Department of Pathology, University of Virginia, 415 Lane Road, Rm. 3051, MR5 building, P.O. Box 800904, Charlottesville, VA 22908-0904, USA.
J Virol. 2010 Feb;84(4):1741-51. doi: 10.1128/JVI.01803-09. Epub 2009 Dec 9.
Vesicular stomatitis virus (VSV) genomic RNA encapsidated by the nucleocapsid (N) protein is the template for transcription and replication by the viral polymerase. We analyzed the 2.9-A structure of the VSV N protein bound to RNA (T. J. Green, X. Zhang, G. W. Wertz, and M. Luo, Science 313:357-360, 2006) and identified amino acid residues with the potential to interact with RNA via hydrogen bonds. The contributions of these interactions to N protein function were investigated by individually substituting the residues with alanine and assaying the effect of these mutations on N protein expression, on the ability of the N protein to interact with the phosphoprotein (P), and on its ability to encapsidate RNA and generate templates that can support transcription and RNA replication. These studies identified individual amino acids critical for N protein function. Nine nucleotides are associated with each N monomer and contorted into two quasi-helices within the N protein RNA binding cavity. We found that N protein residues that formed hydrogen bond contacts with the nucleotides in quasi-helix 2 were critical to the encapsidation of RNA and the production of templates that can support RNA synthesis. Individual hydrogen bond interactions between the N protein and the nucleotides of quasi-helix 1 were not essential for ribonucleoprotein (RNP) template function. Residue R143 forms a hydrogen bond with nucleotide 9, the nucleotide that extends between N monomers. R143A mutant N protein failed to encapsidate RNA and to support RNA synthesis and suppressed wild-type N protein function. These studies show a direct correlation between viral RNA synthesis and N protein residues structurally positioned to interact with RNA.
水疱性口炎病毒（VSV）基因组 RNA 被核衣壳（N）蛋白包裹，是病毒聚合酶进行转录和复制的模板。我们分析了 VSV N 蛋白与 RNA 结合的 2.9-A 结构（T. J. Green、X. Zhang、G. W. Wertz 和 M. Luo，Science 313:357-360，2006），并鉴定出了与 RNA 通过氢键相互作用的潜在氨基酸残基。通过单独用丙氨酸取代这些残基，并检测这些突变对 N 蛋白表达、N 蛋白与磷蛋白（P）相互作用的能力以及其包裹 RNA 的能力和产生支持转录和 RNA 复制的模板的能力的影响，研究了这些相互作用对 N 蛋白功能的贡献。这些研究确定了 N 蛋白功能的关键单个氨基酸。每个 N 单体都与 9 个核苷酸相关联，并在 N 蛋白 RNA 结合腔中扭曲成两个准螺旋。我们发现，与准螺旋 2 中的核苷酸形成氢键的 N 蛋白残基对 RNA 的包裹和产生支持 RNA 合成的模板至关重要。N 蛋白与准螺旋 1 的核苷酸之间的单个氢键相互作用对于核糖核蛋白（RNP）模板功能并非必需。残基 R143 与核苷酸 9 形成氢键，核苷酸 9 延伸在 N 单体之间。R143A 突变 N 蛋白无法包裹 RNA 并支持 RNA 合成，并抑制野生型 N 蛋白的功能。这些研究表明病毒 RNA 合成与结构上定位于与 RNA 相互作用的 N 蛋白残基之间存在直接相关性。
