Mechanisms of genome packaging and replication by a filamentous dsRNA virus

2017年发现的炭疽菌丝状病毒1（CcFV-1）被认为是离群值 在dsRNA病毒中，由于其丝状形态。典型的dsRNA病毒具有球形的 二十面体衣壳，其内部包装有多个病毒聚合酶复合物拷贝，用于原位转录 病毒基因组这些dsRNA病毒在进入宿主期间不完全脱壳，从而允许dsRNA的转录。 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， hosts.相比之下，CcFV-1颗粒呈现具有12.2kb基因组的弯曲细丝的形状， 组织成八个基因片段，具有十个预测的开放阅读框（ORF），包括P1，一个RNA- 依赖性RNA聚合酶（RdRP）; P3，一种假定的S-腺苷甲硫氨酸（SAM）依赖性甲基转移酶; P4，衣壳蛋白（CP）和其他七种功能未知的蛋白质。 这项资助的首要目标是确定CcFV-1如何在细胞内组织其基因组dsRNA。 病毒颗粒，CcFV-1是否具有病毒体相关的转录活性，如果是，丝状病毒如何与CcFV-1结合， CcFV-1衣壳提供聚合酶进入dsRNA基因组而不完全脱壳和拆卸。 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，我们已经获得了初步的冷冻电镜 P1的重建显示S形分子。CcFV-1 P1和P3形成稳定的复合物，其也是合适的 冷冻电镜分析此外，我们将获得由Dr. 中国华中农业大学的徐文星。总而言之，这些数据不仅提供了一个强有力的 理论依据，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个 独立但互补的目标。目的1阐明CcFV-1病毒的结构组成 通过解决丝状VLP和感染性病毒粒子的结构，我们假设 CcFV-1的螺旋衣壳保护其dsRNA基因组在所有的抗病毒过程中不触发细胞抗病毒反应， 病毒复制的各个阶段。目的二是确定CcFV-1的结构和生化特性 聚合酶复合体中心假设是CcFV-1聚合酶复合物应该具有RdRP， 甲基转移酶和鸟苷酰转移酶活性，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。目的3：探讨CcFV-1衣壳蛋白与病毒的相互作用 和聚合酶复合体。我们将分析感染性病毒颗粒的RNA转录活性， 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的蛋白质- 蛋白质和/或蛋白质-RNA相互作用。从我们的研究结果将导致新的范式，适用于 CcFV-1和其他丝状dsRNA病毒。

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.