Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA.
Program in Cellular and Molecular Medicine, Boston Children's Hospital and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
J Virol. 2020 Jun 16;94(13). doi: 10.1128/JVI.00115-20.
Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the L, L, and L, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (P) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription Recombinant virus VSV-P exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that P diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized defective interfering particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of P We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation. All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.
无分段负链 (NNS) RNA 病毒拥有一种核糖核蛋白模板，其中基因组 RNA 被封闭在核衣壳蛋白 (N) 的同聚物中。病毒 RNA 依赖性 RNA 聚合酶 (RdRP) 位于大约 250 kDa 的大蛋白 (L) 内，同时还具有非常规的 mRNA 加帽酶：GDP:多核糖核苷酸转移酶 (PRNT) 和双特异性 mRNA 帽甲基转移酶 (MT)。为了进入 N-RNA 模板并协调 L、L 和 L，需要一种寡聚磷酸蛋白 (P)。水疱性口炎病毒 (VSV) P 是二聚体，其寡聚化结构域 (OD) 将两个主要无序区域隔开，然后是一个球状 C 末端结构域，该结构域与模板结合。P 还负责在基因组复制过程中将新的 N 原聚体带到新生 RNA 上。我们发现缺乏 OD (P) 的 VSV P 是单体，但在支持 mRNA 转录方面与野生型 P 没有区别。重组病毒 VSV-P 表现出明显的后代病毒产生动力学延迟。光漂白后荧光恢复表明，P 在病毒复制隔间内的扩散速度比野生型快 6 倍。VSV 的一种特征明确的缺陷干扰颗粒 (DI-T) 仅具有 RNA 复制能力，在 P 的存在下，需要更高水平的 N 来驱动 RNA 复制。我们得出结论，P 寡聚化不是 mRNA 合成所必需的，但通过促进 RNA 封装来增强基因组复制。所有 NNS RNA 病毒，包括狂犬病、麻疹、呼吸道合胞病毒、尼帕和埃博拉等人类病原体，都拥有一种必需的 L 蛋白辅助因子，该因子需要进入 N-RNA 模板并协调 L 的各种酶活性。聚合酶辅助因子具有相似的模块化组织，即可溶性 N 结合域和模板结合域，中间由一个寡聚化结构域隔开。使用 NNS RNA 病毒基因表达的原型，水疱性口炎病毒 (VSV)，我们确定了 P 寡聚化的重要性。我们发现，VSV P 的寡聚化对于病毒 mRNA 合成的任何步骤都不是必需的，但对于有效的 RNA 复制是必需的。我们提供的证据表明，这可能是通过在 RNA 复制过程中，新生 RNA 链从聚合酶中退出时将可溶性 N 加载到其上的阶段发生的。干扰 P 的寡聚化可能是一种干扰 NNS RNA 病毒复制的通用策略。
