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

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.

Murine cytomegalovirus degrades MHC class II to colonize the salivary glands

“…We show that MCMV, like HCMV, degrades MHC II in infected cells, and that this requires M78, a multi-membrane spanning viral protein with homology to chemokine receptors but without canonical signalling motifs. M78 - MCMV shows reduced virus production from infected macrophages, and poorly colonizes the SG [ 20 – 22 ]. Myeloid cells infected by M78 - but not wild-type (WT) MCMV expressed MHC II in vivo , and CD4 + T cell loss significantly reversed the M78-dependent defect in SG colonization.…”

Anti-herpesvirus CD4+ T cell function

“…4.16c). This result confirms that, M78 is not only required for infection and virus dissemination to the salivary gland as previously described (162) but also for evading CD4 + T cells. C57BL/6 (IA + / -) and MHC II -(IA -/ -) mice infected i.p with 3x10 4 p.f.u/100µL WT or M78b or i.n c with 3x10 4 p.f.u/30µL WT, M78or M78 cΔ155 under anaesthesia.…”

“…As previously shown, M78virus had no replication defect in fibroblasts cells but replicated slightly less than WT in epithelial cells (162). This suggests M78 could have an epithelial infection defect.…”

“…The C-terminus of M78 was critical for rapid constitutive endocytosis of M78 and CCR5 (162,186). To determine whether the C-terminus was required for MHC II downregulation, the C-terminal mutant lacking 155 amino acids from the C-terminus with an HA tag to the N-terminus of M78 (HAM78 cΔ155) was tested.…”

“…M78 C-terminus was previously shown to be necessary for MCMV dissemination in immunocompetent C57BL/6 mice (162). To determine whether the C-terminal mutant of M78 would be rescued by CD4 + T cell loss, IA + /and IA -/mice were infected via the i.n route with WT, M78and the C-terminal mutant (M78 ∆ c155).…”