![]() Despite its functional importance and emergence as a mutational hotspot, the N protein has not been widely studied because of the absence of simple and safe cell-based assays. However, most mutations in circulating variants occur outside of the S gene and are thus inaccessible by this approach ( 8).Īll SARS-CoV-2 variants of interest or concern defined by the World Health Organization contain at least one mutation with >50% penetrance within a seven–amino acid span (199 to 205) in the nucleocapsid (N) protein, which is required for replication, RNA binding, packaging, stabilization, and release ( 8). Current studies employ spike (S) protein–pseudotyped lentivirus systems for evaluation of S-mediated ACE2 receptor binding and cell entry ( 6, 7). ![]() Furthermore, technical challenges impede efforts to generate mutant infectious clones of SARS-CoV-2 ( 1– 5). Understanding the molecular determinants of enhanced infectivity is central to vaccine and therapeutic development, but research is hindered because SARS-CoV-2 can be studied only in a biosafety level 3 (BSL-3) laboratory. The COVID-19 pandemic is a leading cause of death globally, owing to the ongoing emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility. SC2-VLPs provide a platform for rapid testing of viral variants outside of a biosafety level 3 setting and demonstrate N mutations and particle assembly to be mechanisms that could explain the increased spread of variants, including B.1.617.2 (Delta, which contains the R203M mutation). In SC2-VLPs, four nucleocapsid (N) mutations found universally in more-transmissible variants independently increased messenger RNA delivery and expression ~10-fold, and in a reverse genetics model, the serine-202→arginine (S202R) and arginine-203→methionine (R203M) mutations each produced >50 times as much virus. In this study, we show that SARS-CoV-2 virus-like particles (SC2-VLPs) can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and at multiple steps in the viral life cycle. Doudna, Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Visualization, Writing - original draft, Writing - review & editingĮfforts to determine why new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants demonstrate improved fitness have been limited to analyzing mutations in the spike (S) protein with the use of S-pseudotyped particles. Melanie Ott, Methodology, Project administration, Resources, Supervision, Validation, Hayashi, Investigation, Resources, Writing - review & editing, ![]() Khalid, Conceptualization, Investigation, Methodology, Resources, Supervision, Validation, , † Takako Tabata, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review & editing, # Taha, Conceptualization, Investigation, Methodology, Resources, Validation, Visualization, Writing - original draft, Writing - review & editing, # Syed, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing, #
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