Vázquez M I, Rivas G, Cregut D, Serrano L, Esteban M
Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, 28049 Madrid, Spain.
J Virol. 1998 Dec;72(12):10126-37. doi: 10.1128/JVI.72.12.10126-10137.1998.
The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third alpha-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.
痘苗病毒14千道尔顿蛋白（由A27L基因编码）在病毒生物学中发挥重要作用，参与病毒与细胞以及细胞与细胞之间的融合。该蛋白位于细胞内成熟病毒形式的表面，对于细胞释放细胞外被膜病毒以及病毒传播至关重要。序列分析预测该蛋白存在四个区域：从氨基酸1至28的无结构区域、从残基29至37的螺旋区域、从残基44至72的三股卷曲螺旋区域以及C末端的亮氨酸拉链基序。对纯化的野生型蛋白和几种突变形式（缺失上述一个或多个区域或存在点突变）进行的圆二色光谱、分析超速离心和化学交联研究，支持了上述对14千道尔顿蛋白的结构划分。第71和72位的两个相邻半胱氨酸残基与14千道尔顿蛋白三聚体的形成无关。螺旋轮上a和d位置的疏水残基以及相邻位置e和g的带电氨基酸的位置表明，三股卷曲螺旋区域中的疏水和离子相互作用参与寡聚体形成。构建三螺旋束模型和分子动力学支持了这一推测。用在大肠杆菌中表达的纯化蛋白以及来自感染期间不产生14千道尔顿蛋白的病毒（VVindA27L）感染的细胞的细胞质提取物进行的结合试验表明，21千道尔顿蛋白（由A17L基因编码）是特异性病毒结合伴侣，并确定推定的亮氨酸拉链（14千道尔顿蛋白C末端预测的第三个α螺旋）为参与蛋白结合的区域。在用表达14千道尔顿蛋白突变形式的质粒载体转染动物细胞并感染VVindA27L后，这些发现在体内得到证实。我们发现14千道尔顿蛋白的结构组织与其他融合蛋白相似，如流感病毒的血凝素和人类免疫缺陷病毒的gp41，只是存在一个蛋白锚定结构域而非跨膜结构域。基于我们的观察结果，我们建立了14千道尔顿蛋白的结构模型。
