral RNA, forming the viral ribonucleocomplexes (vRNPs) needed for infection initiation (1). Using arenavirus infection and CRISPR/Cas9 knockout studies, we have shown that TRIM25 significantly restricts arenavirus replication, that TRIM25 re-localises to NP-containing viral replication sites and that NP- overexpression is sufficient to induce this re-localisation.TRIM25 was recently identified as key cofactor of the zinc-antiviral protein, ZAP, that targets viral RNAs containing CpG dinucleotides leading to promotion of viral RNA degradation and/or inhibition of viral RNA translation (2). We have demonstrated that ZAP also potently inhibits arenavirus replication and as TRIM25 is also an RNA binding protein, and it remains to be determined how TRIM25 and ZAP activity is co-modulated by their RNA interactions. The molecular structure of TRIM25 is an anti-parallel dimer formed through its coiled-coil domain, that can further multimerise through dimerisation of its N-terminal RING domain, containing E3-ubiquitin ligase activity. The ZAP and other ligand-binding C-terminal PRYSPRY domain is located on either side of the coiled-coil (2).We hypothesise that TRIM25 may multimerise around the helical vRNP complexes, driving disassembly and exposing RNA sites for ZAP recruitment and subsequent RNA degradation.Thus, this project aims to use a combination of structural biology (expertise in the Borkar group), biochemistry and molecular virology techniques to:Characterise how TRIM25 interacts with arenavirus NP protein and with arenavirus viral RNAs with and without ZAP through mutagenesis and arenavirus replication experimentsRecombinantly express and purify TRIM25 and ZAP proteins alone, in complex and in the presence of arenaviral RNAVisualise by electron microscopy and/or X-ray crystallography apo-structures and TRIM25- NP and TRIM25-NP-RNA-ZAP complexes, incorporating specific mutations that modify RNA and cofactor binding.Given the molecular tractability of this antiviral mechanism across diverse RNA and DNA viruses, findings from this work will provide key evidence that could transform our understanding of innate immune mechanisms and influence therapeutic design.