The predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mRNA synthesis, genome replication, and virion biogenesis
The predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mRNA synthesis, genome replication, and virion biogenesis
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DOI：
10.1128/jvi.74.11.5213-5223.2000
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发表时间：
2000-06-01
影响因子：
5.4
通讯作者：
Snijder, EJ
中科院分区：
文献类型：
--
作者：
van Dinten, LC;van Tol, H;Snijder, EJ
马动脉炎病毒 (EAV)，原型动脉病毒，是一种正链 RNA 病毒，以两个含有 1,727 和 3,175 个氨基酸的大多蛋白形式表达其复制酶。驱动 EAV 基因组复制和亚基因组 mRNA 转录的功能性复制酶亚基（非结构蛋白）是通过广泛的蛋白水解加工产生的。亚基因组 mRNA 转录涉及一个不寻常的不连续步骤，并生成用于结构蛋白表达的 mRNA。此前，携带单个复制酶点突变 (Ser-2429-->Pro) 的突变体 EAV030F 的表型表明 nsp10 复制酶亚基 (51 kDa) 参与病毒 RNA 合成，特别是亚基因组 mRNA 转录。 nsp10 包含一个 N 端（推定）金属结合结构域 (MBD)，位于 Ser-2429-->Pro 突变的上游，并且在其 C 端部分具有解旋酶活性。我们现在已经相当详细地分析了 nsp10 的 N 末端结构域。在 EAV 感染性 cDNA 克隆竞赛中测试了总共 38 个突变体，其中大多数携带特定的单点突变。仅观察到对病毒基因组复制和亚基因组 mRNA 转录的不同影响。总的来说，我们的结果表明，MBD 区域，特别是一组 13 个保守的 Cgs 和 His 残基（被认为参与锌结合）对于病毒 RNA 合成至关重要。根据这些数据和比较序列分析，我们推测 MBD 可能采用一种相当不寻常的锌结合模式，这可能导致多达四个锌阳离子与该结构域结合。含有残基 Ser-2429 mag 的区域起着“铰链间隔区”的作用，它将 MBD 连接到 nsp10 的其余部分。该区域的几个突变特别影响亚基因组 mRNA 的合成。此外，MBD 突变体之一具有复制和转录能力，但不产生感染性子代病毒。这表明 nsp10 参与了病毒粒子生物发生的一个尚未确定的步骤。
Equine arteritis virus (EAV), the prototype Arterivirus, is a positive-stranded RNA virus that expresses its replicase in the form of two large polyproteins of 1,727 and 3,175 amino acids. The functional replicase subunits (nonstructural proteins), which drive EAV genome replication and subgenomic mRNA transcription, are generated by extensive proteolytic processing. Subgenomic mRNA transcription involves an unusual discontinuous step and generates the mRNAs for structural protein expression, Previously, the phenotype of mutant EAV030F, which carries a single replicase point mutation (Ser-2429-->Pro), had implicated the nsp10 replicase subunit (51 kDa) in viral RNA synthesis, and in particular in subgenomic mRNA transcription. nsp10 contains an N-terminal (putative) metal-binding domain (MBD), located just upstream of the Ser-2429-->Pro mutation, and a helicase activity in its C-terminal part. We hare now analyzed the N-terminal domain of nsp10 in considerable detail. A total of 38 mutants, most of them carrying specific single point mutations, were tested in the contest of an EAV infectious cDNA clone. Variable effects on viral genome replication and subgenomic mRNA transcription mere observed. In general, our results indicated that the MBD region, and in particular a set of 13 conserved Cgs and His residues that are assumed to be involved in zinc binding, is essential for viral RNA synthesis. On the basis of these data and comparative sequence analyses, we postulate that the MBD may employ a rather unusual mode of zinc binding that could result in the association of up to four zinc cations with this domain. The region containing residue Ser-2429 mag play the role of "hinge spacer," which connects the MBD to the rest of nsp10. Several mutations in this region specifically affected subgenomic mRNA synthesis. Furthermore, one of the MBD mutants was replication and transcription competent but did not produce infectious progeny virus. This suggests that nsp10 is involved in an as yet unidentified step of virion biogenesis.