Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.
Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.
复制标题
DOI：
10.1186/1743-422x-7-6
复制
发表时间：
2010-01-15
期刊：
影响因子：
4.8
通讯作者：
Niedrig M
中科院分区：
文献类型：
--
作者：
Müller MA;van der Hoek L;Voss D;Bader O;Lehmann D;Schulz AR;Kallies S;Suliman T;Fielding BC;Drosten C;Niedrig M
人类致病性冠状病毒NL 63（hCoV-NL 63）是一种1组（α）冠状病毒，通常与呼吸道感染有关。除了已知的非结构蛋白和结构蛋白，所有冠状病毒都有一个或多个辅助蛋白，其功能大多未知。我们的研究集中在hCoV-NL 63开放阅读框3（ORF 3），这是冠状病毒中高度保守的辅助蛋白。对hCoV-NL 63 ORF 3的225个氨基酸序列的计算机分析预测了三重跨膜蛋白。通过免疫荧光和Western印迹分析证实在感染的CaCo-2和LLC-MK2细胞中的表达。在内质网/高尔基中间室（ERGIC）内检测到该蛋白，其中冠状病毒组装和出芽发生。使用重组ORF 3蛋白转染Huh-7细胞的亚细胞定位研究显示，在ERGIC，高尔基体和溶酶体室发生。通过不同标记的包膜（E）、膜（M）和核衣壳（N）蛋白的荧光显微镜，显示ORF 3蛋白与E和M在ERGIC内广泛共定位。使用N-末端FLAG标记的ORF 3蛋白和C-末端特异性的抗血清，我们验证了所提出的拓扑结构的细胞外N-末端和胞质C-末端。通过体外翻译分析和随后的内切糖苷酶H消化，我们发现ORF 3蛋白在N-末端被N-糖基化。对纯化的病毒颗粒的分析表明ORF 3蛋白被掺入病毒体中，因此是额外的结构蛋白。这项研究是第一个广泛的表达分析组1 hCoV-ORF 3蛋白。我们给出的证据表明，ORF 3蛋白是一个结构N-糖基化和病毒粒子掺入蛋白。
Human pathogenic coronavirus NL63 (hCoV-NL63) is a group 1 (alpha) coronavirus commonly associated with respiratory tract infections. In addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. Our study focuses on hCoV-NL63 open reading frame 3 (ORF 3) which is a highly conserved accessory protein among coronaviruses. In-silico analysis of the 225 amino acid sequence of hCoV-NL63 ORF 3 predicted a triple membrane-spanning protein. Expression in infected CaCo-2 and LLC-MK2 cells was confirmed by immunofluorescence and Western blot analysis. The protein was detected within the endoplasmatic reticulum/Golgi intermediate compartment (ERGIC) where coronavirus assembly and budding takes place. Subcellular localization studies using recombinant ORF 3 protein transfected in Huh-7 cells revealed occurrence in ERGIC, Golgi- and lysosomal compartments. By fluorescence microscopy of differently tagged envelope (E), membrane (M) and nucleocapsid (N) proteins it was shown that ORF 3 protein colocalizes extensively with E and M within the ERGIC. Using N-terminally FLAG-tagged ORF 3 protein and an antiserum specific to the C-terminus we verified the proposed topology of an extracellular N-terminus and a cytosolic C-terminus. By in-vitro translation analysis and subsequent endoglycosidase H digestion we showed that ORF 3 protein is N-glycosylated at the N-terminus. Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein. This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein. We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.