Alios BioPharma, Inc. a Janssen Pharmaceutical Company of Johnson & Johnson, South San Francisco, California, United States of America.
Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
PLoS Pathog. 2018 Feb 9;14(2):e1006889. doi: 10.1371/journal.ppat.1006889. eCollection 2018 Feb.
Paramyxoviruses represent a family of RNA viruses causing significant human diseases. These include measles virus, the most infectious virus ever reported, in addition to parainfluenza virus, and other emerging viruses. Paramyxoviruses likely share common replication machinery but their mechanisms of RNA biosynthesis activities and details of their complex polymerase structures are unknown. Mechanistic and functional details of a paramyxovirus polymerase would have sweeping implications for understanding RNA virus replication and for the development of new antiviral medicines. To study paramyxovirus polymerase structure and function, we expressed an active recombinant Nipah virus (NiV) polymerase complex assembled from the multifunctional NiV L protein bound to its phosphoprotein cofactor. NiV is an emerging highly pathogenic virus that causes severe encephalitis and has been declared a global public health concern due to its high mortality rate. Using negative-stain electron microscopy, we demonstrated NiV polymerase forms ring-like particles resembling related RNA polymerases. We identified conserved sequence elements driving recognition of the 3'-terminal genomic promoter by NiV polymerase, and leading to initiation of RNA synthesis, primer extension, and transition to elongation mode. Polyadenylation resulting from NiV polymerase stuttering provides a mechanistic basis for transcription termination. It also suggests a divergent adaptation in promoter recognition between pneumo- and paramyxoviruses. The lack of available antiviral therapy for NiV prompted us to identify the triphosphate forms of R1479 and GS-5734, two clinically relevant nucleotide analogs, as substrates and inhibitors of NiV polymerase activity by delayed chain termination. Overall, these findings provide low-resolution structural details and the mechanism of an RNA polymerase from a previously uncharacterized virus family. This work illustrates important functional differences yet remarkable similarities between the polymerases of nonsegmented negative-strand RNA viruses.
副粘病毒科代表了一组引起重大人类疾病的 RNA 病毒。其中包括麻疹病毒，这是迄今为止报道的最具传染性的病毒，此外还有副流感病毒和其他新兴病毒。副粘病毒可能具有共同的复制机制，但它们的 RNA 生物合成活性机制和其复杂聚合酶结构的细节尚不清楚。副粘病毒聚合酶的机制和功能细节将对理解 RNA 病毒复制以及开发新的抗病毒药物产生广泛影响。为了研究副粘病毒聚合酶的结构和功能，我们表达了一种活性重组尼帕病毒（NiV）聚合酶复合物，该复合物由多功能 NiV L 蛋白与磷酸蛋白辅因子结合而成。NiV 是一种新兴的高致病性病毒，会引起严重的脑炎，并因其高死亡率而被宣布为全球公共卫生关注。通过负染电子显微镜，我们证明 NiV 聚合酶形成类似于相关 RNA 聚合酶的环状颗粒。我们确定了保守的序列元件，这些元件驱动 NiV 聚合酶识别 3'-末端基因组启动子，从而引发 RNA 合成的起始、引物延伸和过渡到延伸模式。多聚腺苷酸化是 NiV 聚合酶停滞的结果，为转录终止提供了机制基础。它还表明，在肺炎病毒和副粘病毒之间，启动子识别存在分歧的适应。由于缺乏针对 NiV 的可用抗病毒疗法，我们试图通过延迟链终止来鉴定 R1479 和 GS-5734 的三磷酸形式，这两种临床相关核苷酸类似物，作为 NiV 聚合酶活性的底物和抑制剂。总的来说，这些发现提供了一种以前未被表征的病毒家族的 RNA 聚合酶的低分辨率结构细节和机制。这项工作说明了未分段负链 RNA 病毒聚合酶之间存在重要的功能差异，但也存在显著的相似性。
