Nsp3 Ubl1-orchestrated dephosphorylation of N protein promotes coronaviral subgenomic RNA synthesis
- 1Cleveland Clinic Florida Research and Innovation Center
Abstract
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/{gamma} 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/{gamma} enables coronavirus subgenomic RNA replication and highlight a potentially targetable axis for antiviral intervention.
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BibTeX
@misc{zhu2026nsp3,
title = {{Nsp3 Ubl1-orchestrated dephosphorylation of N protein promotes coronaviral subgenomic RNA synthesis}},
author = {J. Zhu and C. Chiang and M. Gack},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.25.754410},
url = {https://www.biorxiv.org/content/10.64898/2026.09.25.754410v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Nsp3 Ubl1-orchestrated dephosphorylation of N protein promotes coronaviral subgenomic RNA synthesis
AU - J. Zhu
AU - C. Chiang
AU - M. Gack
PY - 2026
DA - 2026/09/25
PB - bioRxiv
DO - 10.64898/2026.09.25.754410
UR - https://www.biorxiv.org/content/10.64898/2026.09.25.754410v1
AB - 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/{gamma} 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/{gamma} enables coronavirus subgenomic RNA replication and highlight a potentially targetable axis for antiviral intervention.
ER -