{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T02:57:34Z","timestamp":1790477854142,"version":"4.1.0"},"posted":{"date-parts":[[2026,9,26]]},"group-title":"Microbiology","reference-count":0,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T00:00:00Z","timestamp":1790380800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"National Institutes of Health","award":["AI087846"],"award-info":[{"award-number":["AI087846"]}]},{"name":"National Institutes of Health","award":["AI169444"],"award-info":[{"award-number":["AI169444"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,26]]},"abstract":"<jats:p>The coronavirus nucleocapsid (N) protein is indispensable for the viral lifecycle as part of the viral Replication-Transcription Complex together with Nsp3.  Recent research demonstrated that phosphorylation of N by several host kinases intricately regulates its functions during infection. However, the mechanisms that control N dephosphorylation, and its physiological consequence, remain poorly understood. Here, we show that SARS-CoV-2 Nsp3 is a key orchestrator of N dephosphorylation by recruiting the phosphatase PP1\u03b1\/\u03b3 via a conserved PP1-binding motif in the Ubl1 domain. Disruption of this motif abolishes N dephosphorylation and selectively impairs subgenomic RNA synthesis. Comparative interactome proteomics analysis of phospho-mimic vs. phospho-deficient N, together with functional validation, revealed that the host splicing factor SRSF1 cooperates with unphosphorylated N to promote subgenomic RNA transcription. These findings uncover an unrecognized mechanism in which Nsp3-guided N dephosphorylation by PP1\u03b1\/\u03b3 enables coronavirus subgenomic RNA replication and highlight a potentially targetable axis for antiviral intervention.<\/jats:p>","DOI":"10.64898\/2026.09.25.754410","type":"posted-content","created":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T02:05:14Z","timestamp":1790474714000},"source":"Crossref","is-referenced-by-count":0,"title":["Nsp3 Ubl1-orchestrated dephosphorylation of N protein promotes coronaviral subgenomic RNA synthesis"],"prefix":"10.64898","author":[{"given":"Junji","family":"Zhu","sequence":"first","affiliation":[{"name":"Cleveland Clinic Florida Research and innovation center;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cindy","family":"Chiang","sequence":"additional","affiliation":[{"name":"Cleveland Clinic Florida Research and Innovation Center"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michaela","family":"Gack","sequence":"additional","affiliation":[{"name":"Cleveland Clinic Florida Research and Innovation Center"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.09.25.754410","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T02:05:14Z","timestamp":1790474714000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.09.25.754410"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,26]]},"references-count":0,"URL":"https:\/\/doi.org\/10.64898\/2026.09.25.754410","relation":{},"subject":[],"published":{"date-parts":[[2026,9,26]]},"subtype":"preprint"}}