Abstract Background The nucleocapsid (N) protein of coronavirus harbors a conserved serine/arginine (SR)–rich motif whose phosphorylation by GSK-3 is essential for viral transcription and replication. Our previous studies in Severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and the JHM strain of mouse hepatitis virus revealed a phosphorylation-to-dephosphorylation transition during the viral life cycle, with newly synthesized N highly phosphorylated and virion-associated N hypophosphorylated. Here, we characterize this transition in SARS-CoV-2 and define its functional significance. Methods Utilizing high-resolution gel analysis and the phospho-specific antibody, the phosphorylation levels of SARS-CoV-2 N proteins in the virus-infected Calu-3 cells and secreted virions from culture supernatants were compared between different viral strains. This phosphorylation-to-dephosphorylation transition was also verified in the SARS-CoV-2 virus-like particle (SC2-VLP) platform expressing N, membrane (M), envelope, and spike proteins. In this VLP system, the substantial contribution of N dephosphorylation status to particle secretion, either by blocking GSK-3 activity or phospho-related mutants, was assessed. With different viral structural protein-expressing clones and phosphatase inhibitors, we characterized the key viral factor and host phosphatase to mediate the N dephosphorylation process. Furthermore, we evaluated the antiviral efficacy of phosphatase inhibitors using the virus-infected Calu-3 culture. Results Our results showed that the phosphorylation change of SR motif in N was observed across multiple strains and recapitulated in the SC2-VLP system. GSK-3 inhibition or a phospho-deficient N mutant, but not a phospho-mimetic one, enhanced VLP release, indicating that N dephosphorylation promotes virion secretion. Mechanistically, the M protein, via its C-terminal domain, interacts with phosphorylated N to recruit protein phosphatase 2A (PP2A) to the ERGIC, facilitating N dephosphorylation. Inhibition of PP2A, either by inhibitors or siRNA, impaired the M-induced N dephosphorylation, thereby suppressing viral assembly and progeny virion production. Blockade of PP2A also indirectly reduced N phosphorylation through the Akt-mediated inhibition of GSK-3, resulting in decreased genomic RNA synthesis. Conclusions Collectively, these findings establish N dephosphorylation as a critical regulatory step in SARS-CoV-2 assembly and virion release. In ERGIC, this M protein-mediated process recruits host PP2A to dephosphorylate N, thus supporting PP2A as a promising pan-coronavirus antiviral target. Graphical Abstract
Wang et al. (Fri,) studied this question.