Efficient Fusion at Neutral pH by Human Immunodeficiency Virus gp41 Trimers Containing the Fusion Peptide and Transmembrane Domains

Abstract

Human immunodeficiency virus (HIV) is membrane-enveloped, and an initial infection step is joining/fusion of viral and cell membranes. This step is catalyzed by gp41, which is a single-pass integral viral membrane protein. The protein contains an ∼170-residue ectodomain located outside the virus that is important for fusion and includes the fusion peptide (FP), N-helix, loop, C-helix, and viral membrane-proximal external region (MPER). The virion initially has noncovalent complexes between three gp41 ectodomains and three gp120 proteins. A gp120 contains ∼500 residues and functions to identify target T-cells and macrophages via binding to specific protein receptors of the target cell membrane. gp120 moves away from the gp41 ectodomain, and the ectodomain is thought to bind to the target cell membrane and mediate membrane fusion. The secondary and tertiary structures of the ectodomain are different in the initial complex with gp120 and the final state without gp120. There is not yet imaging of gp41 during fusion, so the temporal relationship between the gp41 and membrane structures is not known. This study describes biophysical and functional characterization of large gp41 constructs that include the ectodomain and transmembrane domain (TM). Significant fusion is observed of both neutral and anionic vesicles at neutral pH, which reflects the expected conditions of HIV/cell fusion. Fusion is enhanced by the FP, which in HIV/cell fusion likely contacts the host membrane, and the MPER and TM, which respectively interfacially contact and traverse the HIV membrane. Initial contact with vesicles is made by protein trimers that are in a native oligomeric state that reflects the initial complex with gp120 and also is commonly observed for the ectodomain without gp120. Circular dichroism data support helical structure for the N-helix, C-helix, and MPER and nonhelical structure for the FP and loop. Distributions of monomer, trimer, and hexamer states are observed by size-exclusion chromatography (SEC), with dependences on solubilizing detergent and construct. These SEC and other data are integrated into a refined working model of HIV/cell fusion that includes dissociation of the ectodomain into gp41 monomers followed by folding into hairpins that appose the two membranes, and subsequent fusion catalysis by trimers and hexamers of hairpins. The monomer and oligomer gp41 states may therefore satisfy dual requirements for HIV entry of membrane apposition and fusion.

Figure 1.

(A) Schematic diagrams of full-length HIV gp41 and the four truncated constructs of the present study with domains and corresponding colors: FP ≡ fusion peptide, red; N-helix, blue; loop, grey; C-helix, green; MPER ≡ membrane-proximal external-region, pink; TM ≡ transmembrane domain, orange; and endo = endodomain, white. The four constructs have non-native SGGRGG replacing native residues 582–627. (B) Amino acid sequences with colors matching segments in panel A and the non-native C-terminal G6LEH6 or G8LEH6 in black. The H6 is for Co²⁺-affinity chromatography and the G6LE/G8LE are necessary spacers for exposure of the H6 tag. The sequence is from the HXB2 laboratory strain of HIV.

Figure 2.

SDS-PAGE of the purified HM (MW = 13.7 kDa), HM_TM (MW = 16.7 kDa), FP_HM (MW = 16.5 kDa), and FP_HM_TM (MW = 18.9 kDa).

Figure 3.

Circular dichroism spectra at ambient temperature of samples containing ~10 μM protein in different buffer + detergent solutions: (A) 10 mM Tris at pH 7.4 and 0.2% SDS; (B) 20 mM phosphate at pH 7.4 and 0.25% DPC; and (C) 20 mM acetate at pH 4.0 and 0.25% DPC. All spectra for a single buffer + detergent condition were acquired on the same day. The 0.20% SDS is ~5 × CMC and the 0.25% DPC is ~8 × CMC.

Figure 4.

Circular dichroism in 0.2% SDS at pH 7.4 at 25, 60, and 90 ^oC. Differences between the ambient-temperature spectra of the same construct for these data vs. Fig. 3A may be partly due to use of different CD instruments.

Figure 5.

θ₂₂₂ vs. temperature in 0.2% SDS at pH 7.4. All spectra for a single construct were acquired on the same day.

Figure 6.

SEC of gp41 constructs under the following conditions: (A) 10 mM Tris at pH 7.4, 150 mM NaCl, 0.2% SDS, and ambient temperature; (B) 20 mM phosphate at pH 7.4, 150 mM NaCl, and 0.25% DPC at 4 °C; and (C) 20 mM acetate at pH 4.0, 150 mM NaCl, and 0.25% DPC at 4 °C. SEC was obtained with a Superdex 200-increase column, 1 mg/mL protein loading with ~10-fold dilution in the column, and A280 detection. The arrows in the plots are at the elution volumes of the MW standards, and some of the peaks are identified with dashed lines and with MW’s calculated from interpolation between MW standards.

Figure 7.

Vesicle fusion assays of gp41 proteins. Fusion was initiated by addition of an aliquot of protein stock solution at 0 s, and subsequent fusion was monitored by increased fluorescence associated with inter-vesicle lipid mixing. The stock contained 40 μM protein in buffer at pH 7.4 with 0.2% SDS, and the protein + vesicle mixture contained [protein] = 0.5 μM, [POPC+POPG+Chol] = 225 μM, and vesicle molar compositions and pH’s: (A) POPC:Chol = 2:1 at pH 3.2; (B) POPC:POPG:Chol = 8:2:5 at pH 3.2; (C) POPC:Chol = 2:1 at pH 7.4; and (D) POPC:POPG:Chol = 8:2:5 at pH 7.4. Fusion was not observed for any vesicle composition after addition of an aliquot of buffer + 0.2% SDS without protein. All data were obtained on the same day with the same protein stocks. The assay dead time was ~5 s. Small negative-values of fusion in a few cases were due to decreased fluorescence associated with the volume increase from addition of the protein aliquot.

Figure 8.

Long-time fusion extents (after ~600 s) based on the Fig. 7 data with protein:total lipid mole ratio = 1:450. Replicate data were acquired on different days with the same protein stocks and vesicles, and exhibited ±2% typical variation in extents among assay replicates. Fig. S6 shows extents from experiments using different protein stocks and vesicle preparations, and also comparative fusion extents at protein:lipid ratios = 1:450 and 1:225.

Figure 9.

Structural model of FP_HM_TM based on circular dichroism spectra of the four constructs, and other data. A monomer is shown for clarity but the model should be valid for trimers and hexamers. For a trimer, each interior N-helix of a parallel bundle contacts two exterior C-helices, and each exterior C-helix contacts two interior N-helices. Approximate residue numbers are displayed.

Figure 10.

Schematic illustrating (A) trimer and (B) monomer respectively favored in the absence and presence of peptide inhibitor. Panel B displays “C34” inhibitor which contains C-helix residues 628–661. C34 binding to the N-helix may require dissociation of the C-helix from the N-helix. The sequence color coding matches Figures 1 and 9, and loops between structured regions are not displayed for clarity. The FP’s from different trimers or monomers adopt antiparallel β sheet structure, and the trimeric TM bundle is based on a TM peptide structure.^, Fusion is enhanced in panel A vs. B because of greater clustering of membrane-perturbing protein regions in the trimer vs. monomer. This enhancement exists for the displayed hemifusion state as well as membrane states that precede hemifusion.