Structural and Functional Characterization of the Secondary Mutation N126K Selected by Various HIV-1 Fusion Inhibitors

Abstract

Peptides derived from the C-terminal heptad repeat (CHR) region of HIV-1 gp41 is potent viral membrane fusion inhibitors, such as the first clinically approved peptide drug T20 and a group of newly-designed peptides. The resistance profiles of various HIV-1 fusion inhibitors were previously characterized, and the secondary mutation N126K in the gp41 CHR was routinely identified during the in vitro and in vivo selections. In this study, the functional and structural relevance of the N126K mutation has been characterized from multiple angles. First, we show that a single N126K mutation across several HIV-1 isolates conferred mild to moderate cross-resistances. Second, the N126K mutation exerted different effects on Env-mediated HIV-1 entry and cell-cell fusion. Third, the N126K mutation did not interfere with the expression and processing of viral Env glycoproteins, but it disrupted the Asn126-based glycosylation site in gp41. Fourth, the N126K mutation was verified to enhance the thermal stability of 6-HB conformation. Fifth, we determined the crystal structure of a 6-HB bearing the N126K mutation, which revealed the interhelical and intrahelical interactions underlying the increased thermostability. Therefore, our data provide new information to understand the mechanism of HIV-1 gp41-mediated cell fusion and its resistance mode to viral fusion inhibitors.

tANCHOR: A Novel Mammalian Cell Surface Peptide Display System

“…To demonstrate efficient display and glycosylation in HEK 293T cells of a heterologous protein anchored with CD63, we used a vector expressing the ectodomain of HIV-1 known to contain four canonical N-glycosylation sites [21]. Significant N-glycosylation leads to smeared protein bands in a Western blot [22][23][24][25]. As illustrated in Figure 3C, expression of the 60 kDa (calculated molecular weight of the protein) CD63 LEL-gp41 ecto -mCherry resulted in such a smeared band.…”

In Vitro Selection and Characterization of HIV-1 Variants with Increased Resistance to LP-40, Enfuvirtide-Based Lipopeptide Inhibitor

“…The fusion activities of Env mutants were measured based on a dual-split-protein (DSP) system [ 32 , 33 ]. On the first day, 2 × 10 5 /mL HEK293T cells were seeded into a 96-well plate and 293FT cells were seeded into a 6 cm culture dish, and then these cells were incubated at 37 °C with 5% CO 2 .…”

“…The expression and processing profile of Env mutants in the cell and the incorporation and cleavage of Env mutants in pseudotype particles were examined using a Western blotting assay as described previously [ 32 ]. To acquire the cell lysates, on the first day, 4 × 10 5 /mL HEK293T cells were seeded in a 6-well plate; on the next day, the Env-coding plasmid was transfected into the HEK293T cells, and HEK293T cells were harvested after 48 h. These transfected cells were lysed for 30 min on ice and centrifuged at 20,000× g at 4 °C for 1 h to remove insoluble materials.…”