The N-terminus of the RNA polymerase from infectious pancreatic necrosis virus is the determinant of genome attachment.
The N-terminus of the RNA polymerase from infectious pancreatic necrosis virus is the determinant of genome attachment.
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DOI：
10.1371/journal.ppat.1002085
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发表时间：
2011-06
期刊：
影响因子：
6.7
通讯作者：
Grimes JM
中科院分区：
文献类型：
--
作者：
Graham SC;Sarin LP;Bahar MW;Myers RA;Stuart DI;Bamford DH;Grimes JM
传染性胰腺坏死病毒（IPNV）的RNA依赖性RNA聚合酶VP 1是负责病毒RNA转录和基因组复制的单一多肽。序列分析鉴定IPNV VP 1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IPNV VP 1的结构，其以载脂蛋白形式的分辨率为2.3 nm，并以2.2 nm的分辨率与催化活化金属镁结合。我们发现，重组表达的VP 1是高度活跃的RNA转录和复制，产生游离和聚合酶连接的RNA产物。IPNV VP 1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）和模板非依赖性自身鸟苷酰化活性。VP 1的N-末端与活性位点裂缝相互作用，我们发现N-末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∶聚合酶复合物的发生提供了一种机制。传染性胰腺坏死病毒（IPNV）具有高度传染性，可引起鱼类严重疾病。由于密集的饲养条件，它已成为鲑鱼和鳟鱼养殖业的一个严重问题。IPNV，像许多其他病毒一样，使用本身由病毒基因组编码的蛋白质（“聚合酶”）复制其基因组。不寻常的是，在感染性IPNV颗粒中，发现聚合酶与病毒基因组化学连接。我们已经确定了IPNV聚合酶的原子结构，使用X-射线晶体学，揭示了一些显着的差异相比，其他良好的特征病毒聚合酶的蛋白质链的折叠。通过突变蛋白质开头的一个氨基酸残基，我们展示了与病毒基因组的化学连接是如何被破坏的。这为病毒基因组与体内观察到的聚合酶的连接提供了一种优雅的机制。
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. Infectious pancreatic necrosis virus (IPNV) is highly contagious and causes severe disease in fish. As a result of intensive rearing conditions it has become a serious problem for the salmon and trout farming industries. IPNV, like many other viruses, replicates its genome using a protein (a ‘polymerase’) that is itself encoded by the viral genome. Unusually, in infectious IPNV particles the polymerase is found chemically linked to the viral genome. We have determined the atomic structure of IPNV polymerase using X-ray crystallography, revealing some significant differences in the fold of the protein chain compared to other well-characterized viral polymerases. By mutating an amino acid residue at the beginning of the protein we show how the chemical linkage to the viral genome can be disrupted. This provides an elegant mechanism for the attachment of the viral genome to the polymerase observed in vivo.
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DOI：
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发表时间：
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影响因子：
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