The six-helix bundle of human immunodeficiency virus Env controls pore formation and enlargement and is initiated at residues proximal to the hairpin turn

Abstract

Residues that create the grooves of the human immunodeficiency virus type 1 (HIV-1) Env triple-stranded coiled coil (HR1) and the residues that pack into the grooves (HR2) to complete the formation of the six-helix bundle (6HB) were mutated. The extent and kinetics of fusion as well as pore enlargement were measured for each mutant. Mutations near the hairpin turns of each monomer of the 6HB were more important than those far from the turn, for both HR1 and HR2. This result is consistent with the idea that binding of HR2 to the HR1 grooves is initiated near the hairpin turn of each monomer. Mutations at the distal portions also reduced fusion, albeit to a smaller extent. An intermediate of fusion (temperature-arrested state [TAS]) was formed, and the consequences of mutation were compared; a mutant that exhibited less fusion also showed slower kinetics from TAS. This suggests that formation of the bundle is a rate-limiting step downstream of the intermediate state. The rate of enlargement of a fusion pore also correlated with the extent and kinetics of fusion. The rate of pore enlargement was severely reduced by mutation. This supports our prior conclusion that formation of the 6HB occurs after pore creation and strongly suggests that the free energy released by bundle formation is directly used to promote pore growth.

FIG. 1.

Depiction of a 6HB. Three HR1 segments, one from each gp41 monomer, combine to form a central triple-stranded coiled coil. Three HR2 segments of gp41 pack into the grooves to complete the bundle (six-circle figure). Residues at positions “a” and “d” of HR2 pack into the grooves. All were mutated, one at a time, to alanine. The “e” and “g” of HR1 create the groove. Residues at these positions were mutated to alanine, except for residue A558 (a “g” position) which is naturally alanine.

FIG. 2.

Extent of fusion before and after addition of CPZ. (A) Fusion extent for mutants of HR2 displayed according to increasing residue number. (B) Extent of fusion for mutants of HR1 shown in order of residue number. Black bars are percentages of bound Ef/Tg cells that exhibit transfer of calcein and CMAC. White bars show the percentages of bound cells exhibiting dye transfer after the addition of CPZ. Dye spread was monitored within 1 min after adding CPZ. Only mutations with open bars were tested with CPZ. Error bars are SEM.

FIG. 3.

Promotion of aqueous dye spread by the addition of CPZ requires prior advancement of the fusion process. Addition of CPZ after a 2-h incubation of Ef and Tg cells did not induce further aqueous dye spread (column 1). Including 100 nM C34 during the 2-h incubation abolished the ability of CPZ to induce dye spread (column 2). The addition of CPZ after cell-cell incubation for 1 h and 50 min followed by addition of C34 did not lead to further dye spread (column 3). For Ef cells expressing the G572A mutant, the addition of CPZ after a 2-h cell-cell incubation led to a significant increase in aqueous dye spread (column 4). For the G572A mutant, the inclusion of C34 during cell-cell incubation abolished both fusion and the ability of CPZ to further promote fusion (column 5). Adding C34 after the cell-cell incubation did not affect the subsequent ability of CPZ to promote fusion (column 6).

FIG. 4.

HIV Env is cleaved into subunits. Membrane proteins from cells expressing HIV Env (or mutants) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then labeled with an anti-HIV gp41 antibody (2F5). The bands shown were the most prominent on the gel; minor bands of dimerized gp41 and larger aggregates were also present. Bands are not present for the L663A and W666A mutants because the recognition site of 2F5 includes these positions (4). The contrast in the image has been uniformly heightened to aid visualization here. Quantification of all bands, however, was performed using the original images.

FIG. 5.

Kinetics of fusion from TAS. Extents of fusion for all mutants were normalized to 1. (A) HR2 mutants. (B) HR1 mutants. Insets within panels A and B show expanded views of fusion kinetics over the first minute.

FIG. 6.

Correlation of extents and kinetics of fusion. Kinetics of fusion were measured from TAS and parameterized as the time for half the fusion events to occur. (A) HR2 mutants. (B) HR1 mutants.

FIG. 7.

Growth of fusion pores for mutations within HR2. Top panels show calcein fluorescence of the Tg cell as a function of time for all cell pairs tested for each mutant. Each curve tracks the results from a single cell pair. The rate of change in fluorescence is a measure of the rate of change in pore size. Lower left panel, average fluorescence of Tg cells versus time; lower right panel, the extent of cells for which the small dye calcein (black bars) and the large dye CMFDA (open bars) transferred from Ef to Tg cells. Error bars are the standard errors of the means. Measurements for each mutant were performed at least eight times.

FIG. 8.

Extent of fusion displayed after aligning N- and C-terminal residues according to a standard numbering system (19). The hairpin turn is comprised of residues 597 to 609. The mutated residues for which the extent of fusion is shown are underlined.