Petit Chad M, Melancon Jeffrey M, Chouljenko Vladimir N, Colgrove Robin, Farzan Michael, Knipe David M, Kousoulas K G
Division of Biotechnology and Molecular Medicine (BIOMMED), School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
Virology. 2005 Oct 25;341(2):215-30. doi: 10.1016/j.virol.2005.06.046. Epub 2005 Aug 15.
The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion.
严重急性呼吸综合征冠状病毒（SARS-CoV）是严重急性呼吸综合征（SARS）的病原体。SARS-CoV刺突（S）糖蛋白在病毒进入和病毒诱导的细胞间融合过程中介导膜融合事件。为了描绘SARS-CoV S糖蛋白的功能结构域，在瞬时表达实验中研究了单点突变、聚簇到赖氨酸和聚簇到丙氨酸的突变以及羧基末端截短。S糖蛋白氨基末端七肽重复序列的卷曲螺旋结构域、预测的融合肽或相邻但不同区域的诱变，严重损害了S介导的细胞间融合，而细胞内运输和细胞表面表达未受到不利影响。令人惊讶的是，17个氨基酸的羧基末端截短显著增加了S糖蛋白介导的细胞间融合，表明末端17个氨基酸调节了S的融合特性。相比之下，分别去除两个富含半胱氨酸的胞内结构域基序中的一个或两个的26或39个氨基酸的截短，与野生型S相比抑制了细胞融合。17和26个氨基酸的缺失对S细胞表面表达没有不利影响，而39个氨基酸的截短抑制了S细胞表面表达，表明膜近端富含半胱氨酸的基序在S细胞表面表达中起重要作用。S糖蛋白羧基末端酸性氨基酸簇的诱变以及酸性簇内预测的磷酸化位点的修饰表明，该氨基酸基序可能在S在细胞表面的保留中发挥功能作用。这项遗传分析表明，SARS-CoV S糖蛋白包含调节细胞融合的细胞外结构域以及在细胞内运输、细胞表面表达和细胞融合中起作用的不同胞内结构域。
