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We screened the proteins of SARS-CoV-2 for ubiquitination and identified the RNA-dependent RNA polymerase (RdRp) NSP12 as being ubiquitinated, which has not previously been reported. Importantly, we confirmed that NSP12 is ubiquitinated during SARS-CoV-2 infection, and the NSP12 proteins of different coronaviruses are differentially associated with ubiquitin chains. SARS-CoV-2 NSP12 was primarily associated with K63-linked polyubiquitin chains, which increased SARS-CoV-2 NSP12 stability relative to OC43 NSP12, which was associated with K48 chains. Additionally, we identify the atypical E3 ubiquitin ligase MYCBP2 as a mediator of serine ubiquitination of SARS-CoV-2 NSP12 on serine-564 and observe that MYCBP2 promotes SARS-CoV-2 replication in cell-based assays. Substitution of this single residue (S564A) in CoV-2 NSP12 prevented recovery of infectious virus in three independent attempts, precluding direct genetic validation of its role in viral replication. To our knowledge, these findings provide the first evidence of serine ubiquitination of a viral protein and suggest a previously unrecognized role for RdRp ubiquitination in coronavirus biology.<\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Viruses often hijack host post-translational modification systems, including ubiquitination, to enhance the functions of their proteins and evade host innate immune responses. We screened the viral proteins of SARS-CoV-2 and found that NSP2, NSP5, NSP12, NSP15, and NSP16 are ubiquitinated. Focusing on NSP12, the RNA-dependent RNA polymerase (RdRp), we found that it is associated with multiple types of ubiquitin modifications, and RdRp proteins from other coronaviruses are differentially ubiquitinated. We also found that SARS-CoV-2 RdRp undergoes ubiquitination on a serine, rather than a canonical lysine residue, mediated by the atypical E3 ubiquitin ligase, MYCBP2. Consistent with this, MYCBP2 promotes efficient SARS-CoV-2 replication in cell-based assays. To our knowledge, this study provides the first evidence of non-lysine ubiquitination of a viral protein and demonstrates the serine selectivity of MYCBP2 for the first time in a cellular context. These findings raise important questions about the regulatory function of serine NSP12 ubiquitination in coronavirus replication and its potential contribution to viral pathogenicity.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/msphere.00373-26","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T13:01:25Z","timestamp":1788267685000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Serine ubiquitination of the SARS-CoV-2 RdRp by the non-canonical E3 ligase MYCBP2"],"prefix":"10.1128","volume":"11","author":[{"given":"RuthMabel","family":"Boytz","sequence":"first","affiliation":[{"name":"Department of Microbial Pathogenesis, Yale School of Medicine","place":["New Haven, 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