Modification of the HIV-1 Fusion Inhibitor 2P23 via N-Glycosylation Sites Enhances Its Cell Membrane Level and Antiviral Activity

HIV (Human immunodeficiency virus) gene therapy represents a promising strategy to suppress viral replication and potentially achieve long-term control of infection. One of the effective strategies is based on the generation of the genetically modified virus-resistant T cells by expressing peptide inhibitors of viral fusion on the cell membrane. These peptides, such as MT-C34 and 2P23, are derived from HIV gp41 protein and inhibit HIV entry into modified cells. We previously demonstrated in cell-to-cell viral transmission experiments that the 2P23 peptide showed lower protective activity than the MT-C34 peptide. In this study, we hypothesized that the difference in protective activity may arise from the differential N-glycosylation of the peptides. To test this, the 2P23 sequence was modified by introducing the N-glycosylation site (NDT) at the N-, C-, or both termini. These modifications significantly enhanced the plasma membrane expression and protective activity of the 2P23 peptide, with the NDT-2P23-NDT variant being the most potent. The validity of this peptide engineering approach was confirmed with the highly similar HP23L peptide, demonstrating an increased cell membrane expression and antiviral potency of the modified variant. Experiments with the N-glycosylation inhibitor tunicamycin provide indirect evidence that this effect is glycosylation-dependent. These findings suggest that introducing N-glycosylation sites is a versatile approach to improve the potency of membrane-bound antiviral peptides for HIV gene therapy.