{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T18:03:54Z","timestamp":1788804234694,"version":"build-2803163510"},"posted":{"date-parts":[[2026,1,27]]},"group-title":"Microbiology","reference-count":51,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,1,27]],"date-time":"2026-01-27T00:00:00Z","timestamp":1769472000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"NIH National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["R01AI141549"],"award-info":[{"award-number":["R01AI141549"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"NIH National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["R01 AI152579"],"award-info":[{"award-number":["R01 AI152579"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"NIH National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["T32AI170496"],"award-info":[{"award-number":["T32AI170496"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000038","name":"Natural Science and Engineering Research Council of Canada","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"crossref"}]},{"name":"MJ Murdock Foundation"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,1,27]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>The SARS-CoV-2 nucleocapsid (N) protein is essential for viral RNA packaging, replication, and immune modulation. Despite its central role, the mechanistic contributions of its individual domains, the N-terminal domain (NTD), C-terminal domain (CTD), and the intrinsically flexible linker (LINK), remain poorly defined, largely due to the protein\u2019s structural complexity. In this study, we developed a panel of twelve alpaca-derived nanobodies (VHHs) targeting the NTD, CTD, and LINK regions of N. Using ELISA and biolayer interferometry, we characterized their binding affinities, and we mapped their epitopes via hydrogen-deuterium exchange-mass spectrometry (HDX-MS) and structural modeling. When expressed intracellularly, these VHHs inhibited SARS-CoV-2 infection. In vitro, they disrupted phase separation of the N protein, a critical step in viral replication. Strikingly, VHHs targeting each domain independently blocked both phase condensation and viral replication, underscoring the functional importance of all three regions. These findings establish domain-specific VHHs as versatile tools for dissecting N biology, with promising therapeutic potential.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>SARS-CoV-2 and emerging coronaviruses remain a major global health threat, yet critical gaps persist in our understanding of their molecular pathogenesis. The nucleocapsid (N) protein, the most abundantly expressed SARS-CoV-2 antigen, plays essential roles beyond genome packaging, including immune evasion and intracellular organization. Here, we generate and characterize a panel of domain-specific nanobodies (VHHs) that enable precise dissection of N\u2019s functional architecture. Using integrated biochemical, structural, and virological approaches, we uncover distinct mechanisms of viral inhibition, including disruption of phase condensation through a conserved linker region. These findings address long-standing knowledge gaps about a multifunctional viral protein and establish VHHs as powerful, modular tools for probing coronavirus biology, with broad potential for therapeutic, diagnostic, and cell biology applications.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.01.26.701894","type":"posted-content","created":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T02:35:18Z","timestamp":1769567718000},"source":"Crossref","is-referenced-by-count":1,"title":["Mapping SARS-CoV-2 Nucleocapsid Function with Nanobodies"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2983-3386","authenticated-orcid":false,"given":"Jules B.","family":"Reyes-Weinstein","sequence":"first","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, 97239, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2533-7668","authenticated-orcid":false,"given":"Timothy A.","family":"Bates","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, 97239, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-3875-1162","authenticated-orcid":false,"given":"Aidan","family":"Anastas","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, 97239, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayna","family":"Alfahdli","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, 97239, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-4934-3811","authenticated-orcid":false,"given":"Mila","family":"Trank-Green","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, 97239, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8585-8038","authenticated-orcid":false,"given":"Savannah K.","family":"McBride","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, 97239, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle","family":"Garcia","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, 97239, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew A. H.","family":"Parson","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meredith L.","family":"Jenkins","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7904-9859","authenticated-orcid":false,"given":"John E.","family":"Burke","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada"},{"name":"Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada"},{"name":"University of Victoria Genome BC Proteomics Centre, Victoria, BC, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4944-6671","authenticated-orcid":false,"given":"Eric","family":"Barklis","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, 97239, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8575-4164","authenticated-orcid":false,"given":"Fikadu G.","family":"Tafesse","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, 97239, USA"},{"name":"Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, OR, 97239, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026020504364107000_2026.01.26.701894v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/0378-1135(95)92531-F"},{"key":"2026020504364107000_2026.01.26.701894v1.2","doi-asserted-by":"publisher","DOI":"10.1126\/science.302.5653.2045"},{"key":"2026020504364107000_2026.01.26.701894v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(20)30183-5"},{"key":"2026020504364107000_2026.01.26.701894v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.it.2020.04.008"},{"key":"2026020504364107000_2026.01.26.701894v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2021.03.036"},{"key":"2026020504364107000_2026.01.26.701894v1.6","doi-asserted-by":"publisher","DOI":"10.1002\/jmv.28212"},{"key":"2026020504364107000_2026.01.26.701894v1.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.antiviral.2013.12.009"},{"key":"2026020504364107000_2026.01.26.701894v1.8","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1186\/s12985-023-01968-6","article-title":"The SARS-CoV-2 nucleocapsid protein: its role in the viral life cycle, structure and functions, and use as a potential target in the development of vaccines and diagnostics","volume":"20","year":"2023","journal-title":"Virology Journal"},{"key":"2026020504364107000_2026.01.26.701894v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2003.12.195"},{"key":"2026020504364107000_2026.01.26.701894v1.10","doi-asserted-by":"publisher","DOI":"10.1126\/science.abd3629"},{"key":"2026020504364107000_2026.01.26.701894v1.11","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2022.101677"},{"key":"2026020504364107000_2026.01.26.701894v1.12","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-21953-3"},{"key":"2026020504364107000_2026.01.26.701894v1.13","doi-asserted-by":"publisher","DOI":"10.1111\/febs.16239"},{"key":"2026020504364107000_2026.01.26.701894v1.14","first-page":"2832","article-title":"Structural Basis for SARS-CoV-2 Nucleocapsid Protein Recognition by Single-Domain Antibodies","volume":"12","year":"2021","journal-title":"Frontiers in Immunology"},{"key":"2026020504364107000_2026.01.26.701894v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2019.09.115"},{"key":"2026020504364107000_2026.01.26.701894v1.16","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2004.03.154"},{"key":"2026020504364107000_2026.01.26.701894v1.17","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.adg6473"},{"key":"2026020504364107000_2026.01.26.701894v1.18","unstructured":"Syed, A. 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