McGinnes Lori W, Gravel Kathryn, Morrison Trudy G
Department of Molecular Genetics and Microbiology/Program in Immunology and Virology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655.
J Virol. 2002 Dec;76(24):12622-33. doi: 10.1128/jvi.76.24.12622-12633.2002.
Conformational changes in the Newcastle disease virus (NDV) fusion (F) protein during activation of fusion and the role of HN protein in these changes were characterized with a polyclonal antibody. This antibody was raised against a peptide with the sequence of the amino-terminal half of the F protein HR1 domain. This antibody immunoprecipitated both F(0) and F(1) forms of the fusion protein from infected and transfected cell extracts solubilized with detergent, and precipitation was unaffected by expression of the HN protein. In marked contrast, this antibody detected significant conformational differences in the F protein at cell surfaces, differences that depended upon HN protein expression. The antibody minimally detected the F protein, either cleaved or uncleaved, in the absence of HN protein expression. However, when coexpressed with HN protein, an uncleaved mutant F protein bound the anti-HR1 antibody, and this binding depended upon the coexpression of specifically the NDV HN protein. When the cleaved wild-type F protein was coexpressed with HN protein, the F protein bound anti-HR1 antibody poorly although significantly more than F protein expressed alone. Anti-HR1 antibody inhibited the fusion of R18 (octadecyl rhodamine B chloride)-labeled red blood cells to syncytia expressing HN and wild-type F proteins. This inhibition showed that fusion-competent F proteins present on surfaces of syncytia were capable of binding anti-HR1. Furthermore, only antibody which was added prior to red blood cell binding could inhibit fusion. These results suggest that the conformation of uncleaved cell surface F protein is affected by HN protein expression. Furthermore, the cleaved F protein, when coexpressed with HN protein and in a prefusion conformation, can bind anti-HR1 antibody, and the anti-HR1-accessible conformation exists prior to HN protein attachment to receptors on red blood cells.
用多克隆抗体对新城疫病毒（NDV）融合（F）蛋白在融合激活过程中的构象变化以及血凝素神经氨酸酶（HN）蛋白在这些变化中的作用进行了表征。该抗体是针对F蛋白HR1结构域氨基末端一半序列的肽产生的。该抗体从用去污剂溶解的感染和转染细胞提取物中免疫沉淀融合蛋白的F(0)和F(1)形式，并且沉淀不受HN蛋白表达的影响。与之形成显著对比的是，该抗体在细胞表面检测到F蛋白存在明显的构象差异，这些差异取决于HN蛋白的表达。在没有HN蛋白表达的情况下，该抗体对切割或未切割的F蛋白检测极少。然而，当与HN蛋白共表达时，未切割的突变F蛋白与抗HR1抗体结合，并且这种结合取决于特定的新城疫病毒HN蛋白的共表达。当切割的野生型F蛋白与HN蛋白共表达时，F蛋白与抗HR1抗体的结合较差，尽管明显多于单独表达的F蛋白。抗HR1抗体抑制了R18（十八烷基罗丹明B氯化物）标记的红细胞与表达HN和野生型F蛋白的多核细胞的融合。这种抑制表明多核细胞表面存在的具有融合能力的F蛋白能够结合抗HR1。此外，只有在红细胞结合之前添加的抗体才能抑制融合。这些结果表明，未切割的细胞表面F蛋白的构象受HN蛋白表达的影响。此外，当与HN蛋白共表达且处于融合前构象时，切割的F蛋白可以结合抗HR1抗体，并且在HN蛋白附着于红细胞上的受体之前，抗HR1可及的构象就已存在。
