Brunel Joanna, Chopy Damien, Dosnon Marion, Bloyet Louis-Marie, Devaux Patricia, Urzua Erica, Cattaneo Roberto, Longhi Sonia, Gerlier Denis
Centre International de Recherche en Infectiologie, INSERM, U1111, CNRS, UMR5308, Université Lyon 1, ENS Lyon, CERVI, Lyon, France.
Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques UMR 7257, Marseille, France.
J Virol. 2014 Sep;88(18):10851-63. doi: 10.1128/JVI.00664-14. Epub 2014 Jul 9.
The genome of nonsegmented negative-strand RNA viruses is tightly embedded within a nucleocapsid made of a nucleoprotein (N) homopolymer. To ensure processive RNA synthesis, the viral polymerase L in complex with its cofactor phosphoprotein (P) binds the nucleocapsid that constitutes the functional template. Measles virus P and N interact through two binding sites. While binding of the P amino terminus with the core of N (NCORE) prevents illegitimate encapsidation of cellular RNA, the interaction between their C-terminal domains, P(XD) and N(TAIL) is required for viral RNA synthesis. To investigate the binding dynamics between the two latter domains, the P(XD) F497 residue that makes multiple hydrophobic intramolecular interactions was mutated. Using a quantitative mammalian protein complementation assay and recombinant viruses, we found that an increase in P(XD)-to-N(TAIL) binding strength is associated with a slower transcript accumulation rate and that abolishing the interaction renders the polymerase nonfunctional. The use of a newly developed system allowing conditional expression of wild-type or mutated P genes, revealed that the loss of the P(XD)-N(TAIL) interaction results in reduced transcription by preformed transcriptases, suggesting reduced engagement on the genomic template. These intracellular data indicate that the viral polymerase entry into and progression along its genomic template relies on a protein-protein interaction that serves as a tightly controlled dynamic anchor.
Mononegavirales have a unique machinery to replicate RNA. Processivity of their polymerase is only achieved when the genome template is entirely embedded into a helical homopolymer of nucleoproteins that constitutes the nucleocapsid. The polymerase binds to the nucleocapsid template through the phosphoprotein. How the polymerase complex enters and travels along the nucleocapsid template to ensure uninterrupted synthesis of up to ∼ 6,700-nucleotide messenger RNAs from six to ten consecutive genes is unknown. Using a quantitative protein complementation assay and a biGene-biSilencing system allowing conditional expression of two P genes copies, the role of the P-to-N interaction in polymerase function was further characterized. We report here a dynamic protein anchoring mechanism that differs from all other known polymerases that rely only onto a sustained and direct binding to their nucleic acid template.
非节段性负链RNA病毒的基因组紧密包裹在由核蛋白（N）同聚物构成的核衣壳内。为确保连续的RNA合成，病毒聚合酶L与其辅因子磷蛋白（P）形成的复合物会结合构成功能模板的核衣壳。麻疹病毒的P和N通过两个结合位点相互作用。P的氨基末端与N的核心（NCORE）结合可防止细胞RNA的异常包装，而它们的C末端结构域P（XD）和N（TAIL）之间的相互作用是病毒RNA合成所必需的。为了研究后两个结构域之间的结合动力学，对形成多个疏水分子内相互作用的P（XD）的F497残基进行了突变。使用定量哺乳动物蛋白质互补分析和重组病毒，我们发现P（XD）与N（TAIL）结合强度的增加与转录本积累速率减慢有关，而消除这种相互作用会使聚合酶失去功能。使用新开发的允许条件性表达野生型或突变型P基因的系统，发现P（XD）-N（TAIL）相互作用的丧失会导致预先形成的转录酶的转录减少，这表明其与基因组模板的结合减少。这些细胞内数据表明，病毒聚合酶进入并沿着其基因组模板进行的过程依赖于一种蛋白质-蛋白质相互作用，这种相互作用充当了严格控制的动态锚定。
单股负链RNA病毒具有独特的RNA复制机制。只有当基因组模板完全嵌入构成核衣壳的核蛋白螺旋同聚物中时，其聚合酶的连续性才能实现。聚合酶通过磷蛋白与核衣壳模板结合。聚合酶复合物如何进入并沿着核衣壳模板移动，以确保从六个到十个连续基因不间断地合成长达约6700个核苷酸的信使RNA尚不清楚。使用定量蛋白质互补分析和允许条件性表达两个P基因拷贝的双基因-双沉默系统，进一步表征了P与N相互作用在聚合酶功能中的作用。我们在此报告了一种动态蛋白质锚定机制，该机制不同于所有其他已知的仅依赖于与其核酸模板持续直接结合的聚合酶。
