Abstract
Human cytomegalovirus (HCMV) encodes a number of viral proteins with homology to cellular G protein-coupled receptors (GPCRs). These viral GPCRs, including US27, US28, UL33, and UL78, have been ascribed numerous functions during infection, including activating diverse cellular pathways, binding to immunomodulatory chemokines, and impacting virus dissemination. To investigate the role of US28 during virus infection, two variants of the clinical isolate TB40/E were generated: TB40/E-US28YFP expressing a C-terminal yellow fluorescent protein tag, and TB40/E-FLAGYFP in which a FLAG-YFP cassette replaces the US28 coding region. The TB40/E-US28YFP protein localized as large perinuclear fluorescent structures at late times post-infection in fibroblasts, endothelial, and epithelial cells. Interestingly, US28YFP is a non-glycosylated membrane protein throughout the course of infection. US28 appears to impact cell-to-cell spread of virus, as the ΔUS28 virus (TB40/E-FLAGYFP) generated a log-greater yield of extracellular progeny whose spread could be significantly neutralized in fibroblasts. Most strikingly, in epithelial cells, where dissemination of virus occurs exclusively by the cell-to-cell route, TB40/E-FLAGYFP (ΔUS28) displayed a significant growth defect. The data demonstrates that HCMV US28 may contribute at a late stage of the viral life cycle to cell-to-cell dissemination of virus.
Highlights
Human cytomegalovirus (HCMV) is a widespread pathogen that infects a vast majority of the world’s population [1]
To determine if US28 is a glycosylated membrane protein, lysates prepared from TB40/E-US28YFP-infected fibroblasts at various times post-infection were subjected to digestion by either endoglycosidase H (EndoH) or peptide: N-glycosidase F (PNGaseF) (Figure 3c)
In these studies we demonstrate that US28 impacts dissemination of virus by promoting cell-to-cell spread of infectious progeny
Summary
Human cytomegalovirus (HCMV) is a widespread pathogen that infects a vast majority of the world’s population [1]. GPCRs, known as seven-transmembrane domain proteins, are integral membrane receptors that sense extracellular ligands to trigger signal transduction networks and coordinate cellular responses [8]. To date no activating ligands or signaling properties have been attributed to UL78 Another intriguing characteristic accorded to HCMV-encoded GPCRs is their contribution to dissemination of virus in vitro. Extracellular virus produced by the US28 mutant could be neutralized by the addition of HCMV glycoprotein-specific antibodies and spread of TB40/E-FLAGYFP by the cell-to-cell route was abrogated in fibroblasts and epithelial cells. These findings implicate the viral GPCR US28 as a factor contributing to cellular dissemination of HCMV
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