Au K S, Mattion N M, Estes M K
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030.
Virology. 1993 Jun;194(2):665-73. doi: 10.1006/viro.1993.1306.
The single-shelled particle binding domain(s) on NS28 was examined by testing the ability of different truncated forms of NS28 to bind single-shelled particles (ssp). Deletion of amino acids (aa) 161 to 175 of NS28 abolished ssp binding activity. Deletion of the last three aa (173-175) of NS28 diminished, but did not abolish, the ligand binding activity in our assay conditions. An internal deletion of NS28 (aa 110 to 155) also significantly diminished ssp binding activity in standard binding assays. As an alternative approach to study the ssp binding domain on NS28, we mapped the epitope of binding of monoclonal antibody BA/55, which was found to block ssp binding to NS28. Immunoprecipitation experiments done with truncated mutants of NS28 located the epitope of BA/55 to aa 149-160 of NS28, immediately adjacent to or partially overlapping the putative ssp binding domain. Experiments using synthetic peptides mimicking the carboxy end of NS28, found these peptides were not able to compete for ssp binding. Together, these results suggest that the ssp binding site in NS28 (aa 161-172) is highly dependent on the conformational integrity of the cytoplasmic C-terminus of NS28. NS28 truncation mutants also were assayed for interactions with rotavirus VP4 expressed in baculovirus. Amino acids 112 to 148 of NS28 were found to be critical for NS28-VP4 binding. Unexpectedly, aa 149 to 175 not only were nonessential for interaction with VP4, but mutants lacking those aa showed improved binding activity. We hypothesize that the VP4 binding domain may be buried in the NS28 cytoplasmic domain, and that the binding of ssp and VP4 may be an interdependent process that functions in conjunction with triggering of the budding of the whole complex into the endoplasmic reticulum. These results demonstrate the pleiotropic properties of NS28 in the unique rotavirus morphogenetic process.
通过检测NS28不同截短形式结合单壳颗粒（ssp）的能力，对NS28上的单壳颗粒结合结构域进行了研究。删除NS28的161至175位氨基酸（aa）会消除ssp结合活性。在我们的检测条件下，删除NS28的最后三个氨基酸（173 - 175）会降低但不会消除配体结合活性。NS28的内部缺失（110至155位氨基酸）在标准结合试验中也显著降低了ssp结合活性。作为研究NS28上ssp结合结构域的另一种方法，我们绘制了单克隆抗体BA/55的结合表位，发现它能阻断ssp与NS28的结合。用NS28的截短突变体进行的免疫沉淀实验将BA/55的表位定位到NS28的149 - 160位氨基酸，紧邻假定的ssp结合结构域或与之部分重叠。使用模拟NS28羧基末端的合成肽进行的实验发现，这些肽无法竞争ssp结合。总之，这些结果表明NS28中的ssp结合位点（161 - 172位氨基酸）高度依赖于NS28细胞质C末端的构象完整性。还对NS28截短突变体与杆状病毒中表达的轮状病毒VP4的相互作用进行了检测。发现NS28的112至148位氨基酸对于NS28 - VP4结合至关重要。出乎意料的是，149至175位氨基酸不仅对于与VP4的相互作用不是必需的，而且缺少这些氨基酸的突变体显示出增强的结合活性。我们推测VP4结合结构域可能埋藏在NS28细胞质结构域中，并且ssp和VP4的结合可能是一个相互依赖的过程，与整个复合物出芽进入内质网的触发一起发挥作用。这些结果证明了NS28在独特的轮状病毒形态发生过程中的多效性特性。
