Fraile-Ramos A, Kledal T N, Pelchen-Matthews A, Bowers K, Schwartz T W, Marsh M
Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, London WC1E 6BT, United Kingdom.
Mol Biol Cell. 2001 Jun;12(6):1737-49. doi: 10.1091/mbc.12.6.1737.
Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX(3)C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.
在疱疹病毒和痘病毒中发现了编码趋化因子受体样蛋白的基因，这些基因与病毒发病机制有关。在此，我们描述了由US28基因编码的人巨细胞病毒（HCMV）趋化因子受体在转染的HeLa和Cos细胞中瞬时和稳定表达后的细胞分布及运输情况。免疫荧光染色表明，这种病毒蛋白在细胞内核周区域的囊泡结构内细胞内积累，并与内吞细胞器的标记物有重叠。通过免疫金电子显微镜观察，发现US28大多定位于多囊泡内体。该蛋白的一小部分（最多20%）也在细胞表面表达。抗体摄取实验表明，细胞表面的US28会发生组成型非配体依赖性内吞作用。使用碘化配体的生化分析表明，US28能迅速内化。US28的高亲和力配体CX(3)C趋化因子fractalkine，明显通过抑制内化受体的再循环，降低了细胞表面US28的稳态水平。HCMV US28的内吞作用和循环可能在宿主趋化因子的隔离中发挥作用，从而调节抗病毒免疫反应。此外，US28主要在内体膜上的分布可能使其在HCMV组装过程中被整合到病毒包膜中。
