The cytoplasmic C-tail of the mouse cytomegalovirus 7 transmembrane receptor homologue, M78, regulates endocytosis of the receptor and modulates virus replication in different cell types

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_C Delta 155), had a cell-type specific replication phenotype. M78_C Delta 155 had restricted replication in bone marrow macrophages, indistinguishable from an M78-null recombinant. In contrast, M78_C Delta 155 replicated normally or with enhanced titres to wild type virus in other tested cell-types, whereas M78-null was attenuated. Distinct phenotypes for M78_C Delta 155 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_C Delta 155, 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_C Delta 155 and M78-null for replication in macrophages may have contributed to their similar pathogenic phenotypes