Structure of the recombinant RNA polymerase from African Swine Fever Virus
Structure of the recombinant RNA polymerase from African Swine Fever Virus
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DOI：
10.1038/s41467-024-45842-7
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发表时间：
2024-02-21
影响因子：
16.6
通讯作者：
Werner，Finn
中科院分区：
文献类型：
--
作者：
Pilotto，Simona;Sykora，Michal;Werner，Finn
非洲猪瘟病毒是一种核细胞质大DNA病毒，可在猪中引起无法治愈的出血热，对全球粮食安全造成严重影响。ASFV在被感染细胞的细胞质中复制，并编码其自身的转录机制，该机制不依赖于细胞因子，然而，对于该系统在分子水平上的工作原理所知甚少。在这里，我们提出了生产重组ASFV RNA聚合酶的方法，筛选抑制剂的功能分析，以及不同构象状态下ASFV RNAP的高分辨率冷冻电镜结构。ASFV RNAP与RNAPII具有惊人的相似之处，其12个亚基中有9个具有真正的同源物。关键的差异包括ASFV组装平台亚基RPB3和RPB11的融合，以及与真核mRNA cap 2´- o -甲基转移酶1相关的茎亚基vRPB7的异常c端结构域。尽管与细胞RNA聚合酶具有高度的结构保守性，但ASFV RNAP对抑制剂利福平和α -amanitin具有耐药性。低温电镜结构和完全重组RNAP系统共同为低生物安全密闭环境下抗病毒药物的设计、开发和筛选提供了重要的工具。
African Swine Fever Virus is a Nucleo-Cytoplasmic Large DNA Virus that causes an incurable haemorrhagic fever in pigs with a high impact on global food security. ASFV replicates in the cytoplasm of the infected cell and encodes its own transcription machinery that is independent of cellular factors, however, not much is known about how this system works at a molecular level. Here, we present methods to produce recombinant ASFV RNA polymerase, functional assays to screen for inhibitors, and high-resolution cryo-electron microscopy structures of the ASFV RNAP in different conformational states. The ASFV RNAP bears a striking resemblance to RNAPII with bona fide homologues of nine of its twelve subunits. Key differences include the fusion of the ASFV assembly platform subunits RPB3 and RPB11, and an unusual C-terminal domain of the stalk subunit vRPB7 that is related to the eukaryotic mRNA cap 2´-O-methyltransferase 1. Despite the high degree of structural conservation with cellular RNA polymerases, the ASFV RNAP is resistant to the inhibitors rifampicin and alpha-amanitin. The cryo-EM structures and fully recombinant RNAP system together provide an important tool for the design, development, and screening of antiviral drugs in a low biosafety containment environment.