Phosphopeptide enrichment using Phos-tag technology reveals functional phosphorylation of the nucleocapsid protein of SARS-CoV-2

• Published in: Journal of proteomics 2022-03, Vol.255, p.104501-104501, Article 104501
• Main Authors: Ino, Yoko, Nishi, Mayuko, Yamaoka, Yutaro, Miyakawa, Kei, Jeremiah, Sundararaj Stanleyraj, Osada, Makoto, Kimura, Yayoi, Ryo, Akihide
• Format: Article
• Language: English
• Subjects: cell culture; fetal bovine serum; host-pathogen relationships; magnetism; nucleocapsid proteins; Phos-tag; phosphopeptides; Phosphorylation; phylogeny; Pin1; protein phosphorylation; proteomics; SARS-CoV-2; sequence alignment; serine; Severe acute respiratory syndrome coronavirus 2; ultracentrifugation; virus replication
• ISSN: 1874-3919; 1876-7737
• DOI: 10.1016/j.jprot.2022.104501

Phosphorylation of viral proteins serves as a regulatory mechanism during the intracellular life cycle of infected viruses. There is therefore a pressing need to develop a method to efficiently purify and enrich phosphopeptides derived from viral particles in biological samples. In this study, we utilized Phos-tag technology to analyze the functional phosphorylation of the nucleocapsid protein (N protein; NP) of severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral particles were collected from culture supernatants of SARS-CoV-2-infected VeroE6/TMPRSS2 cells by ultracentrifugation, and phosphopeptides were purified by Phos-tag magnetic beads for LC-MS/MS analysis. Analysis revealed that NP was reproducibly phosphorylated at serine 79 (Ser79). Multiple sequence alignment and phylogenetic analysis showed that the Ser79 was a distinct phospho-acceptor site in SARS-CoV-2 but not in other beta-coronaviruses. We also found that the prolyl-isomerase Pin1 bound to the phosphorylated Ser79 in NP and positively regulated the production of viral particles. These results suggest that SARS-CoV-2 may have acquired the potent virus-host interaction during its evolution mediated by viral protein phosphorylation. Moreover, Phos-tag technology can provide a useful means for analyzing the functional phosphorylation of viral proteins. In this study, we aimed to investigate the functional phosphorylation of SARS-CoV-2 NP. For this purpose, we used Phos-tag technology to purify and enrich virus-derived phosphopeptides with high selectivity and reproducibility. This method can be particularly useful in analyzing viral phosphopeptides from cell culture supernatants that often contain high concentrations of fetal bovine serum and supplements. We newly identified an NP phosphorylation site at Ser79, which is important for Pin1 binding. Furthermore, we showed that the interaction between Pin1 and phosphorylated NP could enhance viral replication in a cell culture model. [Display omitted] •We utilize Phos-tag technology to enrich phosphopeptides from SARS-CoV-2 virions in cell culture supernatants.•LC-MS/MS analysis identifies a putative phosphorylation site (Ser79) in viral nucleocapsid protein.•The host factor Pin1 binds to the phosphorylated Ser79 in NP to positively regulates viral particle production.•Phos-tag technology enables identification of phosphorylated viral proteins for further functional analysis.