A novel severe acute respiratory syndrome coronavirus protein U274, is transported to the cell surface and undergoes endocytosis
A novel severe acute respiratory syndrome coronavirus protein U274, is transported to the cell surface and undergoes endocytosis
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DOI：
10.1128/jvi.78.13.6723-6734.2004
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发表时间：
2004-07-01
影响因子：
5.4
通讯作者：
Hong, WJ
中科院分区：
文献类型：
--
作者：
Tan, YJ;Teng, E;Hong, WJ
严重急性呼吸综合征冠状病毒 (SARS-CoV) 基因组包含开放阅读框 (ORF)，其编码的多个基因与所有已知冠状病毒中发现的蛋白质同源。它们是复制酶基因 1a/1b 和四种结构蛋白：核衣壳 (N)、刺突 (S)、膜 (M) 和包膜 (E)，预计这些蛋白质对于病毒的复制至关重要。此外，该基因组还包含其他九个潜在的 ORF，长度从 39 到 274 个氨基酸不等。其中最大的是第二长亚基因组 RNA 的第一个 ORF，该蛋白质（在本研究中称为 U274）由 274 个氨基酸组成，并包含三个假定的跨膜结构域。使用针对 U274 C 末端的特异性抗体，我们发现 U274 在 SARS-CoV 感染的 Vero E6 细胞中表达，除了全长蛋白外，还检测到了其他两种加工形式。通过间接免疫荧光，在转染和感染细胞中，U274 定位于核周区域以及质膜。使用 N 末端 myc 标记的 U274，确认了 U274 的拓扑结构及其在细胞表面的表达。 U274 的细胞质结构域（包含 Yxxphi 和二酸基序）的缺失，消除了其向细胞表面的运输。此外，表达在细胞表面的U274可以将培养基中的抗体内化到细胞中。共沉淀实验还表明，U274 可以与 M、E 和 S 结构蛋白以及 U122（SARS-CoV 特有的另一种蛋白质）特异性相互作用。
The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains open reading frames (ORFs) that encode for several genes that are homologous to proteins found in all known coronaviruses. These are the replicase gene 1a/1b and the four structural proteins, nucleocapsid (N), spike (S), membrane (M), and envelope (E), and these proteins are expected to be essential for the replication of the virus. In addition, this genome also contains nine other potential ORFs varying in length from 39 to 274 amino acids. The largest among these is the first ORF of the second longest subgenomic RNA, and this protein (termed U274 in the present study) consists of 274 amino acids and contains three putative transmembrane domains. Using antibody specific for the C terminus of U274, we show U274 to be expressed in SARS-CoV-infected Vero E6 cells and, in addition to the full-length protein, two other processed forms were also detected. By indirect immunofluorescence, U274 was localized to the perinuclear region, as well as to the plasma membrane, in both transfected and infected cells. Using an N terminus myc-tagged U274, the topology of U274 and its expression on the cell surface were confirmed. Deletion of a cytoplasmic domain of U274, which contains Yxxphi and diacidic motifs, abolished its transport to the cell surface. In addition, U274 expressed on the cell surface can internalize antibodies from the culture medium into the cells. Communoprecipitation experiments also showed that U274 could interact specifically with the M, E, and S structural proteins, as well as with U122, another protein that is unique to SARS-CoV.