{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T01:17:13Z","timestamp":1781659033232,"version":"3.54.5"},"reference-count":64,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2021,6,17]],"date-time":"2021-06-17T00:00:00Z","timestamp":1623888000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000053","name":"National Eye Institute","doi-asserted-by":"publisher","award":["Intramural research program"],"award-info":[{"award-number":["Intramural research program"]}],"id":[{"id":"10.13039\/100000053","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000092","name":"U.S. National Library of Medicine","doi-asserted-by":"publisher","award":["Intramural research program"],"award-info":[{"award-number":["Intramural research program"]}],"id":[{"id":"10.13039\/100000092","id-type":"DOI","asserted-by":"publisher"}]},{"name":"University of Maryland School Pharmacy Mass Spectrometry Center","award":["SOP1841-IQB2014"],"award-info":[{"award-number":["SOP1841-IQB2014"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681\u2013684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses\/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence \u201cwildtype\u201d spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.<\/jats:p>","DOI":"10.3390\/ijms22126490","type":"journal-article","created":{"date-parts":[[2021,6,17]],"date-time":"2021-06-17T11:20:26Z","timestamp":1623928826000},"page":"6490","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA"],"prefix":"10.3390","volume":"22","author":[{"given":"Olga A.","family":"Postnikova","sequence":"first","affiliation":[{"name":"Laboratory of Retinal Cell & Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3685-8602","authenticated-orcid":false,"given":"Sheetal","family":"Uppal","sequence":"additional","affiliation":[{"name":"Laboratory of Retinal Cell & Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weiliang","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, School of Pharmacy Mass Spectrometry Center, University of Maryland, Baltimore, MD 21201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maureen A.","family":"Kane","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, School of Pharmacy Mass Spectrometry Center, University of Maryland, Baltimore, MD 21201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8956-2979","authenticated-orcid":false,"given":"Rafael","family":"Villasmil","sequence":"additional","affiliation":[{"name":"Flow Cytometry Core Facility, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Igor B.","family":"Rogozin","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eugenia","family":"Poliakov","sequence":"additional","affiliation":[{"name":"Laboratory of Retinal Cell & Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1813-5291","authenticated-orcid":false,"given":"T. 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