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

TRIM25和ZAP介导的抗病毒抑制沙粒病毒复制的分子机制

• 批准号: 2885907
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2885907
• 关键词: Molecular; mechanism; TRIM25; ZAP; mediated

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.

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，重点是抑制病毒感染的细胞抗病毒机制和控制这些机制的病毒-宿主相互作用。福斯特小组的重点是最大的家族出血热引起的病毒称为沙粒病毒。这些人畜共患病毒的遗传多样性正在迅速扩大，导致流行地区的年度疫情增加，并在全球范围内发生零星输入性疫情，包括英国最近的病例（1）。了解宿主抗病毒蛋白如何阻断沙粒病毒复制对于解决病毒生命周期过程的现有知识空白至关重要，这是开发有效疫苗和治疗的关键（1）。福斯特实验室最近的蛋白质组学研究确定了沙粒病毒核蛋白NP和抗病毒E3连接酶三重基序蛋白25（TRIM 25）之间的相互作用，是先天性免疫反应的关键成分，可抑制多种致病病毒的复制，但以前对沙粒病毒一无所知（2）。NP是表达最丰富的沙粒病毒蛋白，是病毒感染期间宿主免疫抑制的主要协调者，并使病毒RNA发生变性，形成感染起始所需的病毒核糖核复合物（vRNP）（1）。使用沙粒病毒感染和CRISPR/Cas9敲除研究，我们已经表明TRIM 25显着限制沙粒病毒复制，TRIM 25重新定位于含NP的病毒复制位点，并且NP过表达足以诱导这种重新定位。TRIM 25最近被确定为锌抗病毒蛋白ZAP的关键辅因子靶向含有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也有效地抑制沙粒病毒复制，并且TRIM 25也是RNA结合蛋白，并且仍有待确定TRIM 25和ZAP活性如何通过它们的RNA相互作用共同调节。TRIM 25的分子结构是通过其卷曲螺旋结构域形成的反平行二聚体，其可以通过其N-末端RING结构域的二聚化进一步多聚化，含有E3-泛素连接酶活性。ZAP和其他配体结合的C-末端PRYSPRY结构域位于卷曲螺旋的两侧（2）。我们假设TRIM 25可能在螺旋状vRNP复合物周围多聚化，驱动拆卸并暴露RNA位点用于ZAP募集和随后的RNA降解。因此，本项目旨在结合结构生物学和分子生物学，（博卡集团的专业知识），生物化学和分子病毒学技术，以：通过诱变和沙粒病毒复制来表征TRIM 25如何与沙粒病毒NP蛋白以及与含有和不含ZAP的沙粒病毒RNA相互作用实验性地表达和纯化单独的TRIM 25和ZAP蛋白，通过电子显微镜和/或X射线晶体学观察脱辅基结构和TRIM 25- NP和TRIM 25-NP-RNA-ZAP复合物，结合修饰RNA和辅因子结合的特定突变。鉴于这种抗病毒机制在不同RNA和DNA病毒中的分子可处理性，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。