{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T17:01:02Z","timestamp":1782493262594,"version":"3.54.5"},"reference-count":56,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,11,23]],"date-time":"2022-11-23T00:00:00Z","timestamp":1669161600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell Dev. Biol."],"abstract":"<jats:p>The ongoing SARS-CoV-2\/COVID-19 pandemic caused a global public health crisis. Yet, everyone\u2019s response to SARS-CoV-2 infection varies, and different viral variants confer diverse pathogenicity. Thus, it is imperative to understand how viral determinants contribute to COVID-19. Viral ORF3a protein is one of those viral determinants, as its functions are linked to induction of cell and tissues damages, disease severity and cytokine storm that is a major cause of COVID-19-related death. ORF3a is a membrane-associated protein. Upon synthesis, it is transported from endoplasmic reticulum, Golgi apparatus to plasma membrane and subcellular endomembranes including endosomes and lysosomes. However, how ORF3a is transported intracellularly remains elusive. The goal of this study was to carry out a systematic mutagenesis study to determine the structural relationship of ORF3a protein with its subcellular locations. Single amino acid (aa) and deletion mutations were generated in the putative function-relevant motifs and other regions of interest. Immunofluorescence and ImageJ analyses were used to determine and quantitate subcellular locations of ORF3a mutants in comparison with wildtype ORF3a. The wildtype ORF3a localizes predominantly (Pearson\u2019s coefficients about 0.8) on the membranes of endosomes and lysosomes. Consistent with earlier findings, deletion of the YXX\u03a6 motif, which is required for protein export, retained ORF3a in the Golgi apparatus. Interestingly, mutations in a double glycine (diG) region (aa 187\u2013188) displayed a similar phenotype to the YXX\u03a6 deletion, implicating a similar role of the diG motif in intracellular transport. Indeed, interrupting any one of the two glycine residues such as deletion of a single (dG188), both (dG187\/dG188) or substitution (G188Y) of these residues led to ORF3a retention in the Golgi apparatus (Pearson\u2019s coefficients \u22650.8). Structural analyses further suggest that the diG motif supports a type-II \u03b2-turn between the anti-parallel \u03b24 and \u03b25 sheets and connects to the YXX\u03a6 motif <jats:italic>via<\/jats:italic> hydrogen bonds between two monomers. The diG- YXX\u03a6 interaction forms a hand-in-hand configuration that could facilitate dimerization. Together, these observations suggest a functional role of the diG motif in intracellular transport of ORF3a.<\/jats:p>","DOI":"10.3389\/fcell.2022.1011221","type":"journal-article","created":{"date-parts":[[2022,11,23]],"date-time":"2022-11-23T12:01:32Z","timestamp":1669204892000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":17,"title":["A novel diG motif in ORF3a protein of SARS-Cov-2 for intracellular transport"],"prefix":"10.3389","volume":"10","author":[{"given":"Ruth","family":"Cruz-Cosme","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiantao","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dongxiao","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vidhyanand","family":"Mahase","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bhargava Teja","family":"Sallapalli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peixi","family":"Chang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanjin","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaolei","family":"Teng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard Y.","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiyi","family":"Tang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2022,11,23]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"005244","DOI":"10.1128\/mBio.00524-13","article-title":"Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles","volume":"4","author":"Angelini","year":"2013","journal-title":"mBio"},{"key":"B2","doi-asserted-by":"publisher","first-page":"492198","DOI":"10.1101\/2022.05.17.492198","article-title":"SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to downregulate MHC-I surface expression","volume":"2022","author":"Arshad","year":"2022","journal-title":"bioRxiv"},{"key":"B3","doi-asserted-by":"publisher","first-page":"e42305","DOI":"10.7554\/eLife.42305","article-title":"Insights into AMS\/PCAT transporters from biochemical and structural characterization of a double Glycine motif protease","volume":"8","author":"Bobeica","year":"2019","journal-title":"Elife"},{"key":"B4","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1111\/j.1365-2818.2006.01706.x","article-title":"A guided tour into subcellular colocalization analysis in light microscopy","volume":"224","author":"Bolte","year":"2006","journal-title":"J. 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