Davis-Poynter Nick, Yunis Joseph, Farrell Helen E
Child Health Research Centre, The University of Queensland, Brisbane, Australia.
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia.
PLoS One. 2016 Oct 19;11(10):e0165066. doi: 10.1371/journal.pone.0165066. eCollection 2016.
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和γ疱疹病毒都编码七跨膜受体（7TMR）的病毒同源物，这表明它们具有重要的功能作用。小鼠巨细胞病毒（MCMV）的M78是所有β疱疹病毒中保守的7TMR家族的代表。已发现M78家族成员在组织培养中对病毒复制表现出细胞类型特异性作用，并影响体内病毒发病机制。我们之前报道过，M78（其配体未知）会经历快速的组成型内吞作用。在本研究中，我们研究了M78细胞质C末端在介导内吞作用中的作用以及C末端缺失对复制和发病机制的影响。M78的突变（C末端截短或点突变）和CCR5-M78嵌合体确定了影响内吞作用的两个不同区域。第一个是经典的酸性双亮氨酸基序（DDxxxLL），靠近C末端。第二个区域，其活性被下游序列抑制，包括靠近第7跨膜结构域的假定第8螺旋。表达缺乏大部分C末端的内吞缺陷型M78（M78_CΔ155）的重组MCMV具有细胞类型特异性复制表型。M78_CΔ155在骨髓巨噬细胞中的复制受限，与M78缺失重组体无法区分。相比之下，M78_CΔ155在其他测试细胞类型中正常复制或滴度高于野生型病毒，而M78缺失型则减弱。M78_CΔ155和M78缺失型的不同表型表明C末端缺失导致M78功能障碍，而非功能完全丧失；此外，它们突出了M78在复制过程中的细胞类型特异性作用。对小鼠进行鼻内感染表明，M78_CΔ155与M78缺失型相似，比野生型病毒从肺部清除得更快，并且在唾液腺中的复制严重减弱。可以推测，M78_CΔ155和M78缺失型在巨噬细胞中复制减弱可能导致了它们相似的致病表型。
