{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T20:27:13Z","timestamp":1792787233939,"version":"4.5.0"},"reference-count":35,"publisher":"American Society for Microbiology","license":[{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2022YFC2303501, 2023YFA0915000"],"award-info":[{"award-number":["2022YFC2303501, 2023YFA0915000"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Guangzhou National Laboratory","award":["GZNL2024A01007"],"award-info":[{"award-number":["GZNL2024A01007"]}],"id":[{"id":"https:\/\/ror.org\/03ybmxt82","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Virol"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global health threat, and understanding the molecular events of its life cycle is critical for developing antiviral strategies. The nucleocapsid (N) protein is essential for RNA genome packaging and viral replication; however, the effects of N protein mutations on ribonucleocapsid disassembly or uncoating remain poorly understood. To address this, we developed dual-labeled fluorescent SARS-CoV-2 trans-complemented virus-like particles (DL-trVLPs) by inserting a tetracysteine (TC) tag at the C-terminus of the N protein. The DL-trVLPs enabled real-time visualization of viral RNA and N protein in living cells under BSL-2 conditions. Using single-virus tracking, we directly observed the uncoating events. By combining this approach with site-directed mutagenesis and molecular dynamics simulations, we found that the R203K\/G204R double mutation present in multiple viral variants significantly accelerated uncoating and enhanced intracellular viral RNA accumulation and viral replication. These findings establish a mechanistic link between N protein mutations and uncoating efficiency and provide insights into variant-specific uncoating dynamics. This study not only presents a versatile visual platform for investigating SARS-CoV-2 infection but also identifies a key N protein mutational hotspot that could serve as a target for antiviral intervention.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose persistent global public health challenges. Understanding variant-specific viral uncoating mechanisms, a rate-limiting infection step, is critical for antiviral development. The nucleocapsid (N) protein is essential for viral RNA packaging and replication, but how its mutations regulate ribonucleocapsid disassembly remains unclear, with technical barriers hindering real-time biosafe visualization. We developed a versatile dual-labeled fluorescent trans-complemented virus-like particle (DL-trVLP) system enabling single-virus tracking of viral RNA and N protein dynamics under BSL-2 conditions. Our findings show that the circulating R203K\/G204R mutation accelerates uncoating and enhances viral RNA accumulation and replication, uncovering a mutation-pathogenicity link. This work advances SARS-CoV-2 lifecycle understanding, provides a valuable research tool, and identifies a key antiviral target for combating variants.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.01086-26","type":"journal-article","created":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T12:53:56Z","timestamp":1791291236000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Real-time tracking of dual-labeled fluorescent SARS-CoV-2 reveals that the nucleocapsid mutations R203K\/G204R accelerate viral uncoating"],"prefix":"10.1128","author":[{"given":"Zizheng","family":"Liu","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/023b72294","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Life Science and Technology, Huazhong Agricultural University","place":["Wuhan, China"]},{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0788-557X","authenticated-orcid":true,"given":"Jibin","family":"Zhang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/023b72294","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Life Science and Technology, Huazhong Agricultural University","place":["Wuhan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6226-869X","authenticated-orcid":true,"given":"Qiang","family":"Ding","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03cve4549","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for Infection Biology, School of Medicine, Tsinghua University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenyang","family":"Yang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hangshen","family":"Wu","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shimin","family":"Li","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fujun","family":"Qin","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences","place":["Shenzhen, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1347-3168","authenticated-orcid":true,"given":"Xian-En","family":"Zhang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences","place":["Beijing, China"]},{"id":[{"id":"https:\/\/ror.org\/03hz5th67","id-type":"ROR","asserted-by":"publisher"}],"name":"Faculty of Synthetic Biology, Shenzhen University of Advanced Technology","place":["Shenzhen, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6225-7010","authenticated-orcid":false,"given":"Dian-Bing","family":"Wang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","published-online":{"date-parts":[[2026,10,6]]},"reference":[{"key":"e_1_3_4_2_2","unstructured":"WHO. 2025. 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