Department of Bioengineering, University of California, Los Angeles, Los Angeles, California, USA.
California NanoSystems Institute, University of California, Los Angeles, Los Angeles, California, USA.
J Virol. 2018 Oct 12;92(21). doi: 10.1128/JVI.00774-18. Print 2018 Nov 1.
Reoviruses carry out genomic RNA transcription within intact viruses to synthesize plus-sense RNA strands, which are capped prior to their release as mRNA. The structures of the transcriptional enzyme complex (TEC) containing the RNA-dependent RNA polymerase (RdRp) and NTPase are known for the single-layered reovirus cytoplasmic polyhedrosis virus (CPV), but not for multilayered reoviruses, such as aquareoviruses (ARV), which possess a primed stage that CPV lacks. Consequently, how the RNA genome and TEC respond to priming in reoviruses is unknown. Here, we determined the near-atomic-resolution asymmetric structure of ARV in the primed state by cryo-electron microscopy (cryo-EM), revealing the structures of 11 TECs inside each capsid and their interactions with the 11 surrounding double-stranded RNA (dsRNA) genome segments and with the 120 enclosing capsid shell protein (CSP) VP3 subunits. The RdRp VP2 and the NTPase VP4 associate with each other and with capsid vertices; both bind RNA in multiple locations, including a novel C-terminal domain of VP4. Structural comparison between the primed and quiescent states showed translocation of the dsRNA end from the NTPase to the RdRp during priming. The RNA template channel was open in both states, suggesting that channel blocking is not a regulating mechanism between these states in ARV. Instead, the NTPase C-terminal domain appears to regulate RNA translocation between the quiescent and primed states. Taking the data together, dsRNA viruses appear to have adapted divergent mechanisms to regulate genome transcription while retaining similar mechanisms to coassemble their genome segments, TEC, and capsid proteins into infectious virions. Viruses in the family are characterized by the ability to endogenously synthesize nascent RNA within the virus. However, the mechanisms for assembling their RNA genomes with transcriptional enzymes into a multilayered virion and for priming such a virion for transcription are poorly understood. By cryo-EM and novel asymmetric reconstruction, we determined the atomic structure of the transcription complex inside aquareoviruses (ARV) that are primed for infection. The transcription complex is anchored by the N-terminal segments of enclosing capsid proteins and contains an NTPase and a polymerase. The NTPase has a newly discovered domain that translocates the 5' end of plus-sense RNA in segmented dsRNA genomes from the NTPase to polymerase VP2 when the virus changes from the inactive (quiescent) to the primed state. Conformation changes in capsid proteins and transcriptional complexes suggest a mechanism for relaying information from the outside to the inside of the virus during priming.
呼肠孤病毒在完整的病毒内进行基因组 RNA 转录，以合成正链 RNA 链，这些 RNA 链在作为 mRNA 释放之前被加帽。单层呼肠孤病毒细胞质多角体病毒 (CPV) 的转录酶复合物 (TEC) 结构，包含 RNA 依赖性 RNA 聚合酶 (RdRp) 和 NTPase，已知，但多层呼肠孤病毒，如 Aquareoviruses (ARV)，则没有，因为它具有 CPV 缺乏的引发阶段。因此，RNA 基因组和 TEC 如何在呼肠孤病毒中对引发做出反应尚不清楚。在这里，我们通过冷冻电镜 (cryo-EM) 确定了处于引发状态的 ARV 的近原子分辨率非对称结构，揭示了每个衣壳内 11 个 TEC 的结构及其与 11 个周围双链 RNA (dsRNA) 基因组片段的相互作用，以及与 120 个封闭衣壳壳蛋白 (CSP) VP3 亚基的相互作用。RdRp VP2 和 NTPase VP4 彼此相互作用，并与衣壳顶点相互作用; 两者都在多个位置结合 RNA，包括 VP4 的新 C 端结构域。在引发和静止状态之间的结构比较表明，dsRNA 末端在引发过程中从 NTPase 转移到 RdRp。在两种状态下，RNA 模板通道均打开，表明通道阻塞不是 ARV 这两种状态之间的调节机制。相反，NTPase C 端结构域似乎调节 RNA 在静止和引发状态之间的转移。综合这些数据，dsRNA 病毒似乎已经适应了不同的机制来调节基因组转录，同时保留了相似的机制来共同组装它们的基因组片段、TEC 和衣壳蛋白，形成感染性病毒粒子。家族中的病毒的特征是能够在病毒内内源合成新生 RNA。然而，组装其 RNA 基因组与转录酶形成多层病毒粒子并引发这种病毒粒子进行转录的机制仍知之甚少。通过冷冻电镜和新的不对称重建，我们确定了 Aquareoviruses (ARV) 中处于感染引发状态的转录复合物的原子结构。转录复合物由封闭衣壳蛋白的 N 端片段锚定，包含 NTPase 和聚合酶。NTPase 具有一个新发现的结构域，当病毒从非活性 (静止) 状态变为引发状态时，它将分段 dsRNA 基因组中 + 链 RNA 的 5' 端从 NTPase 转移到聚合酶 VP2。衣壳蛋白和转录复合物的构象变化表明了一种在引发过程中从病毒外部向内部传递信息的机制。
