Localization of HCMV UL33 and US27 in Endocytic Compartments and Viral Membranes

Authors

  • Alberto Fraile-Ramos,

    1. Cell Biology Unit, MRC Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK
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    • These authors contributed equally to this work.

  • Annegret Pelchen-Matthews,

    1. Cell Biology Unit, MRC Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK
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    • These authors contributed equally to this work.

  • Thomas N. Kledal,

    1.  Department of Microbiology and Immunology, Fairchild Science Building, Stanford School of Medicine, Stanford, CA 94305–5124, USA
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  • Helena Browne,

    1. Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
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  • Thue W. Schwartz,

    1. Laboratory for Molecular Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark
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  • Mark Marsh

    Corresponding author
    1. Cell Biology Unit, MRC Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK
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Corresponding author: Mark Marsh,m.marsh@ucl.ac.uk

Abstract

The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine receptor-like proteins. Although important in viral pathogenesis, little is known about the properties or functions of these proteins. We previously reported that US28 is located in endocytic vesicles and undergoes constitutive endocytosis and recycling. Here we studied the cellular distributions and trafficking of two other human cytomegalovirus chemokine receptor-like proteins, UL33 and US27, in transfected and human cytomegalovirus-infected cells. Immunofluorescence staining indicated that UL33 and US27 are located at the cell surface, although the majority of both proteins was seen in intracellular organelles located in the perinuclear region of the cell. The intracellular pools of UL33 and US27 showed overlap with markers for endocytic organelles. Antibody-feeding experiments indicated that cell surface US27 undergoes endocytosis. By immunogold labeling of cryosections and electron microscopy, UL33 was seen to localize to multivesicular bodies (MVBs or multivesicular endosomes). Electron microscopy analysis of human cytomegalovirus-infected cells showed that most virus particles wrapped individually into short membrane cisternae, although virus particles were also occasionally seen within and budding into MVBs. Electron microscopy immunolocalization of viral UL33 and US27 on ultrathin cryosections of human cytomegalovirus-infected cells showed gold particles over the membranes into which virions were wrapping, in small membrane tubules and vesicles and in MVBs. Labeling of the human cytomegalovirus glycoproteins gB and gH indicated that these proteins were also present in the same membrane structures. This first electron microscopy analysis of human cytomegalovirus assembly using immunolabeling suggests that the localization of UL33, US27 and US28 to endosomes may allow these proteins to be incorporated into the viral membrane during the final stages of human cytomegalovirus assembly.

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