Schaal H, Klein M, Gehrmann P, Adams O, Scheid A
Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität, Düsseldorf, Germany.
J Virol. 1995 Jun;69(6):3308-14. doi: 10.1128/JVI.69.6.3308-3314.1995.
An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion glycoproteins.
设计了一种表达载体，用于检测人免疫缺陷病毒1型诱导膜融合时gp41 N端的结构要求。发现gp41 N端编码区的突变会破坏糖蛋白表达，因为这对Rev反应元件（RRE）有有害影响。在env的3'非编码序列中插入额外的RRE，使得无论在天然位置引入RRE的何种突变，都能实现高效的糖蛋白表达。这允许在gp41的N端序列中插入独特的限制性酶切位点SpeI，从而能够通过使用双链合成寡核苷酸方便且高效地对gp41 N端进行突变。构建了N端缺失1至7个氨基酸的突变体。用抗gp41抗体进行的Western免疫印迹分析证实了所有突变体的表达和切割情况。通过电穿孔和显微注射将野生型和突变体表达载体转染HeLa-T4 +细胞系后，监测突变体诱导膜融合的能力。细胞融合活性在缺失3个和4个氨基酸时急剧下降，在缺失5个氨基酸时完全丧失。亲本和突变体表达载体共转染导致细胞融合活性降低。突变糖蛋白的这种显性干扰程度与单独突变体的细胞融合活性下降情况平行。这表明存在融合活性所需的特定N端结构。然而，对于N端的精确长度似乎没有严格要求。这一发现得到了天然人免疫缺陷病毒1型分离株中该区域长度变化的支持，并且与甲型流感病毒和副粘病毒融合糖蛋白类似N端结构长度上明显的严格要求形成对比。
