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HCMV has the largest genome of the human herpesviruses and encodes an array of genes that affect pathogenesis in different cell types. Given the ability of HCMV to replicate in a range of cell types, investigators have begun to identify viral proteins required for cell type\u2013specific replication. There are four proteins encoded by HCMV that are homologous to G protein-coupled receptors (GPCRs); these viral GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In this study, we find that deletion of all four vGPCR genes severely attenuates HCMV replication in primary human salivary gland epithelial cells and ARPE-19 retinal epithelial cells, as evidenced by decreases in viral gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of viral gene expression and virus production. We find that, similar to US28, UL33 constitutively activates G\u03b1q signaling to high levels in epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5\u2032 UTRs to create novel UL33 proteins that are all capable of constitutive G\u03b1q signaling. Taken together, these studies provide novel molecular and biochemical data regarding UL33 expression, subcellular localization, and signaling, and indicate that UL33 activity is essential for efficient HCMV replication in cells of epithelial origin.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Human cytomegalovirus (HCMV) replicates in a number of cell types and tissues\n                      <jats:italic toggle=\"yes\">in vivo,<\/jats:italic>\n                      and the viral genes involved in cell type\u2013specific replication are just beginning to be elucidated. The HCMV-encoded viral G protein-coupled receptors (vGPCRs) UL33, UL78, US27, and US28 are proving to play important roles in multiple aspects of HCMV replication, including the establishment and maintenance of latency. Here, we demonstrate that the HCMV vGPCRs and UL33, in particular, play an important role in driving lytic replication in cells of epithelial origin, including those derived from the salivary gland. This work expands on potential functions of the vGPCRs, will drive future studies to understand mechanistically how they affect tropism, and provides a new target for future therapeutics.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.00664-26","type":"journal-article","created":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T15:10:07Z","timestamp":1788189007000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["The human cytomegalovirus vGPCR UL33 is essential for efficient lytic replication in epithelial cells"],"prefix":"10.1128","volume":"100","author":[{"given":"MacKenzie R.","family":"Freeman","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01e3m7079","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular and Cellular Biosciences, and the Graduate Program in Molecular Genetics, Biochemistry, and Microbiology, University 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