Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion

Abstract

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.

Hexapeptides that interfere with HIV‐1 fusion peptide activity in liposomes block GP41‐mediated membrane fusion

“…FP sequence is absolutely required for gp41 fusogenic activity [10][11][12]. Previous studies have demonstrated that viral FP-induced pore-formation in liposomes is sensitive to physiologically relevant factors such as single blocking mutations [33][34][35][36].…”

Structural characterization of the fusion core in syncytin, envelope protein of human endogenous retrovirus family W

“…The fusion peptide (FP) was located at the N-terminus of syncytin TM subunit and consisted of hydrophobic, glycine-rich residues, which was essential for the initial penetration of the target cell membrane in many enveloped viruses, e.g., HIV-1 [23,24]. The hydrophobic 4-3 heptad repeat (HR) regions located adjacently to the N-and C-terminal portions of syncytin TM subunit were named as NHR and CHR, respectively ( Figs.…”

The FLG Motif in the N‐Terminal Region of Glucoprotein 41 of Human Immunodeficiency Virus Type 1 Adopts a Type‐I β Turn in Aqueous Solution and Serves as the Initiaion Site for Helix Formation

“…This is likely to be the case for the fusion peptide of gp41. Since the three residues N-terminal to the first residue of the helix are crucial to the stability of the helix, the experiments of successive deletion of residues N-terminal to F8 reported by Schaal et al (1995) can be rationalized. Thus, the capacity of gp41 to induce syncytium was greatly reduced as F8 became the fourth residue from the N-terminus of transmembrane glycoprotein as a result of deletion ; a complete interruption of syncytium formation was observed as further deletion rendered F8 the third or the second residue from the Nterminus of transmembrane glycoprotein.…”