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_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.所有β疱疹病毒与γ疱疹病毒均编码七次跨膜受体（seven-transmembrane receptors, 7TMR）的病毒同源物，提示其具备重要的功能意义。小鼠巨细胞病毒（mouse cytomegalovirus, MCMV）的M78蛋白是所有β疱疹病毒中保守的7TMR家族的典型代表。研究发现，M78家族成员在组织培养体系中对病毒复制表现出细胞类型特异性的调控效应，并可在体内影响病毒的致病过程。本课题组此前曾报道，尚无已知配体的M78蛋白会发生快速的组成型内吞作用。本研究中，我们探究了M78胞质C端尾在介导内吞作用中的功能，以及C端尾缺失对病毒复制与致病过程的影响。通过对M78进行C端尾截短突变或点突变，并构建CCR5-M78嵌合蛋白，我们鉴定出两个影响内吞作用的独立区域。第一个区域为位于C端附近的经典酸性双亮氨酸基序（DDxxxLL）；第二个区域包含靠近第七跨膜结构域的推定第八螺旋，其活性可被下游序列所抑制。我们构建了一株表达内吞功能缺陷型M78的重组小鼠巨细胞病毒，该突变体缺失了大部分C端尾（命名为M78_CΔ155），其复制表型具有细胞类型特异性。M78_CΔ155在骨髓巨噬细胞中的复制受到显著限制，该表型与M78基因敲除重组病毒无显著差异。与之相反，在其他受试细胞类型中，M78_CΔ155的复制水平与野生型病毒相当，甚至部分细胞系中滴度有所提升，而M78基因敲除病毒的复制则出现明显减弱。M78_CΔ155与M78基因敲除病毒的表型存在差异，提示C端尾缺失仅导致M78功能异常，而非完全丧失功能；此外，该结果也凸显了M78在病毒复制过程中的细胞类型特异性作用。小鼠鼻内感染实验表明，与M78基因敲除病毒类似，M78_CΔ155可更快地从肺部被清除，且在唾液腺中的复制受到严重减毒。我们推测，M78_CΔ155与M78基因敲除病毒在巨噬细胞中复制能力的减弱，可能是二者致病表型相似的核心原因。