Tudhope Ericka, Donnelly Camilla M, Sethi Ashish, David Cassandra, Williamson Nicholas, Stewart Murray, Forwood Jade K, Gooley Paul R, Moseley Gregory W
Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.
Gulbali Institute, Charles Sturt University, Wagga Wagga, NSW 2678, Australia.
Viruses. 2025 Aug 1;17(8):1075. doi: 10.3390/v17081075.
The rabies virus (RABV) phosphoprotein (P protein) has multiple functions, including acting as the essential non-catalytic cofactor of the viral polymerase (L protein) for genome replication and transcription; the principal viral antagonist of the interferon (IFN)-mediated innate immune response; and the chaperone for the viral nucleoprotein (N protein). Although P protein is known to undergo phosphorylation by cellular kinases, the location and functions of the phosphorylation sites remains poorly defined. Here, we report the identification by mass-spectrometry (MS) of residues of P protein that are modified by phosphorylation in mammalian cells, including several novel sites. Analysis of P protein with phospho-mimetic and phospho-inhibitory mutations of three novel residues/clusters that were commonly identified by MS (Ser48, Ser183/187, Ser217/219/220) indicate that phosphorylation at each of these sites does not have a major influence on nuclear trafficking or antagonistic functions toward IFN signalling pathways. However, phosphorylation of Ser48 in the N-terminus of P protein impaired function in transcription/replication and in the formation of replication structures that contain complexes of P and N proteins, suggestive of altered interactions of these proteins. The crystal structure of P protein containing the S48E phospho-mimetic mutation indicates that Ser48 phosphorylation facilitates the binding of residues 41-52 of P protein into the RNA-binding groove of non-RNA-bound N protein (N), primarily through the formation of a salt bridge with Arg434 of N protein. These data indicate that Ser48 modification regulates the cycling of P-N chaperone complexes that deliver N protein to RNA to enable transcription/replication, such that enhanced interaction due to S48E phospho-mimetic mutation reduces N protein delivery to the RNA, inhibiting subsequent transcription/replication processes. These data are, to our knowledge, the first to implicate phosphorylation of RABV P protein in conserved replication functions of the P gene.
狂犬病病毒（RABV）磷蛋白（P蛋白）具有多种功能，包括作为病毒聚合酶（L蛋白）进行基因组复制和转录所必需的非催化辅助因子；干扰素（IFN）介导的固有免疫反应的主要病毒拮抗剂；以及病毒核蛋白（N蛋白）的伴侣蛋白。尽管已知P蛋白会被细胞激酶磷酸化，但其磷酸化位点的位置和功能仍不清楚。在此，我们报告通过质谱（MS）鉴定了哺乳动物细胞中被磷酸化修饰的P蛋白残基，包括几个新位点。对通过MS共同鉴定出的三个新残基/簇（Ser48、Ser183/187、Ser217/219/220）进行磷酸模拟和磷酸抑制突变的P蛋白分析表明，这些位点的磷酸化对核转运或对IFN信号通路的拮抗功能没有重大影响。然而，P蛋白N端的Ser48磷酸化损害了转录/复制功能以及包含P和N蛋白复合物的复制结构的形成，提示这些蛋白之间的相互作用发生了改变。含有S48E磷酸模拟突变的P蛋白晶体结构表明，Ser48磷酸化主要通过与N蛋白的Arg434形成盐桥，促进P蛋白41 - 52位残基与非RNA结合的N蛋白（N）的RNA结合槽结合。这些数据表明，Ser48修饰调节了将N蛋白递送至RNA以实现转录/复制的P - N伴侣复合物的循环，使得由于S48E磷酸模拟突变导致的增强相互作用减少了N蛋白向RNA的递送，从而抑制了随后的转录/复制过程。据我们所知，这些数据首次表明RABV P蛋白的磷酸化与P基因的保守复制功能有关。
