Mechanisms of genome packaging and replication by a filamentous dsRNA virus

丝状 dsRNA 病毒的基因组包装和复制机制

• 批准号: 10575353
• 负责人: Yizhi Jane Tao
• 金额: $ 22.4万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-09 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10575353
• 关键词: Agriculture; Amino Acids; Antiviral Response; Biochemical; Biochemistry; Birnavirus; Capsid; Capsid Proteins; China; Colletotrichum; Complex; Cryoelectron Microscopy; Data; Defense Mechanisms; Diameter; Double Stranded RNA Virus; Double-Stranded RNA; Engineering; Filament; Genes; Genetic Transcription; Genome; Genomics; Goals; Grant; In Situ; Methyltransferase; Morphology; Open Reading Frames; Penicillium; Picobirnavirus; Plants; Polymerase; Property; Proteins; Publications; RNA; RNA-Directed RNA Polymerase; RNA-Protein Interaction; Recombinant Proteins; Recombinants; Reovirus; Research; Resolution; Rotavirus; S-Adenosylhomocysteine; S-Adenosylmethionine; Sampling; Series; Shapes; Structure; Tea; Techniques; Universities; Viral; Viral Genome; Virion; Virus; Virus Replication; X-Ray Crystallography; dimer; experimental study; fungus; genomic RNA; guanylyltransferase; mRNA capping; monomer; particle; reconstruction; stoichiometry; structural biology; viral RNA

Discovered in 2017, Colletotrichum camelliae filamentous virus 1 (CcFV-1) is considered an outlier among dsRNA viruses due to its filamentous morphology. Typical dsRNA viruses possess a spherically shaped icosahedral capsid with multiple copies of a viral polymerase complex packaged inside for in situ transcription of the viral genome. These dsRNA viruses do not completely uncoat during host entry, allowing transcription of the viral genome to take place within intact capsids while avoiding dsRNA activated defense mechanisms of their hosts. In contrast, CcFV-1 particles assume the shape of a flexuous filament with a 12.2 kb genome that is organized into eight gene segments with ten predicted open reading frames (ORFs) including P1, an RNA- dependent RNA polymerase (RdRP); P3, a putative S-adenosyl methionine (SAM)-dependent methyltransferase; P4, the capsid protein (CP); and seven other proteins of yet unknown functions. The overarching goals of this grant are to determine how CcFV-1 organizes its genomic dsRNA inside the viral particle, whether CcFV-1 has virion-associated transcription activity and, if so, how the filamentous CcFV-1 capsid provides polymerase access to the dsRNA genome without complete uncoating and disassembly. We have already demonstrated that recombinant CcFV-1 CP forms both helical VLPs and small oligomers. Recombinant P1 RdRP forms stable dimers and we have obtained a preliminary cryo-electron microscopy reconstruction of P1 showing a S-shaped molecule. CcFV-1 P1 and P3 form a stable complex that is also suitable for cryo-EM analysis. In addition, we will have access to infectious CcFV-1 virion samples provided by Dr. Wenxing Xu at the Huazhong Agricultural University in China. Altogether, these data not only provide a strong rationale, but also demonstrate the feasibility of our proposed experiments. Our research plan consists of three independent but complementary aims. Aim 1 is to elucidate the structural organization of the CcFV-1 viral particle by solving the structure of both the filamentous VLP and infectious virion. We hypothesize that the helical capsid of CcFV-1 protects its dsRNA genome from triggering cellular antiviral responses during all stages of viral replication. Aim 2 is to determine the structural and biochemical properties of the CcFV-1 polymerase complex. The central hypothesis is that CcFV-1 polymerase complex should possess RdRP, methyltransferase, and guanylyltransferase activities with possibly unique structural features that allow it to transcribe dsRNA packaged in a helical capsid. Aim 3 is explore interactions between the CcFV-1 capsid and the polymerase complex. We will analyze the RNA transcription activity of infectious viral particles and investigate the mechanism of polymerase incorporation into infectious virions. We hypothesize that the CcFV-1 polymerase complex is likely localized to the terminal ends of the viral helical capsid through direct protein- protein and/or protein-RNA interactions. Results from our research will lead to new paradigms that apply to CcFV-1 and other filamentous dsRNA viruses.

2017 年发现的山茶炭疽菌丝状病毒 1 (CcFV-1) 被认为是异常值 由于其丝状形态，它属于 dsRNA 病毒之一。典型的 dsRNA 病毒具有球形 二十面体衣壳，内部包装有多个病毒聚合酶复合物副本，用于原位转录 病毒基因组。这些 dsRNA 病毒在进入宿主期间不会完全脱壳，从而允许转录 病毒基因组发生在完整的衣壳内，同时避免 dsRNA 激活其防御机制 主机。相比之下，CcFV-1 颗粒呈弯曲丝状，具有 12.2 kb 基因组， 组织成 8 个基因片段，具有 10 个预测的开放阅读框 (ORF)，其中包括 P1（一种 RNA- 依赖性RNA聚合酶（RdRP）； P3，一种推定的 S-腺苷甲硫氨酸 (SAM) 依赖性甲基转移酶； P4，衣壳蛋白（CP）；以及其他七种功能尚不清楚的蛋白质。 这笔资助的首要目标是确定 CcFV-1 如何在内部组织其基因组 dsRNA 病毒颗粒，CcFV-1 是否具有病毒粒子相关的转录活性，如果有，丝状病毒如何 CcFV-1 衣壳提供聚合酶进入 dsRNA 基因组，无需完全脱壳和拆卸。 我们已经证明重组 CcFV-1 CP 形成螺旋 VLP 和小寡聚体。 重组P1 RdRP形成稳定的二聚体，我们获得了初步的冷冻电镜照片 P1 的重建显示 S 形分子。 CcFV-1 P1 和 P3 形成稳定的复合物，也适合 用于冷冻电镜分析。此外，我们还将获得由 Dr. 提供的传染性 CcFV-1 病毒粒子样本。 徐文兴，中国华中农业大学教授。总而言之，这些数据不仅提供了强有力的 基本原理，同时也证明了我们提出的实验的可行性。我们的研究计划包括三部分 独立但互补的目标。目标 1 是阐明 CcFV-1 病毒的结构组织 通过解析丝状 VLP 和感染性病毒颗粒的结构来研究颗粒。我们假设 CcFV-1 的螺旋衣壳可保护其 dsRNA 基因组在所有过程中不触发细胞抗病毒反应 病毒复制的阶段。目标 2 是确定 CcFV-1 的结构和生化特性 聚合酶复合物。中心假设是 CcFV-1 聚合酶复合物应该具有 RdRP， 甲基转移酶和鸟苷基转移酶活性可能具有独特的结构特征，使其能够 转录包装在螺旋衣壳中的 dsRNA。目标 3 是探索 CcFV-1 衣壳之间的相互作用 和聚合酶复合物。我们将分析传染性病毒颗粒的 RNA 转录活性并 研究聚合酶掺入感染性病毒粒子的机制。我们假设 CcFV-1 聚合酶复合物可能通过直接蛋白定位于病毒螺旋衣壳的末端。 蛋白质和/或蛋白质-RNA 相互作用。我们的研究结果将带来适用于的新范式 CcFV-1 和其他丝状 dsRNA 病毒。