Distinct functions for the membrane-proximal ectodomain region (MPER) of HIV-1 gp41 in cell-free and cell-cell viral transmission and cell-cell fusion

Abstract

HIV-1 is spread by cell-free virions and by cell-cell viral transfer. We asked whether the structure and function of a broad neutralizing antibody (bNAb) epitope, the membrane-proximal ectodomain region (MPER) of the viral gp41 transmembrane glycoprotein, differ in cell-free and cell-cell-transmitted viruses and whether this difference could be related to Ab neutralization sensitivity. Whereas cell-free viruses bearing W666A and I675A substitutions in the MPER lacked infectivity, cell-associated mutant viruses were able to initiate robust spreading infection. Infectivity was restored to cell-free viruses by additional substitutions in the cytoplasmic tail (CT) of gp41 known to disrupt interactions with the viral matrix protein. We observed contrasting effects on cell-free virus infectivity when W666A was introduced to two transmitted/founder isolates, but both mutants could still mediate cell-cell spread. Domain swapping indicated that the disparate W666A phenotypes of the cell-free transmitted/founder viruses are controlled by sequences in variable regions 1, 2, and 4 of gp120. The sequential passaging of an MPER mutant (W672A) in peripheral blood mononuclear cells enabled selection of viral revertants with loss-of-glycan suppressor mutations in variable region 1, suggesting a functional interaction between variable region 1 and the MPER. An MPER-directed bNAb neutralized cell-free virus but not cell-cell viral spread. Our results suggest that the MPER of cell-cell-transmitted virions has a malleable structure that tolerates mutagenic disruption but is not accessible to bNAbs. In cell-free virions, interactions mediated by the CT impose an alternative MPER structure that is less tolerant of mutagenic alteration and is efficiently targeted by bNAbs.

Keywords: cell–cell transmission; glycoprotein; human immunodeficiency virus (HIV); membrane fusion; mutant; neutralization; viral replication; virus entry.

Figure 1.

Viral replication initiated by cell-free and cell-associated MPER mutant viruses in U87.CD4.CCR5 cells. A, NMR structure of MPER peptide (amino acids 662–683; PDB code 2PV6) in dodecylphosphocholine micelle (38). The MPER peptide forms a kinked helix in the interfacial region (bordered by arcs) with aromatic and hydrophobic side chains penetrating into the hydrophobic phase (stippled). The side chains of amino acids mutated to alanine in this study are shown. B, functional characteristics of W666A, W672A, and I675A AD8 Env mutants described in Bellamy-McIntyre et al. (52). +++, >90% of WT activity, +/−, <2% of WT activity. U87.CD4.CCR5 cells were inoculated with unpseudotyped (C) or VSV G-pseudotyped (D) HIV-1_AD8 particles (50,000 cpm of RT activity per inoculum) and then trypsinized 24 h later to remove residual adsorbed virus. The cells were then replated and cultured for a further 10 days. Mean RT activity ± S.D. of triplicate samples is shown. Representative of two independent experiments. E, day-7 and day-10 culture supernatants obtained from D were filtered through 0.45-μm nitrocellulose filters and then used to infect naive U87.CD4.CCR5 cells. Mean RT activity ± S.D. of triplicate samples is shown. Representative of two independent experiments. F and G, PHA-stimulated PBMCs were inoculated with nonpseudotyped (left panels) or VSV G-pseudotyped (right panels) HIV-1 particles and then washed extensively 24 h later. The cells were replated and cultured for a further 10 days. The p24 content of viral supernatants was determined at the indicated time points by ELISA. Data are representative of two independent experiments. H, infectivity of WT and mutated viruses for TZM-bl cells. Serially diluted virus-containing supernatants were incubated with TZM-bl cells for 2 days prior to lysis and luciferase assay. The mean relative light units (RLU) ± S.E. was obtained from three independent experiments shown. I, nonpseudotyped and VSV G-pseudotyped HIV-1_AD8 virions fail to infect a CD4-negative cell line, Huh7.5. The experiment was conducted as for C and D. Mean RT activity ± S.D. of triplicate samples is shown. J, infection of Huh7.5 cells by VSV G-pseudotyped NL4.3lucR⁻E⁻ luciferase reporter virus. Mean RLU ± S.D. of triplicate samples is shown. No Env, nonpseudotyped reporter particles.

Figure 2.

L8S/S9R matrix mutation blocks viral spread initiated by cell-associated virus. A, L8S/S9R decreases viral infectivity for TZM-bl cells. Serially diluted virus-containing supernatants were incubated with TZM-bl cells for 2 days prior to lysis and luciferase assay. The mean RLU ± S.E. obtained from two independent experiments is shown. B, cell-surface–expressed Env glycoproteins expressed from WT and mutated pAD8 infectious clones retain fusion competence. 293T cells that had been cotransfected with WT or mutated pAD8 infectious clones and pCAG-T7 were cocultured with BHK21 cells that had been cotransfected with pCCR5 and pT4luc. Luciferase activity was measured 16 h later. The mean RLU ± S.E. obtained from three independent experiments is shown. *, p < 0.05 WT versus mutant; two-tailed t test assuming unequal variances. C, L8S/S9R blocks spreading infection initiated by cell-associated virus in U87.CD4.CCR5 cells. As for Fig. 1D, except that the virus content of culture supernatants was measured by p24 ELISA. Data are representative of three independent experiments. D, VSV G-pseudotyped L8S/S9R-containing viruses retain infectivity for TZM-bl cells. Serially diluted virus-containing supernatants were incubated with TZM-bl cells for 2 days prior to lysis and luciferase assay. The mean RLU ± S.E. obtained from two independent experiments is shown.

Figure 3.

Mutations in the CT of gp41 restore infectivity to cell-free MPER mutant virions. A, schematic representation of gp41. HR1, HR2 are heptad repeats 1 and 2 respectively; TMD, transmembrane domain; LLP, lentiviral lytic peptides; Y⁷¹²SPL, dominant tyrosine-based sorting signal; YW, intracellular sorting motifs; LL, C-terminal dileucine endocytosis motif. B, expression of truncated AD8 glycoproteins in 293T cells transfected with WT and mutated Env expression vectors. Cell lysates were subjected to reducing SDS-PAGE and Western blotting with DV-012 polyclonal anti-gp120 serum. C, cell–cell fusion activities of truncated AD8 Env glycoproteins. 293T cells that had been cotransfected with WT or mutated pcDNA3.1AD8env vectors and pCAG-T7 were cocultured with BHK21 cells that had been cotransfected with pCCR5 and pT4luc. Luciferase activity was measured 18 h later. The mean RLU ± S.E. obtained from at least three independent experiments is shown. D and E, single-cycle infectivity of NL4.3LucR⁻E⁻ luciferase reporter viruses pseudotyped with WT and mutated AD8 Env glycoproteins for U87.CD4.CCR5 cells. The cells were assayed for luciferase activity at 52 h post-transfection. Mean RLU ± S.E. from at least three independent experiments is shown. *, p < 0.05; **, p < 0.01; glycoprotein construct with mutated CT versus corresponding construct with unmutated CT; two-tailed t test assuming unequal variances. F, infectivity of WT and mutated HIV-1_AD8 viruses for TZM-bl cells. Serially diluted virus-containing supernatants were incubated with TZM-bl cells for 2 days prior to lysis and luciferase assay. The mean RLU ± S.D. obtained from a representative experiment is shown. G, gp120 content of virions. Virions were pelleted through a sucrose cushion and then subjected to reducing SDS-PAGE and Western blotting with DV012 (upper panel) and HIVIG (lower panel). gp120-incorporation indices ((gp120^mutant pixels ÷ p24^mutant pixels) × (p24^WT pixels ÷ gp120^WT pixels)) are shown below. The image was obtained from a single gel with the splice point indicated.

Figure 4.

Real-time fusion kinetics of MPER mutant Envs. A, 293T effector cells cotransfected with expression vectors encoding β-lactamase plus WT Env or a fusion-incompetent Env mutant (G597A) were cocultured with CCF2-AM–loaded JC53 targets in 96-well plates at 37 °C for 180 min. Individual wells were read for green and blue fluorescence (excitation at 409 nm; emission at 520 and 447 nm, respectively) every 20 min in a FLUOstar plate reader (BMG). The ratio of blue/green fluorescence is shown. B, fusion kinetics of MPER mutants. The assay conditions were as per C. The ratio of blue/green fluorescence was calculated and then normalized to the final extent of fusion. Mean ± S.E. of four independent experiments is shown.

Figure 5.

Distinct effects of W666A in Envs from two T/F isolates. A, U87.CD4.CCR5 cells were inoculated with unpseudotyped (left panel) or VSV G-pseudotyped (right panel) HIV-1 particles and then trypsinized 24 h later to remove residual adsorbed virus. The cells were then replated and cultured for a further 10 days. The p24 content of viral supernatants was determined at the indicated time points by ELISA. Representative of three independent experiments. B, PHA-stimulated PBMCs were inoculated with nonpseudotyped (left panel) or VSV G-pseudotyped (right panel) HIV-1 particles and then washed extensively 24 h later. The cells were replated and cultured for a further 10 days. The p24 content of viral supernatants was determined at the indicated time points by ELISA. Data are representative of two independent experiments. C, infectivity of WT and mutated viruses for TZM-bl cells. Serially diluted virus-containing supernatants were incubated with TZM-bl cells for 2 days prior to lysis and luciferase assay. The mean RLU ± S.E. obtained from six independent experiments is shown. D, gp120 content of virions. Virions were pelleted through a sucrose cushion and then subjected to reducing SDS-PAGE and Western blotting with DV-012 (upper panel) and HIVIG (lower panel). gp120-incorporation indices relative to PRB958-WT are shown below.

Figure 6.

Phenotypic analysis of PRB958-SC45 chimeric Env glycoproteins. A, schematic representation of PRB958-SC45 chimeric Env glycoproteins. The chimeras contain NL4.3 flanking sequences 5′ and 3′ to the indicated KpnI and BamHI sites, respectively. B, infectivity of WT and mutated viruses for TZM-bl cells. Serially diluted virus-containing supernatants were incubated with TZM-bl cells for 2 days prior to lysis and luciferase assay. The mean RLU ± S.E. obtained from at least three independent experiments is shown. C, gp120 content of virions. Virions were pelleted through a sucrose cushion and then subjected to reducing SDS-PAGE and Western blotting with DV012 (upper panel) and HIVIG (lower panel). gp120-incorporation indices are shown below. The data were obtained from three gels, as indicated. The arrow indicates the position of the 150-kDa marker. D, spreading infection in U87.CD4.CCR5 cells initiated by cell-free and cell-associated virus. Data are representative of two independent experiments. E, spreading infection in PHA-stimulated PBMCs initiated by cell-free and cell-associated virus. Data are representative of two independent experiments.

Figure 7.

Neutralization sensitivity of PRB958 and SC45 Envs. A, alignment of MPER sequences. The asterisks indicate contact residues for bNAbs 10E8 (red) and 2F5 (blue). B, 2F5 (blue) and 10E8 (red) contact residues in the context of the lipid micelle-associated MPER peptide (38). C, location of bNAb epitopes in the gp120–gp41 SOSIP.664 trimer (PDB code 5FYJ) (120). The MPER is absent from the structure but would be located at the base of the trimer (50) as indicated by arrow. D, neutralization of cell-free viruses by various bNAbs. U87.CD4.CCR5 cells were incubated with pseudovirus-IgG mixtures for 2 days prior to lysis and assay for luciferase activity. Neutralizing activities were measured in triplicate, and the average percent luciferase activity was determined for each assay. The data are the means ± S.E. obtained from at least two independent experiments. E, neutralization of cell-associated viruses. U87.CD4.CCR5 cells were infected with VSV G-pseudotyped viruses and then trypsinized 24 h later. The cells were seeded in triplicate into 96-well tissue culture plates in the presence of serially diluted bNAbs. Three days later, 50% of the culture supernatant was replaced with fresh medium containing the appropriate dilution of bNAb. The p24 content of viral supernatants was determined at day-7 post-seeding by ELISA. Neutralizing activities were measured in triplicate and reported as the average percent p24 content. The data are representative of three independent experiments. HC84-1 is a control human monoclonal bNAb directed to the E2 glycoprotein of HCV (139).

Figure 8.

In vitro evolution of pAD8-W672A. A, 14-day replication kinetics of WT and mutated AD8 viruses. Virus stocks produced in 293T cells were normalized according to RT activity and used to infect PHA-stimulated PBMCs. RT activity was measured in culture supernatants obtained at days 3, 7, 10, and 14 postinfection. The mean RT activity ± S.D. of duplicate samples is shown. B, U87.CD4.CCR5 cells were inoculated with unpseudotyped (left panel) or VSV G-pseudotyped (right panel) HIV-1_AD8 particles (50,000 cpm of RT activity per inoculum) and then trypsinized 24 h later to remove residual adsorbed virus. The cells were then replated and cultured for a further 10 days. Mean RT activity ± S.D. of triplicate samples is shown. Data are representative of two independent experiments. C, long-term PBMC culture of WT and mutated AD8 viruses. Viruses produced by transfected 293T cells were normalized according to RT activity prior to infection of PHA-stimulated PBMCs. The PBMCs used in each passage were obtained from different donors. Cell-free virus collected at day 10 of each passage was normalized for RT activity and used to infect fresh PHA-stimulated PBMCs. The mean RT activity of duplicate samples is shown. D, frequency of genotypes observed in env clones obtained at days 30 and 60 from the W672A culture. E, alignment of V1 sequences, with potential N-linked glycosylation sites highlighted in green. F, migration of immunoprecipitated ³⁵S-labeled gp120 molecules containing second site mutations in SDS-PAGE. G, 14-day replication kinetics of HIV-1_AD8-W672A viruses ± 2nd site mutations in V1. Virus stocks produced in 293T cells were normalized according to RT activity and used to infect PHA-stimulated PBMCs. RT activity was measured in culture supernatants obtained at days 3, 7, 10, and 14 postinfection. The mean RT activity of duplicate samples is shown.

Figure 9.

Structural modulation of the MPER, a model. Left, depiction of the Env glycoprotein in a cell-free virion. The trimeric gp120–gp41 ectodomain was drawn using the coordinates from PDB code 5FYJ. The MPER (a monomer is shown for clarity) is embedded as a kinked helix in the polar headgroup (light blue)–acyl chain (white) interfacial region of the viral envelope as indicated by NMR studies of MPER peptides associated with membrane mimics (37, 38) and cryo-EM of HIV-1 particles (50). The hydrophobic acyl-chain–interactive face of the MPER helices is colored teal. The N-terminal segment of the CT (707–752) is unstructured in the absence of MA interactions (143) but becomes organized when engaged by MA trimers in hexameric arrays (62–64, 84). In this context, the MPER is in a conformation that is sensitive to mutations such as W666A, W672A, and I675A that potentially alter its interaction with the envelope. The effects of these mutations are mitigated by truncation of the CT or by Y712S, which uncouple Env from Gag due in part to disruption of the YXXL motif. In the SC54 isolate, the bridge formed between the V1-Asn¹⁴² and V4-Asn³⁸⁶ glycans and the V2–β-hairpin–stabilizing effects of the Pro¹⁸³–Tyr¹⁹¹ interaction at the trimer apex confer a mutation-sensitive phenotype to the MPER via allosteric mechanisms. Right, in VS-transmitted virions, the MPER is resistant to mutations and neutralization by bNAb 10E8 perhaps due to an alternative CT conformation (108) and interaction with the MA domain. The various protein domains were drawn with PyMOL using the following coordinates: gp120–gp41 ectodomain, PDB code 5FYJ (120); MPER, PDB code 2PV6 (38); membrane-spanning sequence, PDB code 5JYN (144); CT, PDB code 5VWL (143); MA trimer, PDB code 1HIW (145).