Abstract
Virus homologues of seven-transmembrane receptors (7TMR) are encoded by all beta- and gammaherpesviruses, suggesting important functional roles. M78 of mouse cytomegalovirus (MCMV) is representative of a family of 7TMR conserved in all betaherpesviruses. M78 family members have been found to exhibit cell-type specific effects upon virus replication in tissue culture and to affect virus pathogenesis in vivo. We reported previously that M78, for which no ligands are known, undergoes rapid, constitutive endocytosis. In this study, we have investigated the role of the M78 cytoplasmic C-tail in mediating endocytosis and consequences of C-tail deletion upon replication and pathogenesis. Mutations of M78 (C-tail truncations or point mutations) and CCR5-M78 chimeras identified two distinct regions affecting endocytosis. The first was a classical acidic di-leucine motif (DDxxxLL), located close to the C-terminus. The second region, the activity of which was suppressed by downstream sequences, included the putative 8th helix, located close to the 7th transmembrane domain. A recombinant MCMV expressing an endocytosis-deficient M78, lacking most of the C-tail (M78_CΔ155), had a cell-type specific replication phenotype. M78_CΔ155 had restricted replication in bone marrow macrophages, indistinguishable from an M78-null recombinant. In contrast, M78_CΔ155 replicated normally or with enhanced titres to wild type virus in other tested cell-types, whereas M78-null was attenuated. Distinct phenotypes for M78_CΔ155 and M78-null suggest that the C-tail deletion resulted in M78 dysfunction, rather than complete loss of function; furthermore, they highlight a cell-type specific role of M78 during replication. Infection of mice (intranasal) demonstrated that M78_CΔ155, similar to M78-null, was cleared more rapidly from the lungs than wild type virus and was severely attenuated for replication in salivary glands. It may be speculated that attenuation of both M78_CΔ155 and M78-null for replication in macrophages may have contributed to their similar pathogenic phenotypes.
Highlights
Seven-transmembrane receptors (7TMR), commonly known as G protein-coupled receptors (GPCR), comprise a large superfamily of membrane proteins that regulate diverse cellular activities
Endocytosis was studied in transfected cells using antibody feeding, followed by sequential staining via immunofluorescence of cells pre- and post- permeabilisation, using secondary antibodies conjugated to either AF594 or AF488
Preliminary studies demonstrated that the M78 C-tail was required for efficient endocytosis, since a mutant deleted of 155 amino acid residues from the C-tail (M78_CΔ155: aa 1–316) retained substantial surface staining, compared with full length M78, where surface staining was mostly absent (Fig 1 and S1 Fig)
Summary
Seven-transmembrane receptors (7TMR), commonly known as G protein-coupled receptors (GPCR), comprise a large superfamily of membrane proteins that regulate diverse cellular activities. Homologues of 7TMRs have been identified in all beta- and gammaherpesviruses, promoting interest as potential targets for novel antiviral drugs [1, 2]. The prototypes of the betaherpesvirus 7TMR gene families were initially identified in human cytomegalovirus (CMV), namely 1) US28 and the related US27, 2) UL33 and 3) UL78 [4]. The US28 gene family is found only in Old World primate CMVs, whereas UL33 and UL78 homologues have been identified in all sequenced betaherpesviruses [5]. Many of the herpesvirus 7TMRs are most closely homologous to chemokine receptors and several have been demonstrated to conserve functions such as chemokine binding and G protein-coupled signalling [1,2,3]
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