Lytic Inactivation of Human Immunodeficiency Virus by Dual Engagement of gp120 and gp41 Domains in the Virus Env Protein Trimer.

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Biochemistry. Author manuscript; available in PMC 2017 May 25. Published in final edited form as: Biochemistry. 2016 November 08; 55(44): 6100–6114. doi:10.1021/acs.biochem.6b00570.

Lytic Inactivation of Human Immunodeficiency Virus by Dual Engagement of gp120 and gp41 Domains in the Virus Env Protein Trimer Bibek Parajuli†, Kriti Acharya†, Reina Yu†, Brendon Ngo†, Adel A. Rashad†, Cameron F. Abrams†,‡, and Irwin M. Chaiken*,†

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†Department

of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, United States

‡Department

of Chemical and Biological Engineering, Drexel University, Philadelphia, Pennsylvania 19104, United States

Abstract

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We recently reported the discovery of a recombinant chimera, denoted DAVEI (dual-acting virucidal entry inhibitor), which is able to selectively cause specific and potent lytic inactivation of both pseudotyped and fully infectious human immunodeficiency virus (HIV-1) virions. The chimera is composed of the lectin cyanovirin-N (CVN) fused to the 20-residue membraneproximal external region (MPER) of HIV-1 gp41. Because the Env gp120-binding CVN domain on its own is not lytic, we sought here to determine how the MPER(DAVEI) domain is able to endow the chimera with virolytic activity. We used a protein engineering strategy to identify molecular determinants of MPER(DAVEI) that are important for function. Recombinant mutagenesis and truncation demonstrated that the MPER(DAVEI) domain could be significantly minimized without loss of function. The dependence of lysis on specific MPER sequences of DAVEI, determination of minimal linker length, and competition by a simplified MPER surrogate peptide suggested that the MPER domain of DAVEI interacts with the Env spike trimer, likely with the gp41 region. This conclusion was further supported by observations from binding of the biotinylated MPER surrogate peptide to Env protein expressed on cells, monoclonal antibody competition, a direct binding enzyme-linked immunosorbent assay on viruses with varying numbers of trimeric spikes on their surfaces, and comparison of maximal interdomain spacing in DAVEI to that in high-resolution structures of Env. The finding that MPER(DAVEI) in CVN–MPER

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*

Corresponding Author: Address: 245 N. 15th St., New College Building, Room 11102, Drexel University College of Medicine, Philadelphia, PA 19102. Telephone: +1-215-762-4917. [email protected]. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00570. Index of the Supporting Information (PDF) Additional observations and results (PDF) Author Contributions B.P. designed studies, performed experiments and analyses, and prepared the manuscript. K.A. assisted with subcloning experiments and optimization of the cell-based ELISA. R.Y. assisted with the p24 ELISA experiment and protein characterization. C.F.A and I.M.C. initiated this project. and I.M.C. provided guidance for experimental design, interpretation of data. and preparation of the manuscript. Notes The authors declare no competing financial interest.

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linker sequences can be minimized without loss of virolytic function provides an improved experimental path for constructing size-minimized DAVEI chimeras and molecular tools for determining how simultaneous engagement of gp120 and gp41 by these chimeras can disrupt the metastable virus Env spike.

Graphical abstract

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The infection of host cells by HIV-1 occurs by virus–cell fusion driven by a cascade of interactions and conformational transformations programmed in the virus envelope trimeric glycoprotein (Env). HIV-1 Env is composed of receptor-binding gp120 and membraneanchored gp41 subunits held together by noncovalent interactions.1 gp120 interacts with the receptor (CD4) and coreceptor (CCR5/CXCR4),1 triggering conformational changes in both gp120 and gp41.2,3 After receptor binding, the fusion peptide at the N-terminus of gp41 is exposed and interacts with the target cell membrane to form a prehairpin intermediate that bridges the virus and cell membranes.4 The gp41 in this transient prehairpin reassembles into a six-helix bundle5,6 to bring the two membranes sufficiently close together for virus– cell fusion5–7 and formation of pores that allow viral contents to be released into the cell. The ability of receptor engagement to trigger energy-requiring membrane fusion for entry can be ascribed to the intrinsic metastability of the Env spike protein complex, with six-helix bundle formation providing an energetic driving force for membrane fusion.

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We previously hypothesized that an engineered protein chimera composed of a gp120 ligand fused to MPER could hijack the intrinsic metastability of HIV-1 Env by anchoring on the virus Env spike and imparting sufficient stress on the virus membrane adjacent to the spike protein to disrupt the virus membrane and cause virus poration and consequent irreversible inactivation. We devised the chimeric molecule, termed DAVEI (dual-acting viral entry inhibitor), by cloning the gp120-binding lectin protein cyanovirin-N (CVN) joined to gp41 MPER using a flexible [(Gly4Ser)X] linker.8 DAVEI fusions could block HIV-1 infection in a pseudoviral infection assay at low nanomolar concentrations typical of CVN potency. Importantly, treating viral stocks with DAVEI molecules in the absence of target cells led to dose-dependent virolysis and release of intraluminal p24.8 In contrast to CVN-containing DAVEI, CVN alone binds with high affinity to glycans on gp120 and prevents binding of gp120 to CD4 but does not cause membrane poration.9 Hence, it is inclusion of the MPER domain in DAVEI that causes virus membrane lysis. While the lytic inactivation function of DAVEI is striking, the mechanism by which the MPER(DAVEI) domain allows virolysis has remained undetermined. The MPER peptide on its own has been found to disrupt membranes in model systems,10–12 and initially, we envisioned that the MPER domain in DAVEI might similarly function by binding to the virus membrane. However, competition of DAVEI virolysis with the MPER peptide alone

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occurred at surprisingly low concentrations8 inconsistent with general MPER capping of the membrane surface to prevent DAVEI function. We sought to resolve the role of MPER(DAVEI) in this study. We employed a combined mutagenesis and sequence redesign approach to identify minimal sequence surrogates of the MPER domain of DAVEI required for virolysis. The effects of MPER and linker simplification, together with binding measurements and correlation with Env protein structure, argued that the MPER(DAVEI) domain interacts with Env protein itself. The results obtained open up possibilities for devising smaller DAVEI constructions and also a route to further structure–function analysis of the way in which dual gp120–gp41 engagement causes lytic inactivation of HIV-1.

MATERIALS AND METHODS Reagents

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Modified human osteosarcoma cells (HOS.T4.R5) were a gift from N. Landau. HEK293T cells were purchased from ATCC. The HIV-1 BaL.01 plasmid was a gift from J. M. Garcia. DNA purification was conducted using the Promega miniprep kit (catalog no. A1222). BamHI (catalog no. R0136S) and NdeI (R0111S) restriction enzymes and DNA ligase (catalog no. M0202S) were purchased from New England Biolabs for plasmid digestion and ligation. Mutagenesis was conducted using a Quick Change II XL Site Directed Mutagenesis kit (catalog no. 200522) with reactions conducted using PFU Ultra Polymerase (catalog no. 600380-51) obtained from Agilent technologies. All other reagents were purchased from Sigma-Aldrich unless otherwise specified. Peptide Synthesis

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Peptides were synthesized with a microwave peptide synthesizer (CEM LibertyBlue), using standard Fmoc chemistry on Rink amide resin. All peptides were purified to >95% homogeneity as judged by an analytical reverse phase C18 high-performance liquid chromatography column. Biotinylated-Trp3 peptide was purchased from Scilight-Peptide Inc. The integrity of purified peptides was confirmed by mass spectrometry; observed masses for Trp1, Trp2, Trp3, Trp4, MPER, and Bt-Trp3 were 446.5, 905.2, 1204.6, 2011.3, 2714.9, and 1561.11 Da, respectively, versus expected masses of 447.5, 905.02, 1205.34, 2011.22, 2715.11, and 1559.82 Da, respectively (Supporting Information 11). Plasmid Constructs

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Preparation of DAVEI-L4(ΔMPER) and DAVEI-L4(ΔCRAC) Constructs—Plasmids containing DAVEI-L48 were confirmed for the base sequence. To investigate the virolytic role of the CRAC (cholesterol recognition amino acid consensus) sequence (LWYIK) located at the C-terminus of MPER, two DAVEI-L4 truncated proteins were constructed: DAVEI-L4(ΔCRAC) and DAVEI-L4-(ΔMPER) (Figure 1A). Because the viral membrane contains cholesterol, we hypothesized CRAC(DAVEI)–cholesterol(virus) interaction to be an important aspect of virolysis. Forward primer 5′GAAATAACAGAATGGTAGTAGTGGATAAAATAGTAATAAAAGC-3′ and 5′CATCATCATCATCATCATTAGAAATGGGCAAGTTTGTGG-3′ and their reverse complement primers were used to create DAVEI-L4(ΔCRAC) and DAVEI-L4(ΔMPER), respectively, using a standard mutagenesis protocol from Agilent technologies (Figure 1B). Biochemistry. Author manuscript; available in PMC 2017 May 25.

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Subcloning of DAVEI-L2 and DAVEI-L0 Constructs—BamHI restriction sites were introduced at two different positions within the DAVEI-L4 plasmid for removing glycine-4serine repeats. Primer 5′GGTACCCTGAAATACGAAGGATCCGGTGGCGGAGGGTC-3′ and 5′GGTAGTGGTGGAGGCGGATCCCATCATCATCATCATCAT-3′ and their reverse complements were used to introduce two BamHI restriction sites. Forward primer 5′GAAGGAGATATACATATGAAATACCTGCTGC-3′ and its reverse complement were used to introduce the NdeI restriction site for both of the modified plasmid constructs. The modified plasmids were digested with BamHI and NdeI enzymes for 2 h at 37 °C before being treated with shrimp alkaline phosphatase for 30 min at room temperature. The excised vectors were run on a 1% agarose gel (Supporting Information 8), purified using a gel extraction kit (Qiagen), and religated using T4 ligase (New England Biolabs M0202L). 5′GGAGGGTCGGGCGGAGGTGGATCCGGTGGCGGAGGTGG-3′ and its reverse complement primer and 5′GGTAGTGGTGGAGGCGGATCCCATCATCATCATCATCATC-3′ and its reverse complement primer were used to introduce two BamHI restriction sites for making the DAVEI-L2 construct. Forward primer 5′GAAGGAGATATACATATGAAATACCTGCTGC-3′ and its reverse complement primer were used to introduce the NdeI restriction site for both modified plasmid constructs. Both mutated plasmids were digested with BamHI and NdeI (New England Biolabs) for 2 h at 37 °C before being treated with shrimp alkaline phosphatase for 30 min at room temperature. The excised vectors were run on a 1% agarose gel (Supporting Information 8), purified using a gel extraction kit (Qiagen), and religated using T4 ligase (New England Biolabs M0202L) overnight at room temperature. All ligated products were transformed on XL1-Blue Escherichia coli cells, plated on LB-kanamycin agar plates, and transformed overnight at 37 °C. Positive colonies were selected for miniprep, and plasmids were isolated and sequenced (Figure 4A).

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DAVEI-L4(ΔCRAC) Constructs with the MPER Mutation on Tryptophan—The DAVEI-L4(ΔCRAC) construct has an MPER sequence (lacking the CRAC sequence) at its C-terminus (DKWASLWNWFEITEW) with tryptophan at positions 3, 7, 9, and 15. Alanine scanning mutagenesis was performed to create W15A, W9,15A, W7,9,15A, and W3,7,9,15A mutations to determine the effect of tryptophan residues on the lytic behavior of DAVEI-L4. W3A, W7A, W9A, and W15A mutations were created using 5′CATCATGACAAAGCGGCAAGTTTG-3′, 5′-CAAGTTTGGCGAATTGGTTTG-3′, 5′GGCAAGTTTGTGGAATGCGTTTGAAATAACAGAATGG-3′, and 5′GTTTGAAATAACAGAAGCGTAGTAGTGGATAAAATAG-3′ and their reverse complement primers, respectively. Because W7 and W9 are too close, a combination mutation was formed using 5′-GCAAGTTTGGCGAATGCGTTTGAAATAAC-3′ that had tryptophan to alanine mutations at both positions 7 and 9. Schematic representations for plasmid constructs are shown in Figure 2A. Generation of DAVEI Derivatives with Shortened MPER Sequences—DAVEIL4(ΔCRAC) has four tryptophans at positions 3, 7, 9, and 15. To identify the minimal MPER sequence required for lysis by DAVEI-L4, the MPER sequence was truncated by


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introducing stop codons after each tryptophan. Three truncated proteins, namely, DAVEIL4-1Trp, DAVEI-L4-2Trp, and DAVEI-L4-3Trp, were formed using 5′CATCATCATGACAAATGGTGAAGTTTGTGGAATTGG-3′, 5′GACAAATGGGCAAGTTTGTGGTAGTGGTTTGAAATAAC-3′, and 5′GGCAAGTTTGTGGAATTGGTAGGAAATAACAG-3′ and their reverse complements, respectively, as shown in Figure 3A. The number X in DAVEI-L4-XTrp represents the total number of tryptophans present on the MPER sequence of the DAVEI-L4 derivative (Figure 3A). Protein Expression, Purification, and Validation of DAVEI Fusions


Purified DAVEI-L4(ΔCRAC) and all other derived plasmids were transformed on BL21(DE3) pLysS competent cells (Promega) and plated on LB-kanamycin agar plates for 16–18 h at 37 °C. Positive colonies were isolated and inoculated on 1 mL of LB-kanamycin for 16 h at 30 °C. A 1 mL culture was subcultured to a 4 L culture, grown for 6–8 h at 30 °C, and induced with 1 mM isopropyl D-thiogalactopyranoside (IPTG) when the optical density was in the range of 0.6–0.8. Cells were induced for 16–18 h at 16 °C and a shaking speed of 225 rpm. Grown cells were pelleted by centrifugation, and 60 mL of buffer A (50 mM Na2HPO4, 300 mM NaCl, and 10 mM imidazole) was added to the pellet. The mixture was sonicated using a microtip probe (Misonix Sonicator 3000) for three 1 min intervals at 70 V. To separate the protein from the cell debris, the sonicated mixture was spun down at 10000g for 45 min at 4 °C. The protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen), followed by gel filtration with a 26/60 Superdex 200 prep-grade column (GE Healthcare) using an AKTA fast protein liquid chromatograph (GE Healthcare). The homogeneity of these proteins was assessed with protein eluates run on 18% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels (Supporting Information 9) followed by ELISA analysis. Fractions containing target proteins were concentrated and buffer exchanged with phosphate-buffered saline at pH 7.4 using a 3000 molecular weight cutoff (MWCO) spin filter (Amicon). The final concentration was determined using absorbance at 280 nm and extinction coefficients for various DAVEI constructs. CyanovirinN was produced in E. coli as previously reported.13–15 The ability of fusion proteins to bind YU2 gp120 was analyzed by a sandwich ELISA. First, varying concentrations (500–0.5 ng) of CVN or fusion proteins were adsorbed to wells of 96-well high-binding polystyrene plates (Fisher Scientific) for 12 h at 4 °C. The plates were blocked with 3% bovine serum albumin (BSA) (Research Products International Corp.) in phosphate-buffered saline (PBS) for 2 h at 25 °C; 100 nM purified wild-type YU2 gp120 protein was loaded onto the plates and incubated for 1 h. The plates were washed with 0.1% (v/v) Tween 20 (PBS-T) for 15 min. All subsequent incubation steps were performed in 0.5% BSA in PBS. After a 15 min incubation period, 50 μL of sheep anti-gp120 (dilution factor of 1:3000; Aalto Bioreagents, D7324) was added to the plates for 1 h at room temperature. The plates were washed with PBS-T followed by addition of rabbit anti-sheep secondary antibody (dilution factor of 1:3000; life technologies, PJ209733). The plates were incubated for an additional 1 h at 25 °C and washed three times with PBS-T and one more time with PBS before the addition of OPD. Plates were developed for 30 min in the dark, and the final absorbance was measured at 450 nm using an Infinite m50 (Tecan) plate reader.


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Expression and Purification of gp120 Protein

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YU2 gp120 in the pcDNA3.1 plasmid was purified using a Qiagen MaxiPrep kit (Qiagen). The wild-type (WT) plasmid was transiently transfected into HEK293F cells according to the manufacturer’s protocol (Invitrogen). Five days post-transfection, cells were harvested and spun down, and the supernatant was filtered through 0.2 μm filters. Purification was performed over a 17b antibody column prepared using an NHS-activated Sepharose, HiTrap HP column (GE Healthcare). gp120 was eluted from the column using 0.1 M glycine buffer (pH 2.4). The pH of the eluted protein was rapidly neutralized by addition of 1 M Tris (pH 9.0). Purified proteins were run on 10% SDS gels for detecting the expression of protein. Eluted proteins were immediately dialyzed into 1× PBS and loaded onto a HiLoad 26/60 Superdex 200 HR prepacked gel filtration column (GE) for size exclusion chromatography. Eluted fractions of protein were run on 10% SDS–PAGE gels. Purified monomeric fractions of gp120 were concentrated and flash-frozen at −80 °C.

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Direct Binding of DAVEI Derivatives to gp120 Monomeric Proteins for Functional Validation Recombinantly purified YU2 gp120 protein (50 ng) was immobilized on ELISA plates. Plates were blocked overnight with 3% BSA at 16 °C. Varying concentrations of DAVEI derivatives were added to the plate, and the plate was incubated for 2 h followed by two 30 min washes with 1× PBS. Rabbit anti-CVN (Biosyn Inc.; dilution of 1:3000) was added followed by two washes with 1× PBS. The donkey anti-rabbit HRP conjugate (dilution factor of 1:3000; GE Biosciences, GE NA934V) was used as the secondary antibody, which was detected using an OPD solution. Production of HIV-1 BaL.01 Pseudotype Virus

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Recombinant pseudovirus were produced by cotransfection of two plasmids: (1) envelope plasmid that encodes the BaL.01 gp160 region and (2) backbone sequence corresponding to envelope-deficient pNL4-3 Luc+ Env−.16 Three million HEK293T cells were plated on a T75 flask (Corning Inc.). Cells were cotransfected 24 h after being plated with 4 μg of envelope DNA and 8 μg of backbone DNA, using PEI as a transfection reagent. Medium was changed 24 h post-transfection, and the cells were allowed to grow for an additional 48 h. After 48 h, the cell supernatant containing virus was collected and filtered using a 0.45 μm filter (Corning Inc.). The filtered supernatant was loaded onto a Iodixanol gradient (gradient range from 6 to 20%; Optiprep, Sigma-Aldrich) and centrifuged in a Sw41 Ti rotor (Beckman Coulter) at 110000g for 2 h at 4 °C. The five lower fractions (each 1 mL) were pooled together,17 and 400 μL aliquots were frozen at −80 °C. Purified pseudoviruses were tested for infectivity and p24 content postproduction.

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Direct Binding of DAVEI Derivatives to Trimeric Spikes on BaL.01 Viruses BaL.01 viruses were diluted (1:20) in 1× PBS and coated onto 48-well ELISA plates, sealed with parafilm, and kept at 4 °C overnight. Plates were blocked the following day with 200 μL of 3% BSA in 1× PBS for 2 h at room temperature. Blocking buffer was discarded (by pipetting), and DAVEI derivatives were added to the viruses at varying concentrations and incubated for 30 min. Plates were washed twice with 1× PBS, 10 min each. Viruses were fixed onto the ELISA plates with 100 μL of 1% PFA (paraformaldehyde) for 65% inhibition at 10 nM; 10E8, >60% inhibition at 10 nM (n = 3)] (Figure 8B). Similarly, the gp120−gp41 interface specific antibody (35O22) competed with binding of Bt-Trp3 to the trimeric spike, as well (35O22, >60% inhibition at 100 nM) (Figure 8B). Inhibition of DAVEI-L4-3Trp-Induced Virolysis by gp41 Specific Antibodies

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The cell-based competition ELISA performed on JRFL Env(−)ΔCT suggested gp41 as an interactor for biotinylated Trp3 peptide. Because 35O22, 4E10, and 10E8 competed for binding of biotinylated Trp3 peptide with trimeric spike on JRFL Env(−)ΔCT, we examined the competition of DAVEI-L4-3Trp-induced virolysis in BaL.01 viruses. JRFL gp120 shows an 89.26% sequence identity with the BaL.01 gp120 region (Supporting Information 7), with all eight CVN specific glycosylation sites being conserved in both BaL.01 and JRFL Env(−)ΔCT (Supporting Information 7), thus justifying the comparison. In addition, both BaL.01 and JRFL Env(−)ΔCT show 100% sequence identity in the MPER region (Supporting Information 7) as well as 35O22 binding epitopes (N88, N230, N241, and N625). F105 was taken as a negative control for the virolysis assay. The results obtained showed that 35O22, 4E10, and 10E8 compete with DAVEI-L4-3Trp-induced virolysis in a dose-dependent fashion (IC50 = 196.5 ± 22.4 nM for 35O22, IC50 = 85.5 ± 6.6 nM for 4E10, and IC50 = 25.8 ± 15.2 nM for 10E8) (Figure 9A).

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We inspected the crystal structure of one of the protomers of the JRFL ectodomain trimer (PDB entry 5FUU) to ascertain how inhibition of virolysis might be exhibited by these antibodies (Figure 9B). On the basis of binding site analysis of mAbs, we envision that 4E10 and 10E8 inhibit virolysis because of steric hindrance conferred by these antibodies to MPER(DAVEI) when they interact with the gp41 subdomain. Inhibition by the 35O22 mAb could derive from steric hindrance of MPER(DAVEI) binding, or binding of the CVN component of DAVEI, or both simultaneously, because 35O22 and CVN share a common glycan-binding site on gp120, namely N230 (Figure 9B, colored orange). If DAVEI engages N230 for virolytic function, 35O22 might inhibit virolysis by inhibiting CVN engagement. Similarly, the bulky light and heavy chains of 35O22 could sufficiently block engagement of MPER(DAVEI) to prevent virolysis, as well (Figure 9B). The uncertainty with 35O22 notwithstanding, these overall results reinforce the conclusion that gp41 interacts with the 3Trp moiety of DAVEI-L4-3Trp protein. Validation of Virus with Varying Numbers of Trimeric Spikes on Surfaces

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Viruses with varying amounts of BaL.01 trimeric spikes on their surfaces were purified (Figure 10A) and tested for p24 content. All virus types (BaL.01 and VSV) contained equal amounts of p24 (Supporting Information 10A) at corresponding dilutions. The infectivity of BaL.01 viruses decreased as the number of spikes on the virus surfaces was decreased by lowering the plasmid concentration during transfection (Supporting Information 10B). VSV viruses had infectivity that was 33% higher than that of BaL.01 at 4 μg of plasmid transfection. No infectivity was observed for BaL.01 viruses produced in the absence of the trimer-coding plasmid.


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Virus ELISA with VRC01 and Bt-Trp3 Peptide


In addition to infectivity, recombinantly purified BaL.01 and VSV viruses (Figure 10A) were assayed with the VRC01 antibody to confirm the differential in the expression of functional HIV-1 spikes. VRC01 bound to BaL.01 viruses containing trimeric spikes in a dose-dependent fashion (∼50% binding at a concentration of 10 nM) but not to BaL.01 viruses without trimers or VSV viruses (Figure 10B). Similar experiments were performed with Bt-Trp3 peptide. Bt-Trp3 also bound to BaL.01 viruses with trimers on their surfaces (∼100% binding at a concentration of 1000 nM to BaL.01 viruses with the maximal number of spikes) (Figure 10C). The reduction in the number of spikes reduced the level of binding of Bt-Trp3 (∼55% binding at a concentration of 1000 nM to BaL.01 viruses with a reduced number of spikes) (Figure 10C). No significant binding of Bt-Trp3 was observed for BaL.01 viruses with no trimeric spikes or for VSV viruses (Figure 10C). The lack of competition for binding of Bt-Trp3 to virus Env by gp120 (Figure 8) argues that Bt-Trp3 does not bind to the gp120 components of Env trimers. Hence, the binding of Bt-Trp3 to virus in proportion to the amount of Env spike content, combined with the results from a competition cell-based ELISA, further confirms that binding of the 3Trp moiety occurs with the gp41 domain of trimeric spike.

DISCUSSION


DAVEIs initially designed as fusions of CVN and MPER are potent inhibitors of HIV-1 cell infection and are capable of inducing lytic inactivation of both fully infectious and BaL.01 pseudotype virus.8 Because a mechanistic understanding of DAVEI-induced virolysis has been lacking, we used a protein engineering approach to investigate the role of the MPER domain that is required for the virolytic function of the DAVEIs. We showed that both the CRAC sequence and tryptophan residues in MPER can be compromised without loss of DAVEI function, thus making it unlikely that membrane interaction is required for virolysis by DAVEI. Additionally, spatial assessment of CVN to MPER distance in DAVEI-L2 and glycan-to-membrane distance in the gp120−gp41 protomer of trimeric Env(+)ΔCT suggested that if MPER(DAVEI) interaction with the membrane were required for virolysis, the trimeric spike would require bending for DAVEI-L2MPER to interact with the membrane while CVN binds to gp120 glycans. Because no evidence for such bending of the trimer toward the membrane has been reported to date, we conclude that MPER(DAVEI) interacts with spike protein. These results, combined with results from a direct-binding virus ELISA, a competition cell-based ELISA with antibodies, and competition virolysis experiments, argue in favor of gp41 as the binding site of MPER(DAVEI). While the CVN domain of DAVEI is the source of infection inhibition activity by gp120 interaction, it is association of MPER(DAVEI) with the gp41 subunit that causes virolysis. Furthermore, we determined that the N-terminal nine residues in MPER, DKWASLWNW, provide a minimal molecular signature for virolytic function. The proposed mechanism of MPER in DAVEI as a gp41-interacting domain contrasts with recent observations with MPER-derived peptides or gp41 fusions containing MPER sequences. Membrane-bound gp41 proteins containing fusion peptide and MPER, CHR, and NHR groups have the capacity to induce content leakage from cholesterol-rich liposomes.31


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In fact, MPER peptide alone has the capacity to induce leakage.10–12 Apart from HIV-1 virus, fusion peptide analogues in other viruses such as influenza have also shown vesicular lytic properties,32 preferentially in the trimeric form.33 The membrane disruptive property for both fusion peptide and MPER is attributed to their hydrophobic amino acid sequences. MPER-induced membrane disruption and fusion depend on the presence of an unusually high percentage of tryptophan residues.10–12 These tryptophan residues are segregated along the axial length of α-helical MPER to form interactions with the membrane.26–28 The MPER Trp residues are known to be involved in infectivity34 as well as fusion pore formation and expansion.35 In addition, the cholesterol recognition motif, also known as the CRAC sequence (LWYIK) located at the MPER C-terminus,22–25 has strong affinity for cholesterol, an essential component of the HIV-1 envelope membrane.22,24,25 Mutations or truncations in the CRAC region of virus interrupt interaction with cholesterol and lead to loss of fusion activity.36,37


The results of this work leave open the question of what specific epitopes in gp41 are the lysis-enabling targets for MPER(DAVEI) binding. MPER has been reported to have a tendency to self-associate.31,38 In addition, a recent mutagenesis study by Yi et al.39 reported the possibility of interaction of MPER with the fusion peptide and NHR domain. The observations described above allow the possibility that MPER(DAVEI) could bind to gp41 MPER, FP, or NHR to induce virolysis. Hypothetically, interactions of MPER(DAVEI) with regions of gp41 concurrent with interaction of CVN with gp120 could cause conformational distortion, inducing membrane stress and hence lysis. The “spring-loaded” conformation of gp41 in the unliganded state has long been recognized to hold strong potential energy, which is released during the fusion process.6,40 It may be hypothesized that the strong potential energy of the metastable trimer is exploited by DAVEI molecules to elicit virolysis when gp120 and gp41 are simultaneously engaged. In this hypothesized view, interactions of DAVEI with gp41 can be sterically hindered by antibodies such as 4E10, 10E8, and possibly 35O22, all of which inhibit virolysis (Figure 9). Further work is needed to define the interaction pathway of the lysis-inducing DAVEI engagement with gp120 and gp41 sites on the Env spike. Overall, the results of this work demonstrate that simplifications in both linker and MPER domains can be introduced into DAVEI molecules with retention of lytic function. This leads to the follow-up question of whether the CVN domain of DAVEI also could be simplified and indeed whether other gp120-binding ligands, including smaller molecules, can be substituted. The potential for smaller molecule DAVEI lytic inactivators of HIV-1 remains an intriguing possibility for future investigation.

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Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Acknowledgments We are grateful to Dr. Joseph Sodroski (Professor in the Department of Immunology and Infectious Disease, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA) and Dr. Alon Herschhorn (Department of Cancer


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Immunology and AIDS, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA) for providing us with the JRFL Env(−)ΔCT plasmid construct for cell-based ELISA experiments. Funding Funding for this study was supported by National Institutes of Health Grants R01GM115249 and P01GM056550 provided to I.M.C. and C.F.A.

ABBREVIATIONS

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DAVEI

dual-acting virucidal entry inhibitor

ELISA

enzyme-linked immunosorbent assay

HIV-1

human immunodeficiency virus

CVN

cyanovirin-N

MPER

membrane-proximal external region

References


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29. Chang LC, Bewley CA. Potent inhibition of HIV-1 fusion by cyanovirin-N requires only a single high affinity carbohydrate binding site: characterization of low affinity carbohydrate binding site knockout mutants. J Mol Biol. 2002; 318:1–8. [PubMed: 12054763] 30. Lee JH, Ozorowski G, Ward AB. Cryo-EM structure of a native, fully glycosylated, cleaved HIV-1 envelope trimer. Science (Washington, DC, U S). 2016; 351:1043–1048. 31. Dai Z, Tao Y, Liu N, Brenowitz MD, Girvin ME, Lai JR. Conditional trimerization and lytic activity of HIV-1 gp41 variants containing the membrane-associated segments. Biochemistry. 2015; 54:1589–1599. [PubMed: 25658332] 32. Soltesz SA, Hammer DA. Lysis of Large Unilamellar Vesicles Induced by Analogs of the Fusion Peptide of Influenza Virus Hemagglutinin. J Colloid Interface Sci. 1997; 186:399–409. [PubMed: 9056369] 33. Lau WL, Ege DS, Lear JD, Hammer DA, DeGrado WF. Oligomerization of fusogenic peptides promotes membrane fusion by enhancing membrane destabilization. Biophys J. 2004; 86:272–284. [PubMed: 14695269] 34. Salzwedel K, West JT, Hunter E. A conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for Envmediated fusion and virus infectivity. J Virol. 1999; 73:2469–2480. [PubMed: 9971832] 35. Munoz-Barroso I, Salzwedel K, Hunter E, Blumenthal R. Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion. J Virol. 1999; 73:6089–6092. [PubMed: 10364363] 36. Vishwanathan SA, Thomas A, Brasseur R, Epand RF, Hunter E, Epand RM. Hydrophobic substitutions in the first residue of the CRAC segment of the gp41 protein of HIV. Biochemistry. 2008; 47:124–130. [PubMed: 18081318] 37. Vishwanathan SA, Thomas A, Brasseur R, Epand RF, Hunter E, Epand RM. Large changes in the CRAC segment of gp41 of HIV do not destroy fusion activity if the segment interacts with cholesterol. Biochemistry. 2008; 47:11869–11876. [PubMed: 18937430] 38. Liu J, Deng Y, Dey AK, Moore JP, Lu M. Structure of the HIV-1 gp41 membrane-proximal ectodomain region in a putative prefusion conformation. Biochemistry. 2009; 48:2915–2923. [PubMed: 19226163] 39. Yi HA, Diaz-Rohrer B, Saminathan P, Jacobs A. The membrane proximal external regions of gp41 from HIV-1 strains HXB2 and JRFL have different sensitivities to alanine mutation. Biochemistry. 2015; 54:1681–1693. [PubMed: 25649507] 40. Carr CM, Kim PS. A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell. 1993; 73:823–832. [PubMed: 8500173]

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Figure 1.

Assessment of the role of the CRAC domain in virolysis shown by the DAVEI molecule. (A) Different subdomains of gp41 subunit are highlighted. The amino acid sequence in the MPER region is shown. CRAC residues are colored red. (B) Cartoon representation of two derivatives of DAVEI-L4: DAVEI-L4(ΔCRAC) and DAVEI-L4(ΔMPER). (C) Detection of direct binding of DAVEI-L4, CVN, and DAVEI-L4(ΔCRAC) on BaL.01 viruses with trimers. Monomeric gp120 (50 ng) directly immobilized onto an ELISA plate was taken as a positive control. Data were normalized relative to the positive control. Viruses devoid of


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trimers were taken as a negative control that showed no detectable binding to DAVEI derivatives. (D) HOS.T4.R5 cells were exposed to the HIV-1 BaL.01 pseudovirus with serial dilutions of the DAVEI-L4 derivatives. The inhibitory potencies of DAVEI-L4, CVN, DAVEI-L4(ΔCRAC), and DAVEI-L4(ΔMPER) were 1.2 ± 0.3, 0.9 ± 0.2, 1.1 ± 0.2, and 1.4 ± 0.3 nM, respectively. IC50s were calculated using Origin version 8.1. (E) Release of p24 from HIV-1 BaL.01 pseudovirus upon incubation with DAVEI-L4 derivatives. A sandwich ELISA was conducted in which experimental p24 release was background-subtracted using PBS treated virus and then compared to virus lysed with 1% Triton X-100 (means ± SD; n = 3). The EC50s for DAVEI-L4 and DAVEI-L4(ΔCRAC) protein were 29.8 ± 1.0 and 28.3 ± 2.1 nM, respectively. CVN and DAVEI-L4(ΔMPER) did not show p24 release. EC50s of virolysis of HIV-1 BaL.01 pseudovirus were determined with Origin version 8.1.

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Figure 2.

Assessment of the role of hydrophobic tryptophan residues of MPER in virolysis. (A) Cartoon representation of four derivatives of DAVEI-L4(ΔCRAC). (B) Detection of direct binding of DAVEI-L4(ΔCRAC) and DAVEI-L4(ΔCRAC)W3,7,9,15A tryptophan mutants on BaL.01 viruses with trimers. Monomeric gp120 (50 ng) directly immobilized onto an ELISA plate was taken as a positive control. Viruses devoid of trimers were taken as a negative control that showed no detectable binding to DAVEI derivatives. (C) Inhibition of HIV-1 viral infection by DAVEI-L4(ΔCRAC) derivatives. The inhibitory potencies of DAVEI-


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L4(ΔCRAC) (IC50 = 1.1 ± 0.2 nM), DAVEI-L4(ΔCRAC)W15A (IC50 = 0.4 ± 0.1 nM), DAVEI-L4(ΔCRAC)W9,15A (IC50 = 0.45 ± 0.1 nM), DAVEI-L4(ΔCRAC)-W7,9,15A (IC50 = 0.36 ± 0.1 nM), and DAVEI-L4(ΔCRAC)-W3,7,9,15A (IC50 = 0.78 ± 0.3 nM) are shown [means ± standard deviations (SD); n = 3]. (D) Release of p24 from HIV-1 BaL.01 pseudovirus upon incubation with DAVEI-L4(ΔCRAC) derivatives. EC50s of virolysis were 29.8 ± 1.0, 46.8 ± 5.9, 49.7 ± 5.5, 43.1 ± 3.6, and 122.2 ± 11.6 nM for DAVEI-L4(ΔCRAC) and its single, double, triple, and quadruple mutants, respectively (means ± SD; n = 3).


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Figure 3.

Determination of the minimal MPER sequence (MPERmin) required for virolysis by the DAVEI molecule. (A) Cartoon representation of four derivatives of DAVEI-L4(ΔCRAC). (B) Truncated MPER peptides of corresponding lengths as the protein derivatives that are used as a control for virolysis. (C) Detection of direct binding of DAVEI-L4(ΔCRAC) and DAVEI-L4-3Trp truncates on BaL.01 viruses with trimers. Monomeric gp120 (50 ng) directly immobilized onto an ELISA plate was taken as a positive control, and viruses devoid of trimers were taken as a negative control that showed no detectable binding to


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DAVEI derivatives. (D) Inhibition of HIV-1 viral infection by DAVEI-L4(ΔCRAC) truncated proteins [means ± standard deviations (SD); n = 3]. The inhibitory potencies are as follows: IC50 = 1.1 ± 0.2 nM for DAVEI-L4(ΔCRAC), IC50 = 0.8 ± 0.2 nM for DAVEI-L4-1Trp, IC50 = 2.3 ± 0.1 nM for DAVEI-L4-2Trp, and IC50 = 2.2 ± 0.4 nM for DAVEI-L4-3Trp. (E) Release of p24 from HIV-1 BaL.01 pseudovirus upon incubation with DAVEI-L4-(ΔCRAC) truncations. EC50s for DAVEI-L4(ΔCRAC), DAVEI-L4-3Trp, and DAVEI-L4-2Trp proteins were 28.3 ± 2.1, 36.1 ± 5.1, and 113.8 ± 11.1 nM, respectively, determined using Origin Pro version 8.1 with sigmoidal fits (means ± SD; n = 3). DAVEI-L4-1Trp did not show p24 release.


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Figure 4.

Determination of the minimal linker sequence required for virolysis by DAVEI. (A) Cartoon representation of two linker derivatives of DAVEI-L4: DAVEI-L2 and DAVEI-L0. (B) Detection of direct binding of DAVEI-L4, DAVEI-L2, and DAVEI-L0 on BaL.01 viruses with trimers. Monomeric gp120 (50 ng) directly immobilized onto an ELISA plate was taken as a positive control, and viruses devoid of trimers were taken as a negative control that showed no detectable binding to DAVEI derivatives. (C) Inhibition of HIV-1 viral infection by DAVEI-L4, DAVEI-L2, and DAVEI-L0 [means ± standard deviation (SD); n =


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3]. The inhibitory potencies are as follows: IC50 = 4.3 ± 1.7 nM for DAVEI-L0 and IC50 = 2.1 ± 0.3 nM for DAVEI-L2). (C) The EC50s for DAVEI-L2- and DAVEI-L4-induced virolysis were 182.6 ± 28.5 and 23.6 ± 2.5 nM, respectively, determined using Origin Pro version 8.1 with sigmoidal fits (means ± SD; n = 3). DAVEI-L0 did not induce p24 release at concentrations of up to 2000 nM. PBS-treated virus was used as a negative control, and virus lysed with 1% Triton X-100 was taken as a positive control (means ± SD; n = 3).


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Figure 5.

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Free MPER peptide competition of virolysis induced by DAVEI derivatives. Free MPER and Trp3 peptide were able to compete out the virolysis exhibited by DAVEI-L4 and DAVEIL4-3Trp proteins; 50 nM DAVEI-L4 and DAVEI-L4-3Trp proteins were incubated with BaL.01 virus in the absence or presence of serial dilutions of both MPER and Trp3 peptide. IC50s for inhibition of DAVEI-L4-3Trp-induced virolysis for MPER peptide and Trp3 peptide were 16.8 ± 1.6 and 52.6 ± 5.4 nM, respectively, measured using a p24 ELISA (means ± SD; n = 3).


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Figure 6.

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Spatial comparison of the DAVEI fusion with the ectodomain of the HIV-1 JRFL gp120– gp41 protomer. (A) Simulated structure of DAVEI-L2 constructed computationally by fusion of the CVN molecule (PDB entry 1IIY) with an extended (glycine4-serine) linker and MPER using Maestro version 10.3. (B) Cryo-electron microscopy structure of the gp120–gp41 protomer in the cleaved wild-type HIV-1 JR-FL Env trimer complex (PDB entry 5FUU), with distance measurements made from the center of CVN-binding glycans in gp120 (N332) to the C-terminus of the gp41 ectodomain close to the membrane as shown in the structure (D664).


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Author Manuscript Author Manuscript Figure 7.

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Assessment of the interaction of the trimer with various antibodies and biotinylated Trp3 peptide by a cell-based ELISA. (A) Cartoon representation showing trimer expression on the HEK293T cell surface and its interaction with antibodies. (B) Interaction of gp120 and MPER specific antibodies with trimers expressed on the HEK293T cell surface [F105 (EC50 = 4.5 ± 0.3 nM), 35O22 (EC50 = 6.8 ± 1 nM), 4E10 (EC50 = 9.6 ± 0.5 nM), 10E8 (EC50 = 6.7 ± 0.7 nM), and 2G12 (EC50 = 2.7 ± 0.5 nM)]. F105 does not bind to HEK293T cells not transfected with the JRFL Env(−)ΔCT plasmid. (C) Cartoon representation showing trimer expression on the HEK293T cell surface and its interaction with biotinylated Trp3 peptide. (D) Interaction of biotinylated Trp3 peptide with HEK293T cells transfected with the JRFL Env(−)ΔCT plasmid [biotinylated Trp3 (EC50 = 129 ± 14 nM)]. Biotinylated Trp3 peptide does not bind to cells not transfected with the JRFL Env(−)ΔCT plasmid.


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Figure 8.

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Binding of biotinylated Trp3 peptide with the JRFL Env(−)ΔCT trimer expressed on the HEK293T cell surface in the presence of various mAbs and other proteins. (A) Biotinylated Trp3 peptide competed against gp120 and gp120 specific antibodies, including CVN and DAVEI-L4-3Trp protein, in a cell-based ELISA. Bt-Trp3 peptide binding was competed by DAVEI-L4-3Trp protein but not by F105, gp120, 2G12, or CVN. (B) Biotinylated Trp3 peptide competed against the gp120–gp41 interface specific antibody (35O22) and gp41 specific antibodies (4E10 and 10E8) in a cell-based ELISA. 35O22, 4E10 and 10E8 competed with Bt-Trp3 peptide binding (n = 3).


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Figure 9.

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Inhibition of DAVEI-L4-3Trp-induced virolysis by gp41 specific antibodies. (A) DAVEIL4-3Trp protein (50 nM) was incubated with BaL.01 virus in the absence or presence of serial dilutions of 35O22, 4E10, and 10E8 antibodies. The IC50 values for inhibition of DAVEI-L4-3Trp-induced virolysis for 35O22, 4E10, and 10E8 were 196.5 ± 22.4, 85.5 ± 6.6, and 25.8 ± 15.2 nM, respectively (means ± SD; n = 3). (B) gp120–gp41 protomer bound to the 35O22 antibody, produced by a structural overlay of the crystal structure of the ectodomain of JRFL EnvΔCT (PDB entry 5FUU) with the 35O22-bound crystal structure of SOSIP 664 (clade G x1193.c1, PDB entry 5FYJ). The gp120 subunit is colored gray, and gp41 is colored wheat. Cyanovirin specific glycosylation sites are colored green (N160, N339, N386, N392, N448, N136, and N332), and 35O22 specific glycosylation sites (N88, N241, and N625) are colored red. N230, a common glycosylation site shared by CVN and 35O22, is colored orange, and the tentative binding site of 4E10 and 10E8 (D664) is colored blue. Figures were produced using Pymol graphics version 1.4.1.


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Author Manuscript Author Manuscript Figure 10.

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Direct binding of VRC01 and biotinylated Trp3 peptide to viruses with varying amounts of trimeric spikes. (A) Cartoon representation showing three different types of BaL.01 viruses produced each with varying numbers of trimeric spikes on their surfaces and VSV virus, as well. (B) Interaction of each virus type with the VRC01 antibody. VRC01 binds to BaL.01 viruses having trimers on their surfaces (>50% binding at a concentration of 10 nM) but does not bind to viruses without trimers. No binding of VRC01 was seen with VSV viruses. (C) Interaction of each virus type with Bt-Trp3 peptide. Bt-Trp3 bound to BaL.01 viruses with trimers on their surfaces (∼100% binding at a concentration of 1000 nM with BaL.01 with a maximal number of spikes). A reduction in the number of spikes weakened binding of Bt-Trp3 (∼55% binding at a concentration of 1000 nM with BaL.01 with a reduced number of spikes). No significant binding of VRC01 was seen with BaL.01 viruses with no trimeric spikes or VSV viruses (n = 3).


gp41.

Gp120 in the pathogenesis of human immunodeficiency virus-associated pain.

Apolipoprotein A-1 interacts with the N-terminal fusogenic domains of SIV (simian immunodeficiency virus) GP32 and HIV (human immunodeficiency virus) GP41: implications in viral entry.

Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein.

Lineage-Specific Differences between the gp120 Inner Domain Layer 3 of Human Immunodeficiency Virus and That of Simian Immunodeficiency Virus.

Two immunodominant domains of gp41 bind antibodies which enhance human immunodeficiency virus type 1 infection in vitro.

The control of the antibody isotype response to recombinant human immunodeficiency virus gp120 antigen by adjuvants.

Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies.

Correction: Effects of the I559P gp41 Change on the Conformation and Function of the Human Immunodeficiency Virus (HIV-1) Membrane Envelope Glycoprotein Trimer.

Human immunodeficiency virus-like, nonreplicating, gag-env particles assemble in a recombinant vaccinia virus expression system.

The N-terminal 31 amino acids of human immunodeficiency virus type 1 envelope protein gp120 contain a potential gp41 contact site.

Generation of hybrid genes and proteins by vaccinia virus-mediated recombination: application to human immunodeficiency virus type 1 env.

Effects of the I559P gp41 change on the conformation and function of the human immunodeficiency virus (HIV-1) membrane envelope glycoprotein trimer.

Interleukin 10 mediated by herpes simplex virus vectors suppresses neuropathic pain induced by human immunodeficiency virus gp120 in rats.

Development of Broadly Neutralizing Antibodies and Their Mapping by Monomeric gp120 in Human Immunodeficiency Virus Type 1-Infected Humans and Simian-Human Immunodeficiency Virus SHIVSF162P3N-Infected Macaques.

Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41.

Characterization of the secreted, native gp120 and gp160 of the human immunodeficiency virus type 1.

Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1.

Independent fluctuation of human immunodeficiency virus type 1 rev and gp41 quasispecies in vivo.

Stabilizing the native trimer of HIV-1 Env by destabilizing the heterodimeric interface of the gp41 postfusion six-helix bundle.

Inhibition of viral replication by monoclonal antibodies directed against human immunodeficiency virus gp120.

CD4-binding regions of human immunodeficiency virus envelope glycoprotein gp120 defined by proteolytic digestion.

Inactivation of human immunodeficiency virus by chlorhexidine: the possible role of neutralizers.

A method to analyze the interaction between gp120 of human immunodeficiency virus and CD4.