Anderson K, Stott E J, Wertz G W
Department of Microbiology, University of Alabama, Birmingham 35294.
J Gen Virol. 1992 May;73 ( Pt 5):1177-88. doi: 10.1099/0022-1317-73-5-1177.
The intracellular processing and transport of the respiratory syncytial virus (RSV) fusion (F) glycoprotein was examined by comparing the maturation and stability of wild-type F, uncleaved mutant F and chimeric F glycoproteins expressed by recombinant vaccinia viruses to that of F protein expressed by RSV. One of the recombinant viruses, vF317, expressed F protein (F317) that was processed like the RSV F glycoprotein. F317 was synthesized initially as F0, the uncleaved glycosylated precursor of mature F protein, and formed stable oligomeric structures that were maintained following cleavage of F0 to form the disulphide bond-linked F1 and F2 subunits. Most of the newly synthesized F0 expressed by either RSV or by vF317 was sensitive to treatment with endoglycosidase H (Endo H). Following cleavage of F0, F1 was resistant to Endo H, suggesting that conversion to complex-type sugars, which takes place in the medial Golgi apparatus, occurred simultaneously with or immediately prior to cleavage of F0 into F1 and F2. Another recombinant virus, vF313, synthesized only uncleaved F protein (F313) that comigrated with F0. Uncleaved F313 was expressed as a stable glycosylated protein; however, unlike cleaved F317, its oligosaccharides were not modified to complex forms, as determined from its Endo H sensitivity, and uncleaved F313 did not assemble into stable oligomeric structures. Nucleotide sequence analysis of the cDNA clones encoding F313 and F317 revealed four predicted amino acid sequence differences, none of which were located at the cleavage site. Expression of chimeric F proteins obtained by restriction fragment exchange between the two cDNA clones indicated that two amino acid changes in the F1 domain, located at amino acid residues 301 (Val to Ala) and 447 (Val to Met), resulted in the expression of uncleaved F protein. A change at either of these two amino acid residues, 301 or 447, resulted in the expression of inefficiently cleaved F protein, defining an additional F protein phenotype. Pulse-chase analyses to examine the association of recombinant F glycoproteins with gradient-purified fractionated membranes or with GRP78-BiP, a protein resident in the endoplasmic reticulum (ER) which binds to nascent proteins, revealed that uncleaved F protein (F313) is associated with GRP78-BiP in the ER for a longer time than F317, and little if any F313 was transported to the cell surface. In addition, the uncleaved F protein (F313) was not recognized by a panel of F protein-specific monoclonal antibodies in ELISA or indirect immunofluorescence assays, suggesting that F313 was misfolded and, as a result, not transported properly or cleaved.
通过比较重组痘苗病毒表达的野生型融合（F）糖蛋白、未切割的突变型F糖蛋白和嵌合F糖蛋白与呼吸道合胞病毒（RSV）表达的F蛋白的成熟度和稳定性，研究了RSV F糖蛋白的细胞内加工和运输过程。其中一种重组病毒vF317表达的F蛋白（F317）的加工方式与RSV F糖蛋白相同。F317最初以F0形式合成，F0是成熟F蛋白未切割的糖基化前体，并形成稳定的寡聚结构，在F0切割形成二硫键连接的F1和F2亚基后仍能维持。RSV或vF317表达的大多数新合成的F0对内切糖苷酶H（Endo H）处理敏感。F0切割后，F1对Endo H有抗性，这表明在内质网中发生的向复杂型糖的转化与F0切割成F1和F2同时或在其之前立即发生。另一种重组病毒vF313只合成了与F0迁移率相同的未切割F蛋白（F313）。未切割的F313以稳定的糖基化蛋白形式表达；然而，与切割后的F317不同，根据其对Endo H的敏感性确定，其寡糖未被修饰为复杂形式，且未切割的F313未组装成稳定的寡聚结构。对编码F313和F317的cDNA克隆进行核苷酸序列分析，发现有四个预测的氨基酸序列差异，其中没有一个位于切割位点。通过两个cDNA克隆之间的限制性片段交换获得的嵌合F蛋白的表达表明，F1结构域中位于氨基酸残基301（缬氨酸突变为丙氨酸）和447（缬氨酸突变为甲硫氨酸）的两个氨基酸变化导致了未切割F蛋白的表达。这两个氨基酸残基（301或447）中的任何一个发生变化都会导致F蛋白切割效率低下，从而定义了另一种F蛋白表型。脉冲追踪分析用于检测重组F糖蛋白与梯度纯化的分级膜或与GRP78-BiP（一种驻留在内质网（ER）中与新生蛋白结合的蛋白）的结合，结果显示未切割的F蛋白（F313）在内质网中与GRP78-BiP的结合时间比F317长，并且几乎没有F313转运到细胞表面。此外，在ELISA或间接免疫荧光试验中，一组F蛋白特异性单克隆抗体未识别出未切割的F蛋白（F313），这表明F313发生了错误折叠，因此未被正确转运或切割。
