Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.
Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.
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DOI：
10.1371/journal.ppat.1001101
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发表时间：
2010-09-16
期刊：
影响因子：
6.7
通讯作者：
Cusack S
中科院分区：
文献类型：
--
作者：
Reguera J;Weber F;Cusack S
布尼亚病毒是分段RNA病毒的一个大家族，与流感病毒一样，它们使用帽抢夺机制进行转录，病毒RNA依赖性RNA聚合酶（L蛋白）使用宿主mRNA的核酸内切裂解衍生的短帽引物来转录病毒mRNA。最近的研究表明，流感病毒的抢帽子核酸内切酶位于 PA 聚合酶亚基的离散 N 末端结构域中。在这里，我们从结构和功能上表征了拉克罗斯正布尼亚病毒 (LACV) L 蛋白中类似的核酸内切酶。我们表达了 LACV L 蛋白的 N 端片段，发现残基 1-180 具有金属结合和二价阳离子依赖性核酸酶活性，类似于流感病毒核酸内切酶的活性。该结构域的 2.2 Å 分辨率 X 射线晶体结构证实了 LACV 和流感核酸内切酶具有相似的整体折叠和相同的两个金属结合活性位点。 LACV核酸内切酶的体外活性可以通过活性位点的点突变或通过结合2,4-二氧代-4-苯基丁酸（DPBA）（一种已知的流感病毒核酸内切酶抑制剂）来消除。结合 DPBA 的晶体结构显示抑制剂螯合两个活性位点锰离子。这种核酸内切酶在帽依赖性转录中的重要作用通过在全长 LACV L 蛋白中发生相同点突变后细胞中 RNP 重建系统中转录活性的丧失而得到证明。使用基于结构的序列比对，我们发现类似的核酸内切酶几乎肯定存在于基本上所有已知的负链和抢帽分段RNA病毒的L蛋白或PA聚合酶亚基的N端，包括沙粒病毒（2个片段）、布尼亚病毒（3个片段）、细病毒（4-6个片段）和正粘病毒（6-8个片段） 段）。这种对应关系，连同众所周知的 L 蛋白和流感 PB1 亚基中心区域的保守聚合酶基序的映射，表明 L 蛋白可能在结构上和功能上等同于按 PA-PB1-PB2 顺序的三个正粘病毒聚合酶亚基的串联体。此外，我们对与 LACV 核酸内切酶结合的已知流感核酸内切酶抑制剂的结构表明，可以使用基于结构的优化来开发针对潜在广谱分段 RNA 病毒（其中几种是严重或新兴的人类、动物和植物病原体）的化合物。布尼亚病毒是一个RNA病毒大家族，包括严重的人类、动物和植物病原体。病毒RNA依赖性RNA聚合酶（L蛋白）负责病毒RNA的复制和转录，但除了其中心聚合酶结构域外，其特征尚不清楚。与流感病毒聚合酶一样，布尼亚病毒 L 蛋白采用帽抢夺机制来转录病毒 mRNA，通过这种机制，宿主 mRNA 被内切核酸酶切割，作为短帽引物的来源。最近，流感聚合酶核酸内切酶被定位于 PA 亚基 N 末端。在这里，我们通过生化和晶体结构测定表明，拉克罗斯正布尼亚病毒L蛋白的N末端存在类似的二锰依赖性核酸酶，其功能是帽依赖性转录所必需的。通过序列分析，我们表明相似的核酸内切酶特征基序存在于几乎所有已知的分段RNA、抢帽病毒中，包括沙粒病毒、布尼亚病毒、细病毒和正粘病毒。这表明这些病毒的聚合酶可能共享一个保守的全局结构，L-蛋白相当于正粘病毒 PA-PB1-PB2 亚基的串联。我们还建议可以开发针对此类病毒核酸内切酶结构域的广谱药物，例如我们的 LACV 核酸内切酶与已知流感核酸内切酶抑制剂复合的结构。
Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV) L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 Å resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA), a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments), bunyaviruses (3 segments), tenuiviruses (4–6 segments), and orthomyxoviruses (6–8 segments). This correspondence, together with the well-known mapping of the conserved polymerase motifs to the central regions of the L-protein and influenza PB1 subunit, suggests that L-proteins might be architecturally, and functionally equivalent to a concatemer of the three orthomyxovirus polymerase subunits in the order PA-PB1-PB2. Furthermore, our structure of a known influenza endonuclease inhibitor bound to LACV endonuclease suggests that compounds targeting a potentially broad spectrum of segmented RNA viruses, several of which are serious or emerging human, animal and plant pathogens, could be developed using structure-based optimisation. Bunyaviruses are a large family of RNA viruses that include serious human, animal and plant pathogens. The viral RNA-dependent RNA polymerase (L-protein) is responsible for replication and transcription of the viral RNA, but apart from its central polymerase domain, it is poorly characterized. Like influenza virus polymerase, bunyavirus L-proteins employ a cap-snatching mechanism to transcribe viral mRNAs, by which host mRNAs are endonucleolytically cleaved as a source of short capped primers. Influenza polymerase endonuclease has recently been located at the PA subunit N-terminus. Here we show biochemically and by crystal structure determination that a similar two-manganese dependent nuclease exists at the N-terminus of La Crosse orthobunyavirus L-protein, whose function is required for cap-dependent transcription. By sequence analysis we show that similar endonuclease signature motifs exist in almost all known segmented RNA, cap-snatching viruses including arenaviruses, bunyaviruses, tenuiviruses and orthomyxoviruses. This suggests that the polymerases of these viruses might share a conserved global architecture with the L-protein being equivalent to a concatenation of the orthomxyovirus PA-PB1-PB2 subunits. We also propose that broad spectrum drugs targeting the endonuclease domain of such viruses could be developed, as exemplified by our structure of the LACV endonuclease complexed with a known influenza endonuclease inhibitor.
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