{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T19:03:24Z","timestamp":1790622204603,"version":"4.1.0"},"posted":{"date-parts":[[2026,9,23]]},"group-title":"Microbiology","reference-count":48,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"National Institutes of Health","award":["CA251275"],"award-info":[{"award-number":["CA251275"]}]},{"name":"National Institutes of Health","award":["AG084065"],"award-info":[{"award-number":["AG084065"]}]},{"name":"National Institutes of Health","award":["EY029169"],"award-info":[{"award-number":["EY029169"]}]},{"name":"National Institutes of Health","award":["AG089561"],"award-info":[{"award-number":["AG089561"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,23]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Cholesterol is a major structural component of the plasma membrane and a key allosteric regulator of G protein-coupled receptors (GPCRs), yet its role in controlling constitutive receptor activity remains poorly understood. Virally encoded GPCRs provide an ideal system to address this question because many exhibit constitutive signaling that promotes viral persistence and pathogenesis, although how excessive receptor activation is restrained remains unknown. Here, we identify a previously unrecognized cholesterol-dependent allosteric mechanism by which the oncogenic Kaposi\u2019s sarcoma-associated herpesvirus (KSHV) GPCR ORF74 constrains its constitutive activity. CryoEM structural analysis reveals a cholesterol-binding pocket formed by transmembrane helices 3, 5, and 6 that is present only in the inactive receptor. Cholesterol binding restrains the outward movement of transmembrane helix 6, stabilizes the inactive conformation, and suppresses spontaneous receptor activation, as supported by molecular dynamics simulations. Mechanistically, replacement of the canonical DRY motif with a non-canonical VRY motif exposes the conserved R143\n                  <jats:sup>3.50<\/jats:sup>\n                  residue, creating a membrane-facing cholesterol-binding interface that couples membrane cholesterol to receptor conformational control. Consistent with this model, cellular cholesterol depletion enhances ORF74 signaling, whereas disruption of cholesterol binding impairs receptor stabilization. Together, our findings uncover a previously unrecognized mechanism by which a viral GPCR exploits host membrane cholesterol to regulate its constitutive activity, suggesting that persistent viruses optimize constitutive signaling by coupling receptor activity to host lipid-dependent allosteric regulation.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>SIGNIFICANCE<\/jats:title>\n                  <jats:p>Constitutively active viral G protein-coupled receptors (GPCRs) promote viral persistence and oncogenesis, yet how excessive signaling is restrained has remained unknown. We identify a cholesterol-binding allosteric pocket that stabilizes the inactive conformation of the Kaposi\u2019s sarcoma-associated herpesvirus GPCR ORF74, thereby suppressing constitutive receptor activity. Unexpectedly, the non-canonical VRY motif previously linked to constitutive signaling also creates the cholesterol-responsive interface, coupling intrinsic receptor activity to the host membrane environment. These findings reveal a mechanism by which a persistent virus exploits host membrane cholesterol to optimize rather than maximize signaling output and establish membrane cholesterol as a direct regulator of constitutive GPCR signaling with potential therapeutic implications.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.22.753489","type":"posted-content","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T04:55:16Z","timestamp":1790225716000},"source":"Crossref","is-referenced-by-count":0,"title":["Host membrane cholesterol constrains constitutive signaling of the oncogenic KSHV GPCR ORF74"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3023-1040","authenticated-orcid":false,"given":"Jun Bae","family":"Park","sequence":"first","affiliation":[{"name":"Department of Microbial Sciences in Health, Cleveland Clinic Research, Cleveland Clinic, Cleveland, Ohio, USA"},{"name":"Global Center for Pathogen and Human Health Research, Cleveland Clinic Research, Cleveland Clinic, Cleveland, Ohio, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2190-9518","authenticated-orcid":false,"given":"Amita Rani","family":"Sahoo","sequence":"additional","affiliation":[{"name":"Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Annabelle","family":"Ryu","sequence":"additional","affiliation":[{"name":"Department of Microbial Sciences in Health, Cleveland Clinic Research, Cleveland Clinic, Cleveland, Ohio, USA"},{"name":"Global Center for Pathogen and Human Health Research, Cleveland Clinic Research, Cleveland Clinic, Cleveland, Ohio, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2958-0403","authenticated-orcid":false,"given":"Matthias","family":"Buck","sequence":"additional","affiliation":[{"name":"Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4559-8774","authenticated-orcid":false,"given":"Jae U.","family":"Jung","sequence":"additional","affiliation":[{"name":"Department of Microbial Sciences in Health, Cleveland Clinic Research, Cleveland Clinic, Cleveland, Ohio, USA"},{"name":"Global Center for Pathogen and Human Health Research, Cleveland Clinic Research, Cleveland Clinic, Cleveland, Ohio, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026092811401565000_2026.09.22.753489v1.1","doi-asserted-by":"crossref","unstructured":"M. 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