Phosphorylation-Related Crosstalk Between Distant Regions of the Core Region of the Coat Protein Contributes to Virion Assembly of Plum Pox Virus

Abstract

Eukaryotic proteins are often targets of posttranslational modifications (PTMs). Capsid protein (CP) of plum pox virus (PPV), a member of genus Potyvirus, has been reported to be prone to phosphorylation in four serines at the N-terminal region. CP phosphorylation has been proposed to influence PPV infection by regulating CP accumulation in coordination with a second PTM, O-GlcNAcylation. In this study, a further proteomic characterization of PPV CP phosphorylation revealed additional phospho-targets, thus evidencing even greater complexity of the network of PTMs affecting this protein. In particular, two new phosphorylation targets, T254 and T313, at protein distal core, appear to be highly relevant for infection. Although abolishing phosphorylation at these positions does not have a severe effect on infectivity or viral accumulation, phospho-mimicking at either of these targets disrupts cell-to-cell movement. Strand-specific reverse transcription-quantitative PCR analysis and fractionation by centrifugation in a continuous sucrose gradient enabled us to conclude that such a deleterious effect is not related to failures in replication but is a consequence of inaccurate virion assembly. The analysis of spontaneous compensatory mutations at the CP core identified in a multiple phospho-mimicking mutant disclosed a functional dialogue between distant phospho-targets, which was further supported by an in silico PPV virion model, built on the watermelon mosaic virus atomic structure. Therefore, whereas joint and opposite action of O-GlcNAcylation and phosphorylation at the N-terminal disordered protrusion of CP appears to regulate protein stability, we propose that phosphorylations at the core region control assembly and disassembly of viral particles.

Common and Strain-Specific Post-Translational Modifications of the Potyvirus Plum pox virus Coat Protein in Different Hosts

“…Indeed, the CP of PPV displays both O-GlcNAcylations and phosphorylations, while that of PVA is only phosphorylated. Moreover, PPV and PVA CP phosphorylations have shown specific peculiarities [26,38,39]. In this work, we deepened into the characterization of PTMs affecting PPV CP, showing they are not exclusive to the protein assembled in virions and occur not only in experimental herbaceous plants but also in natural woody PPV hosts.…”

“…and Arabidopsis thaliana [37] and phosphorylation in Nicotiana spp. [38,39]. However, until now, it was unknown whether these modifications also affect PPV CP in natural PPV woody hosts.…”

“…Former results had shown that R-CP was targeted by phosphorylation in PPV-infected N. benthamiana at specific serines (S25, S81, S101, and S118) and, more sporadically, at threonines (T71, T106, T254, and T313) [38,39]. To determine whether CP phosphorylation is extensible to PPV natural hosts, PPV virions were purified from P. persica seedlings infected by agroinfiltration with pSN-PPV-5 BD-GFP and analyzed by LC-MS/MS after trypsin/Lys-C digestion and phospho-peptide enrichment.…”

“…CP from virions of the PPV R isolate, which belongs to strain D, has been shown to be extensively O-GlcNAcylated by the O-GlcNAc transferase (OGT) SECRET AGENT (SEC) along the N-terminal region of the protein [30,36,37]. PPV CP also displays a complex phosphorylation pattern, which spans both N terminus and the core region of the protein [38,39]. Neither O-GlcNAcylation nor phosphorylation of CP is essential for virus viability, but both appear to contribute to infection efficiency [30,37,38].…”

“…Neither O-GlcNAcylation nor phosphorylation of CP is essential for virus viability, but both appear to contribute to infection efficiency [30,37,38]. It has been proposed that dynamic phosphorylation and O-GlcNAcylation at N terminus of the protein regulate CP stability, thus providing appropriate CP amounts for each phase of viral infection, while phosphorylation at core region controls viral particle assembly/disassembly [39].…”