Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
PLoS Pathog. 2011 Jun;7(6):e1002085. doi: 10.1371/journal.ppat.1002085. Epub 2011 Jun 23.
The RNA-dependent RNA polymerase VP1 of infectious pancreatic necrosis virus (IPNV) is a single polypeptide responsible for both viral RNA transcription and genome replication. Sequence analysis identifies IPNV VP1 as having an unusual active site topology. We have purified, crystallized and solved the structure of IPNV VP1 to 2.3 Å resolution in its apo form and at 2.2 Å resolution bound to the catalytically-activating metal magnesium. We find that recombinantly-expressed VP1 is highly active for RNA transcription and replication, yielding both free and polymerase-attached RNA products. IPNV VP1 also possesses terminal (deoxy)nucleotide transferase, RNA-dependent DNA polymerase (reverse transcriptase) and template-independent self-guanylylation activity. The N-terminus of VP1 interacts with the active-site cleft and we show that the N-terminal serine residue is required for formation of covalent RNA:polymerase complexes, providing a mechanism for the genesis of viral genome:polymerase complexes observed in vivo.
传染性胰脏坏死病毒（IPNV）的 RNA 依赖性 RNA 聚合酶 VP1 是一种单一多肽，负责病毒 RNA 的转录和基因组复制。序列分析将 IPNV VP1 鉴定为具有不寻常的活性位点拓扑结构。我们已经纯化、结晶并解析了无辅因子形式的 IPNV VP1 结构，分辨率为 2.3 Å，以及结合了催化激活金属镁的复合物结构，分辨率为 2.2 Å。我们发现重组表达的 VP1 具有高度的 RNA 转录和复制活性，产生游离和聚合酶结合的 RNA 产物。IPNV VP1 还具有末端（脱氧）核苷酸转移酶、RNA 依赖性 DNA 聚合酶（逆转录酶）和无模板的自身鸟苷酰化活性。VP1 的 N 端与活性位点裂隙相互作用，我们表明 N 端丝氨酸残基是形成共价 RNA:聚合酶复合物所必需的，为体内观察到的病毒基因组:聚合酶复合物的产生提供了一种机制。
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