Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength.
Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength.
复制标题
调节PXD在基因间区域对麻疹病毒转录的重新定位，以调节Ntail结合强度。
DOI：
10.1371/journal.ppat.1006058
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发表时间：
2016-12
期刊：
影响因子：
6.7
通讯作者：
Gerlier D
中科院分区：
文献类型：
--
作者：
Bloyet LM;Brunel J;Dosnon M;Hamon V;Erales J;Gruet A;Lazert C;Bignon C;Roche P;Longhi S;Gerlier D
麻疹病毒 (MeV) 和所有副粘病毒科成员依靠复杂的聚合酶机制来确保病毒转录和复制。它们的聚合酶将磷蛋白 (P) 和 L 蛋白结合起来，赋予所有必要的酶活性。为了持续进行，聚合酶使用由基因组 RNA 制成的核衣壳作为模板，该核衣壳完全包裹在核蛋白 (N) 的连续寡聚物中。聚合酶进入基因组 3' 端的核衣壳，此处是转录和复制的启动子所在。六个基因的转录顺序发生。这意味着在每个基因间区域 (IGR) 结束并重新启动 mRNA 合成。我们在此探讨了 P (XD) 的 X 结构域与 N 蛋白 (NTAIL) C 末端区域的结合在多大程度上参与了顺序转录过程中维持 P/L 复合物锚定到核衣壳模板上的过程。 NTAIL 上 XD 结合位点中引入的氨基酸取代导致了广泛的结合亲和力，这是通过结合大肠杆菌和人类细胞中的蛋白质互补测定以及等温滴定量热法来确定的。分子动力学模拟表明，XD 与 NTAIL 的结合涉及复杂的氢键网络，两个单独的氨基酸取代对该网络的破坏显着降低了结合亲和力。使用新设计的高灵敏度双荧光素酶报告基因小基因组测定，发现通过五种麻疹病毒 IGR 重新启动的效率与 NTAIL/XD KD 相关。相关地，还发现表达 N 变体的重组病毒的 P 转录物积累率和 F/N 转录物比率与 NTAIL 与 XD 的结合强度相关。总而言之，我们的数据支持 XD 与 NTAIL 结合在维持 P/L 复合物的正确锚定方面发挥着关键作用，从而确保每个基因间区域的转录重新启动。聚合酶 L、磷蛋白 P 和核蛋白 N 三种蛋白质相互作用，确保麻疹病毒（副粘病毒科成员）的转录和复制。规则的核蛋白阵列可保护病毒基因组 RNA。由此产生的核衣壳构成了由 L 和 P 组成的聚合酶复合物使用的 RNA 合成模板，后者确保 L 锚定在核衣壳上。我们在此报告了 P (XD) 的 C 端 X 结构域和 N (NTAIL) 本质上无序的 C 端尾部 (NTAIL) 的结合亲和力、在基因间区域重新启动转录的能力以及重组病毒的病毒转录物的积累率之间的相关性。因此，我们认为 NTAIL/XD 相互作用有助于维持聚合酶复合物锚定在核衣壳上，同时终止上游转录本并在每个基因间区域重新启动下游转录本。因此，必须控制 NTAIL/XD 相互作用强度，以便将病毒转录梯度保持在最佳效率窗口内。在副粘病毒科的许多成员中，病毒 P 和 N 蛋白之间这种相互作用模式的保守性反映了它们的聚合酶机制所受到的主要进化限制之一。
Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3’end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region. Three proteins, the polymerase L, the phosphoprotein P and the nucleoprotein N, interplay to ensure transcription and replication of measles virus, a member of the Paramyxoviridae family. A regular array of nucleoprotein shields the viral genomic RNA. The resulting nucleocapsid constitutes the template of RNA synthesis used by the polymerase complex made of L and P, with the latter ensuring L anchoring onto the nucleocapsid. We herein report a correlation between the binding affinity of the C-terminal X domain of P (XD) and the intrinsically disordered C-terminal tail of N (NTAIL), the ability to reinitiate the transcription at the intergenic regions and the accumulation rate of viral transcripts from recombinant viruses. We therefore propose that the NTAIL/XD interaction contributes to maintaining the polymerase complex anchored onto the nucleocapsid while ending the upstream transcript and re-initiating the downstream transcript at every intergenic region. As such, the NTAIL/XD interaction strength must be controlled so as to keep the viral transcription gradient within an optimal efficiency window. The conservation of this mode of interaction between the viral P and N proteins in many members of the Paramyxoviridae family reflects one of the major evolution constraints to which their polymerase machinery is subjected.