{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T04:16:39Z","timestamp":1791432999842,"version":"4.3.3"},"reference-count":68,"publisher":"American Society for Microbiology","issue":"11","license":[{"start":{"date-parts":[[2025,11,12]],"date-time":"2025-11-12T00:00:00Z","timestamp":1762905600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,11,12]],"date-time":"2025-11-12T00:00:00Z","timestamp":1762905600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01AI164698"],"award-info":[{"award-number":["R01AI164698"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2025,11,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      In herpesviruses, the fusogenic activity of the conserved glycoproteins gB and gH is regulated by their cytoplasmic (or intraviral) tails. However, prior to this work, their regulatory mechanisms had only been investigated in\n                      <jats:italic toggle=\"yes\">Alpha-<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Gamma-<\/jats:italic>\n                      but not in\n                      <jats:italic toggle=\"yes\">Betaherpesvirinae<\/jats:italic>\n                      . Here, we developed a plasmid-transfection-based split-luciferase assay as a quantitative platform for measuring cell-cell fusion mediated by glycoproteins from human cytomegalovirus (HCMV), a member of\n                      <jats:italic toggle=\"yes\">Betaherpesvirinae<\/jats:italic>\n                      . Using this assay, we confirmed that glycoproteins gB, gH, gL, and gO, along with the PDGFR\u03b1 receptor, are necessary and sufficient for efficient cell-cell fusion in two distinct HCMV strains, TR and AD169. To investigate the roles of the cytoplasmic tails of HCMV TR gB and gH in membrane fusion, we generated a series of truncation and point mutants analogous to those that have hyper- or hypo-fusogenic phenotypes in herpes simplex virus 1 (HSV-1). We found that, similarly to HSV-1, the C-terminal amphipathic helix in the HCMV gB cytoplasmic tail restricts fusion, whereas the entire HCMV gH cytoplasmic tail is required for fusion activation. However, the structure of the HCMV gB cytoplasmic tail and its interactions with the cytoplasmic tail of gH might be different from HSV-1. We hypothesize that while in HCMV\u2014as in HSV-1\u2014the cytoplasmic tails of HCMV gB and gH function as an inhibitory clamp and an activating wedge, their structures and interactions differ from HSV-1, implicating potential mechanistic differences in fusion regulation between herpesvirus subfamilies.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Herpesviruses promote membrane fusion during infection using a complex multi-component membrane fusion machinery. Proper spatiotemporal deployment of such machinery is subject to precise regulatory inputs. One of these is the regulation of the fusogenic activity of the conserved herpesviral glycoproteins gB and gH by their cytoplasmic (or intraviral) domains. This regulatory mechanism has been investigated in\n                        <jats:italic toggle=\"yes\">Alpha-<\/jats:italic>\n                        and\n                        <jats:italic toggle=\"yes\">Gamma-<\/jats:italic>\n                        but not yet in\n                        <jats:italic toggle=\"yes\">Betaherpesvirinae<\/jats:italic>\n                        . Here, we combined mutagenesis targeting the cytoplasmic tails of HCMV gB and gH with a plasmid-transfection-based split-luciferase assay for measuring cell-cell fusion, which we developed here. By testing a panel of truncation mutants, we showed that inhibitory and activating regulatory regions in gB and gH, respectively, are conserved in HCMV. However, none of the single point mutants of gB had expected phenotypes, suggesting that the cytoplasmic tails of HCMV gB and gH have distinct structures and interactions. This study introduces a robust and sensitive\n                        <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                        cell-cell fusion assay for probing HCMV fusion mechanism and shows that despite sequence conservation, the cytoplasmic domains of gB and gH may regulate fusion by distinct mechanisms. This knowledge may inform future efforts in rational vaccine design and antiviral development.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01905-25","type":"journal-article","created":{"date-parts":[[2025,9,22]],"date-time":"2025-09-22T13:03:12Z","timestamp":1758546192000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Mutational dissection of HCMV gB and gH cytoplasmic tails highlights conserved and divergent features of fusion regulation"],"prefix":"10.1128","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4102-6528","authenticated-orcid":false,"given":"Chanyoung","family":"Lee","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05wvpxv85","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Biology and Microbiology, Tufts University School of Medicine","place":["Boston, USA"]},{"id":[{"id":"https:\/\/ror.org\/05wvpxv85","id-type":"ROR","asserted-by":"publisher"}],"name":"Graduate Program in Molecular Microbiology, Graduate School of Biomedical Sciences, Tufts University School of Medicine","place":["Boston, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hannah K.","family":"DiBella","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/049v69k10","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological and Biomedical Science, Rowan University","place":["Glassboro, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3113-6958","authenticated-orcid":true,"given":"Ekaterina E.","family":"Heldwein","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05wvpxv85","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Biology and Microbiology, Tufts University School of Medicine","place":["Boston, USA"]},{"id":[{"id":"https:\/\/ror.org\/05wvpxv85","id-type":"ROR","asserted-by":"publisher"}],"name":"Graduate Program in Molecular Microbiology, Graduate School of Biomedical Sciences, Tufts University School of Medicine","place":["Boston, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-virology-111821-093413"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2015.03.043"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2009.02.012"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-020-00448-w"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.3390\/v14020296"},{"key":"e_1_3_3_7_2","first-page":"16","article-title":"Congenital cytomegalovirus: impact on child health","volume":"35","author":"Schleiss MR","year":"2018","unstructured":"Schleiss MR. 2018. 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