REQUIREMENT OF N-TERMINAL AMINO-ACID-RESIDUES OF GP41 FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE 1-MEDIATED CELL-FUSION
REQUIREMENT OF N-TERMINAL AMINO-ACID-RESIDUES OF GP41 FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE 1-MEDIATED CELL-FUSION
复制标题
DOI：
10.1128/jvi.69.6.3308-3314.1995
复制
发表时间：
1995-06-01
影响因子：
5.4
通讯作者：
SCHEID, A
中科院分区：
文献类型：
--
作者：
SCHAAL, H;KLEIN, M;SCHEID, A
设计表达载体来测试人类免疫缺陷病毒1型诱导的膜融合的gp41 N末端的结构要求。 gp41 N 末端编码区域的突变被发现会破坏糖蛋白的表达，因为它会对 Rev 反应元件 (RRE) 产生有害影响。在 env 的 3'-非编码序列中插入额外的 RRE 使得有效的糖蛋白表达成为可能，而不管在自然位置中引入 RRE 的突变如何。这允许在 gp41 N 端序列中插入独特的限制性位点 SpeI，从而允许通过使用双链合成寡核苷酸对 gp41 N 端进行方便且有效的突变。构建了N末端1至7个氨基酸缺失的突变体。所有突变体的表达和切割均通过使用抗gp41抗体的Western免疫印迹分析来证实。通过电穿孔和显微注射用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。删除3个和4个氨基酸后，细胞融合活性的效率急剧下降，删除5个氨基酸后，细胞融合活性的效率完全丧失。亲本和突变体表达载体的共转染导致细胞融合活性降低。突变体糖蛋白的显着干扰程度与突变体单独的细胞融合活性的降低平行。这表明存在融合活性所需的特定 N 末端结构。然而，对于N末端的精确长度似乎没有严格的要求。这一发现得到了天然人类免疫缺陷病毒 1 型分离株中该区域的长度变化的支持，并且与甲型流感病毒和副粘病毒融合糖蛋白的类似 N 末端结构的长度的明显严格性形成对比。
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.