Zhan Jingyu, Chakraborty Shatabdi, Sethi Ashish, Mok Yee-Foong, Yan Fei, Moseley Gregory W, Gooley Paul R
Department of Biochemistry and Pharmacology, University of Melbourne, 3010, Parkville, VIC, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 3010, Parkville, VIC, Australia.
Department of Biochemistry and Pharmacology, University of Melbourne, 3010, Parkville, VIC, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 3010, Parkville, VIC, Australia; Australian Nuclear Science Technology Organisation, The Australian Synchrotron, 800 Blackburn Rd, 3168, Clayton, VIC, Australia.
Antiviral Res. 2025 Feb;234:106075. doi: 10.1016/j.antiviral.2024.106075. Epub 2024 Dec 28.
The Phosphoprotein (P protein) of the rabies virus has multiple roles in virus replication. A critical function is to act as a cofactor in genome replication and mRNA production through binding via its N-terminal region to the L protein, the essential enzyme for mRNA and genome synthesis/processing, and via its C-terminal domain (P) to the N protein and viral RNA (N-RNA) ribonucleoprotein complex. The binding site of the P on the N protein is a disordered loop that is expected to be phosphorylated at Ser389. This interface may provide novel targets for antiviral approaches. Following an alanine scan of the peptide we selected two single site mutations that showed improved affinity and combined these mutations with a phosphomimetic (S389E) to produce double and triple mutants in the context of linear and cyclic peptides of the disordered loop, with the goal of generating a competitive peptide against the N-RNA complex. To assess the binding properties of the peptides we characterized their thermodynamics identifying complex properties of improved enthalpy but with compensating entropy for mutants and cyclized peptides. Nevertheless, a triple mutant shows 3.5-fold stronger affinity for P than the full-length S389E N protein. Structural characterization of the triple mutant suggests the improved affinity may be due to trapping a favoured β-strand structure for binding to the P. This novel peptide may serve as a template for the future design of antivirals.
狂犬病病毒的磷蛋白（P蛋白）在病毒复制中具有多种作用。其关键功能是作为一种辅助因子参与基因组复制和mRNA生成，通过其N端区域与L蛋白（mRNA和基因组合成/加工的必需酶）结合，以及通过其C端结构域（P）与N蛋白和病毒RNA（N-RNA）核糖核蛋白复合物结合。P蛋白在N蛋白上的结合位点是一个无序环，预计在Ser389处会发生磷酸化。该界面可能为抗病毒方法提供新的靶点。在对该肽进行丙氨酸扫描后，我们选择了两个显示出亲和力提高的单点突变，并将这些突变与一个拟磷酸化突变（S389E）相结合，在无序环的线性和环状肽背景下产生双突变体和三突变体，目的是生成一种与N-RNA复合物竞争的肽。为了评估这些肽的结合特性，我们对它们的热力学进行了表征，确定了突变体和环化肽具有改善的焓但伴有补偿熵的复杂特性。然而，一个三突变体对P的亲和力比全长S389E N蛋白强3.5倍。三突变体的结构表征表明，亲和力的提高可能是由于捕获了一种有利于与P结合的β-链结构。这种新型肽可能为未来抗病毒药物的设计提供模板。
