{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,17]],"date-time":"2026-05-17T12:37:44Z","timestamp":1779021464835,"version":"3.51.4"},"reference-count":105,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T00:00:00Z","timestamp":1768262400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01 AI153216"],"award-info":[{"award-number":["R01 AI153216"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["T32 GM144293"],"award-info":[{"award-number":["T32 GM144293"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["P41 GM128577"],"award-info":[{"award-number":["P41 GM128577"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["RM1 GM149374"],"award-info":[{"award-number":["RM1 GM149374"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"name":"National Science Foundation","award":["MCB-1817712"],"award-info":[{"award-number":["MCB-1817712"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The SARS-CoV-2 nucleocapsid protein (Np) is essential for viral RNA replication and genomic RNA packaging. Phosphorylation of Np within its central Ser-Arg-rich (SRR) linker is proposed to modulate these functions. To gain mechanistic insights into these distinct roles, we performed in vitro biophysical and biochemical studies using recombinantly expressed ancestral Np and phosphomimetic SRR variants. Limited-proteolysis showed minor cleavage differences between wild-type (WT) and phosphomimetic Np, but no major structure or stability changes in the N- and C-terminal domains were observed by circular dichroism spectroscopy and differential scanning fluorimetry, respectively. Mass photometry (MP) revealed that WT Np dimerized more readily than phosphomimetic variants. Crosslinking-MP showed that WT Np formed discrete complexes on viral 5\u2032 UTR stem-loop (SL) 5 RNA, whereas phosphomimetic Np assembled preferentially on SL1\u20134. WT Np bound non-specifically to all RNAs tested primarily via hydrophobic interactions, whereas phosphomimetic Np showed selectivity for SARS-CoV-2-derived RNAs despite binding more electrostatically. A major difference was observed in the binding kinetics; WT Np compacted and irreversibly bound single-stranded DNA, whereas phosphomimetic Np displayed reduced compaction and fast on\/off binding kinetics. These mechanistic insights support a model where phosphorylated Np functions in RNA replication and chaperoning, while non-phosphorylated Np facilitates genomic RNA packaging. The findings also help to explain infectivity differences and clinical outcomes associated with SRR linker variants.<\/jats:p>","DOI":"10.3390\/v18010105","type":"journal-article","created":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T10:13:51Z","timestamp":1768299231000},"page":"105","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Mechanism of SARS-CoV-2 Nucleocapsid Protein Phosphorylation-Induced Functional Switch"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6424-5371","authenticated-orcid":false,"given":"Megan S.","family":"Sullivan","sequence":"first","affiliation":[{"name":"Center for Retroviral Research, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"},{"name":"Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8561-1833","authenticated-orcid":false,"given":"Michael","family":"Morse","sequence":"additional","affiliation":[{"name":"Department of Physics, Northeastern University, Boston, MA 02115, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kaylee","family":"Grabarkewitz","sequence":"additional","affiliation":[{"name":"Center for Retroviral Research, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"},{"name":"Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dina","family":"Bayachou","sequence":"additional","affiliation":[{"name":"Center for Retroviral Research, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"},{"name":"Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4651-5264","authenticated-orcid":false,"given":"Ioulia","family":"Rouzina","sequence":"additional","affiliation":[{"name":"Center for Retroviral Research, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"},{"name":"Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"},{"name":"Department of Physics, Northeastern University, Boston, MA 02115, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vicki","family":"Wysocki","sequence":"additional","affiliation":[{"name":"Center for Retroviral Research, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"},{"name":"Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"},{"name":"School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3219-376X","authenticated-orcid":false,"given":"Mark C.","family":"Williams","sequence":"additional","affiliation":[{"name":"Department of Physics, Northeastern University, Boston, MA 02115, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0354-4172","authenticated-orcid":false,"given":"Karin","family":"Musier-Forsyth","sequence":"additional","affiliation":[{"name":"Center for Retroviral Research, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"},{"name":"Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.virusres.2004.05.002","article-title":"Characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus","volume":"105","author":"He","year":"2004","journal-title":"Virus Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"11827","DOI":"10.1021\/bi0609319","article-title":"Carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: Self-association analysis and nucleic acid binding characterization","volume":"45","author":"Luo","year":"2006","journal-title":"Biochemistry"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1007\/s11373-005-9035-9","article-title":"Modular organization of SARS coronavirus nucleocapsid protein","volume":"13","author":"Chang","year":"2006","journal-title":"J. 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