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

这个联合项目跨越了福斯特、博卡和邓纳姆小组的病毒学和结构生物学研究项目，重点研究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-宿主相互作用。福斯特小组专注于引起出血热的最大病毒家族，即沙粒病毒。这些人畜共患病毒的遗传多样性正在迅速扩大，导致流行地区每年爆发的疫情增加，以及全球范围内散发的输入性疫情，包括最近在英国发生的病例(1)。了解宿主抗病毒蛋白如何阻断沙粒病毒复制对于解决当前病毒生命周期过程的知识缺口至关重要，而病毒生命周期过程是开发有效疫苗和治疗方法的关键(1)。福斯特实验室最近的蛋白质组学研究发现，沙粒病毒核蛋白NP与抗病毒E3连接酶三方基元蛋白25 （TRIM25）之间存在相互作用，TRIM25是先天免疫反应的关键组成部分，可抑制多种致病性病毒的复制，但此前对沙粒病毒未知(2)。NP是最丰富表达的沙粒病毒蛋白，是病毒感染期间宿主免疫抑制的主要协调者，并封装病毒RNA，形成感染起始所需的病毒核糖核复合物（vRNPs）(1)。通过沙粒病毒感染和CRISPR/Cas9敲除研究，我们发现TRIM25显著限制了沙粒病毒的复制，TRIM25重新定位到含有NP的病毒复制位点，并且NP-过表达足以诱导这种重新定位。TRIM25最近被确定为锌抗病毒蛋白ZAP的关键辅助因子，ZAP靶向含有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，ZAP也能有效地抑制沙粒病毒的复制，而且TRIM25也是一种RNA结合蛋白，TRIM25和ZAP的活性是如何通过它们的RNA相互作用共同调节的还有待确定。TRIM25的分子结构是一种反平行二聚体，通过其螺旋结构域形成，可以通过其n端RING结构域的二聚化进一步聚合，具有e3 -泛素连接酶活性。ZAP和其他配体结合的c端PRYSPRY结构域位于螺旋状螺旋的两侧(2)。我们假设TRIM25可能在螺旋vRNP复合物周围多聚合，驱动分解并暴露RNA位点以供ZAP招募和随后的RNA降解。因此，该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，通过诱变和沙粒病毒复制实验，表征TRIM25如何与沙粒病毒NP蛋白以及带有和不带有ZAP的沙粒病毒rna相互作用。通过电子显微镜和/或x射线晶体学观察载脂蛋白结构和TRIM25-NP和TRIM25-NP-RNA- zap复合物，其中包含修饰RNA和辅因子结合的特异性突变。鉴于这种抗病毒机制在不同RNA和DNA病毒中的分子可追踪性，这项工作的发现将提供关键证据，可以改变我们对先天免疫机制的理解，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.