Domain Of Gp41 Research Articles

HIV-1 gp41 Transmembrane Domain Interacts with the Fusion Peptide: Implication in Lipid Mixing and Inhibition of Virus–Cell Fusion

Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion peptide (FP) at the late fusion stages, but there are no direct experimental data to support this hypothesis. Here, we investigated the TMD focusing on two regions: the N-terminal containing the GxxxG motif and the C-terminal containing the GLRI motif, which is conserved among the TMDs of HIV and the T-cell receptor. Studies utilizing the ToxR expression system combined with synthetic peptides and their fluorescent analogues derived from TMD revealed that the GxxxG motif is important for TMD self-association, whereas the C-terminal region is for its heteroassociation with FP. Functionally, all three TMD peptides induced lipid mixing that was enhanced significantly upon mixing with FP. Furthermore, the TMD peptides inhibited virus-cell fusion apparently through their interaction with their endogenous counterparts. Notably, the R2E mutant (in the GLRI) was significantly less potent than the two others. Overall, our findings provide experimental evidence that HIV-1 TMD contributes to membrane assembly and function of the HIV-1 envelope. Owing to similarities between functional domains within viruses, these findings suggest that the TMDs and FPs may contribute similarly in other viruses as well.

Determining the mechanism of membrane permeabilizing peptides: Identification of potent, equilibrium pore-formers

To enable selection and characterization of highly potent pore-forming peptides, we developed a set of novel assays to probe 1) the potency of peptide pores at very low peptide concentration; 2) the presence or absence of pores in membranes after equilibration; 3) the interbilayer exchangeability of pore-forming peptides; and 4) the degree to which pore-forming peptides disrupt the bilayer organization at equilibrium. Here, we use these assays to characterize, in parallel, six membrane-permeabilizing peptides belonging to multiple classes. We tested the antimicrobial peptides LL37 and dermaseptin S1, the well-known natural lytic peptides melittin and alamethicin, and the very potent lentivirus lytic peptides LLP1 and LLP2 from the cytoplasmic domain of HIV GP41. The assays verified that that the antimicrobial peptides are not potent pore formers, and form only transient permeabilization pathways in bilayers which are not detectable at equilibrium. The other peptides are far more potent and form pores that are still detectable in vesicles after many hours. Among the peptides studies, alamethicin is unique in that it is very potent, readily exchanges between vesicles, and disturbs the local bilayer structure even at very low concentration. The equally potent LLP peptides do not exchange readily and do not perturb the bilayer at equilibrium. Comparison of these classes of pore forming peptides in parallel using the set of assays we developed demonstrates our ability to detect differences in their mechanism of action. Importantly, these assays will be very useful in high-throughput screening where highly potent pore-forming peptides can be selected based on their mechanism of action.

The Cytoplasmic Domain of the HIV-1 Glycoprotein gp41 Induces NF-κB Activation through TGF-β-Activated Kinase 1

The human and simian immunodeficiency viruses (HIV and SIV) primarily infect lymphocytes, which must be activated for efficient viral replication. We show that the cytoplasmic domain of the transmembrane glycoprotein gp41 (gp41CD) of both HIV-1 and SIV induces activation of NF-κB, a cellular factor important for proviral genome transcription and lymphocyte activation. This NF-κB activating property localized to a region 12-25 (SIV) or 59-70 (HIV-1) residues from the gp41 membrane-spanning domain. An siRNA-based screen of 42 key NF-κB regulators revealed that gp41CD-mediated activation occurs through the canonical NF-κB pathway via TGF-β-activated kinase 1 (TAK1). TAK1 activity was required for gp41CD-mediated NF-κB activation, and HIV-1-derived gp41CD physically interacted with TAK1 through the same region required for NF-κB activation. Importantly, an NF-κB activation-deficient HIV-1 mutant exhibited increased dependence on cellular activation for replication. These findings demonstrate an evolutionarily conserved role for gp41CD in activating NF-κB to promote infection.

Passively Transmitted gp41 Antibodies in Babies Born from HIV-1 Subtype C-Seropositive Women: Correlation between Fine Specificity and Protection

HIV-exposed, uninfected (EUN) babies born to HIV-infected mothers are examples of natural resistance to HIV infection. In this study, we evaluated the titer and neutralizing potential of gp41-specific maternal antibodies and their correlation with HIV transmission in HIV-infected mother-child pairs. Specific gp41-binding and -neutralizing antibodies were determined in a cohort of 74 first-time mother-child pairs, of whom 40 mothers were infected with HIV subtype C. Within the infected mother cohort, 16 babies were born infected and 24 were PCR negative and uninfected at birth (i.e., exposed but uninfected). Thirty-four HIV-uninfected and HIV-unexposed mother-child pairs were included as controls. All HIV-positive mothers and their newborns showed high IgG titers to linear epitopes within the HR1 region and to the membrane-proximal (MPER) domain of gp41; most sera also recognized the disulfide loop immunodominant epitope (IDE). Antibody titers to the gp41 epitopes were significantly lower in nontransmitting mothers (P < 0.01) and in the EUN babies (P < 0.005) than in HIV-positive mother-child pairs. Three domains of gp41, HR1, IDE, and MPER, elicited antibodies that were effectively transmitted to EUN babies. Moreover, in EUN babies, epitopes overlapping the 2F5 epitope (ELDKWAS), but not the 4E10 epitope, were neutralization targets in two out of four viruses tested. Our findings highlight important epitopes in gp41 that appear to be associated with exposure without infection and would be important to consider for vaccine design.

HIV gp41 Trans-Membrane Domain Promotes both Stalk and Fusion Pore Formation in Poly(Ethylene-) Glycol Mediated Membrane Fusion

The trans-membrane domain (TMD) of gp41 is essential for efficient fusion between HIV-1 and its host cell in vivo. HIV virus with gp41 mutated by R696L is reported to be defective in infectivity and fusion (Helseth, J Vir, 1990, 6314), so we have examined both native and R696L gp41 TMDs’ effects on PEG-mediated fusion of PC/PE/SM/CH (35/30/15/20) SUVs. Lipid mixing (LM), contents mixing (CM) and leakage (L) time courses were fitted globally to a 3-state sequential model (Weinreb & Lentz, BJ, 2007, 4012), from which we obtained estimates of rate constants for conversion between states as well as probabilities of LM, CM and L for each state. The WT peptide increased the rates of stalk (k1) and fusion pore (k3) formation in a cooperative fashion (maximum effect seen at ∼ 0.25mol% peptide or 6 peptides/vesicle). R696L peptide had no effect on k3 and a very minor effect on k1. At this concentration, CD spectroscopy showed the WT peptide to be ∼ 39% helix, 39% unordered, and 22% β-sheet, but to increase in β- and decrease in α-content at high peptide/lipid ratios up to 1/50. Neither peptide affected the extent of content mixing, but the R696L mutant actually inhibited the extent of lipid mixing. The native but not the mutant peptide increased the probability of content mixing in the stalk intermediate. Studies with hexadecane as a space-filling agent showed that the TMD peptide was unlikely to promote fusion by this mechanism. Analysis of transition state thermodynamics suggests that the TMD may disrupt interfacial packing so as to promote penetration of lipid acyl chains into the inter-bilayer space as a means of catalyzing stalk formation. Supported by NIGMS grant 32707 to BRL.

Capturing a Fusion Intermediate of Influenza Hemagglutinin with a Cholesterol-conjugated Peptide, a New Antiviral Strategy for Influenza Virus

We previously described fusion-inhibitory peptides that are targeted to the cell membrane by cholesterol conjugation and potently inhibit enveloped viruses that fuse at the cell surface, including HIV, parainfluenza, and henipaviruses. However, for viruses that fuse inside of intracellular compartments, fusion-inhibitory peptides have exhibited very low antiviral activity. We propose that for these viruses, too, membrane targeting via cholesterol conjugation may yield potent compounds. Here we compare the activity of fusion-inhibitory peptides derived from the influenza hemagglutinin (HA) and show that although the unconjugated peptides are inactive, the cholesterol-conjugated compounds are effective inhibitors of infectivity and membrane fusion. We hypothesize that the cholesterol moiety, by localizing the peptides to the target cell membrane, allows the peptides to follow the virus to the intracellular site of fusion. The cholesterol-conjugated peptides trap HA in a transient intermediate state after fusion is triggered but before completion of the refolding steps that drive the merging of the viral and cellular membranes. These results provide proof of concept for an antiviral strategy that is applicable to intracellularly fusing viruses, including known and emerging viral pathogens.

3D6 and 4B3: Recombinant expression of two anti-gp41 antibodies as dimeric and secretory IgA

Background Sexually transmitted diseases are predominantly acquired via mucosal membranes of the rectal or genital tract during sexual intercourse. This major port of virus entry is naturally defended by the humoral immune response, with immunoglobulin A (IgA) as the primary antibody class to elicit mucosal immunity. Dimeric IgA (dIgA) reaches the luminal side of mucosal tissues by transcytosis through epithelial cells lining the mucosa. In a first step, dIgAs specifically bind to the basolaterally expressed polymeric immunoglobulin receptor (pIgR) on epithelial cells. For release of IgAs on the luminal side the extracellular portion, termed secretory component (SC), remains attached to the antibody to form secretory IgA (sIgA) [1,2]. One example of a sexually transmitted disease is the human immunodeficiency virus (HIV) which annually infects several million individuals on a global scale and potentially leads to the acquired immunodeficiency syndrome (AIDS). Although current therapies can reduce disease progression in infected individuals, no cure is yet available or within reach in near future. As a consequence increased attention is now being paid to develop drugs that could prevent virus acquisition. 3D6 and 4B3 are two monoclonal antibodies (mAb) which have originally been isolated as IgG1 isotype from seroconverted HIV-1 patients and bind to the principal immunodominant domain of gp41. In the course of this project both mAbs were isotype switched to IgA1. Recombinant CHO cell lines were established for the production of 3D6 and 4B3 as dimeric as well as secretory IgA. While dIgA were expressed by a single cell line, sIgA are produced by a biochemical association of dIgA with SC. Both dIgA and sIgA variants were characterized and the contribution of the heavily glycosylated SC on IgA stability will be investigated.

Evidence against Extracellular Exposure of a Highly Immunogenic Region in the C-Terminal Domain of the Simian Immunodeficiency Virus gp41 Transmembrane Protein

The generally accepted model for human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein topology includes a single membrane-spanning domain. An alternate model has been proposed which features multiple membrane-spanning domains. Consistent with the alternate model, a high percentage of HIV-1-infected individuals produce unusually robust antibody responses to a region of envelope, the so-called "Kennedy epitope," that in the conventional model should be in the cytoplasm. Here we show analogous, robust antibody responses in simian immunodeficiency virus SIVmac239-infected rhesus macaques to a region of SIVmac239 envelope located in the C-terminal domain, which in the conventional model should be inside the cell. Sera from SIV-infected rhesus macaques consistently reacted with overlapping oligopeptides corresponding to a region located within the cytoplasmic domain of gp41 by the generally accepted model, at intensities comparable to those observed for immunodominant areas of the surface component gp120. Rabbit serum raised against this highly immunogenic region (HIR) reacted with SIV envelope in cell surface-staining experiments, as did monoclonal anti-HIR antibodies isolated from an SIVmac239-infected rhesus macaque. However, control experiments demonstrated that this surface staining could be explained in whole or in part by the release of envelope protein from expressing cells into the supernatant and the subsequent attachment to the surfaces of cells in the culture. Serum and monoclonal antibodies directed against the HIR failed to neutralize even the highly neutralization-sensitive strain SIVmac316. Furthermore, a potential N-linked glycosylation site located close to the HIR and postulated to be outside the cell in the alternate model was not glycosylated. An artificially introduced glycosylation site within the HIR was also not utilized for glycosylation. Together, these data support the conventional model of SIV envelope as a type Ia transmembrane protein with a single membrane-spanning domain and without any extracellular loops.

Membrane-Proximal External HIV-1 gp41 Motif Adapted for Destabilizing the Highly Rigid Viral Envelope

Electron microscopy structural determinations suggest that the membrane-proximal external region (MPER) of glycoprotein 41 (gp41) may associate with the HIV-1 membrane interface. It is further proposed that MPER-induced disruption and/or deformation of the lipid bilayer ensue during viral fusion. However, it is predicted that the cholesterol content of this membrane (∼45 mol %) will act against MPER binding and restructuring activity, in agreement with alternative structural models proposing that the MPER constitutes a gp41 ectodomain component that does not insert into the viral membrane. Here, using MPER-based peptides, we test the hypothesis that cholesterol impedes the membrane association and destabilizing activities of this gp41 domain. To that end, partitioning and leakage assays carried out in lipid vesicles were combined with x-ray reflectivity and grazing-incidence diffraction studies of monolayers. CpreTM, a peptide combining the carboxyterminal MPER sequence with aminoterminal residues of the transmembrane domain, bound and destabilized effectively cholesterol-enriched membranes. Accordingly, virion incubation with this peptide inhibited cell infection potently but nonspecifically. Thus, CpreTM seems to mimic the envelope-perturbing function of the MPER domain and displays antiviral activity. As such, we infer that CpreTM bound to cholesterol-enriched membranes would represent a relevant target for anti-HIV-1 immunogen and inhibitor development.

Adoption of an "open" envelope conformation facilitating CD4 binding and structural remodeling precedes coreceptor switch in R5 SHIV-infected macaques.

A change in coreceptor preference from CCR5 to CXCR4 towards the end stage disease in some HIV-1 infected individuals has been well documented, but the reasons and mechanisms for this tropism switch remain elusive. It has been suggested that envelope structural constraints in accommodating amino acid changes required for CXCR4 usage is an obstacle to tropism switch, limiting the rate and pathways available for HIV-1 coreceptor switching. The present study was initiated in two R5 SHIVSF162P3N-infected rapid progressor macaques with coreceptor switch to test the hypothesis that an early step in the evolution of tropism switch is the adoption of a less constrained and more “open” envelope conformation for better CD4 usage, allowing greater structural flexibility to accommodate further mutational changes that confer CXCR4 utilization. We show that, prior to the time of coreceptor switch, R5 viruses in both macaques evolved to become increasingly sCD4-sensitive, suggestive of enhanced exposure of the CD4 binding site and an “open” envelope conformation, and this correlated with better gp120 binding to CD4 and with more efficient infection of CD4low cells such as primary macrophages. Moreover, significant changes in neutralization sensitivity to agents and antibodies directed against functional domains of gp120 and gp41 were seen for R5 viruses close to the time of X4 emergence, consistent with global changes in envelope configuration and structural plasticity. These observations in a simian model of R5-to-X4 evolution provide a mechanistic basis for the HIV-1 coreceptor switch.

Epitopes immediately below the base of the V3 loop of gp120 as targets for the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals.

Epitopes that drive the initial autologous neutralizing antibody response in HIV-1-infected individuals could provide insights for vaccine design. Although highly strain specific, these epitopes are immunogenic, vulnerable to antibody attack on infectious virus, and could be involved in the ontogeny of broadly neutralizing antibody responses. To delineate such epitopes, we used site-directed mutagenesis, autologous plasma samples, and autologous monoclonal antibodies to map the amino acid changes that led to escape from the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals. Additional mapping of the epitopes was accomplished by using alanine scanning mutagenesis. Escape in the two individuals occurred by different pathways, but the responses in both cases appeared to be directed against the same region of gp120. In total, three amino acid positions were identified that were independently associated with autologous neutralization. Positions 295 and 332 are located immediately before and after the N- and C-terminal cysteines of the V3 loop, respectively, the latter of which affected an N-linked glycan that was critical to the neutralization epitope. Position 415 affected an N-linked glycan at position 413 in the C terminus of V4 that might mask epitopes near the base of V3. All three sites lie in close proximity on a four-stranded antiparallel sheet on the outer domain of gp120. We conclude that a region just below the base of the V3 loop, near the coreceptor binding domain of gp120, can be a target for autologous neutralization.

Probing the HIV gp120 Envelope Glycoprotein Conformation by NMR

The HIV envelope glycoprotein gp120 plays a critical role in virus entry, and thus, its structure is of extreme interest for the development of novel therapeutics and vaccines. To date, high resolution structural information about gp120 in complex with gp41 has proven intractable. In this study, we characterize the structural properties of gp120 in the presence and absence of gp41 domains by NMR. Using the peptide probe 12p1 (sequence, RINNIPWSEAMM), which was identified previously as an entry inhibitor that binds to gp120, we identify atoms of 12p1 in close contact with gp120 in the monomeric and trimeric states. Interestingly, the binding mode of 12p1 with gp120 is similar for clades B and C. In addition, we show a subtle difference in the binding mode of 12p1 in the presence of gp41 domains, i.e. the trimeric state, which we interpret as small differences in the gp120 structure in the presence of gp41.

Stabilization of HIV-1 Envelope in the CD4-bound Conformation through Specific Cross-linking of a CD4 Mimetic

CD4 binding on gp120 leads to the exposure of highly conserved regions recognized by the HIV co-receptor CCR5 and by CD4-induced (CD4i) antibodies. A covalent gp120-CD4 complex was shown to elicit CD4i antibody responses in monkeys, which was correlated with control of the HIV virus infection (DeVico, A., Fouts, T., Lewis, G. K., Gallo, R. C., Godfrey, K., Charurat, M., Harris, I., Galmin, L., and Pal, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 17477-17482). Because the inclusion of CD4 in a vaccine formulation should be avoided, due to potential autoimmune reactions, we engineered small sized CD4 mimetics (miniCD4s) that are poorly immunogenic and do not induce anti-CD4 antibodies. We made covalent complexes between such an engineered miniCD4 and gp120 or gp140, through a site-directed coupling reaction. These complexes were recognized by CD4i antibodies as well as by the HIV co-receptor CCR5. In addition, they elicit CD4i antibody responses in rabbits and therefore represent potential vaccine candidates that mimic an important HIV fusion intermediate, without autoimmune hazard.

A Chimeric HIV-1 Envelope Glycoprotein Trimer with an Embedded Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) Domain Induces Enhanced Antibody and T Cell Responses

An effective HIV-1 vaccine should ideally induce strong humoral and cellular immune responses that provide sterilizing immunity over a prolonged period. Current HIV-1 vaccines have failed in inducing such immunity. The viral envelope glycoprotein complex (Env) can be targeted by neutralizing antibodies to block infection, but several Env properties limit the ability to induce an antibody response of sufficient quantity and quality. We hypothesized that Env immunogenicity could be improved by embedding an immunostimulatory protein domain within its sequence. A stabilized Env trimer was therefore engineered with the granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted into the V1V2 domain of gp120. Probing with neutralizing antibodies showed that both the Env and GM-CSF components of the chimeric protein were folded correctly. Furthermore, the embedded GM-CSF domain was functional as a cytokine in vitro. Mouse immunization studies demonstrated that chimeric Env(GM-CSF) enhanced Env-specific antibody and T cell responses compared with wild-type Env. Collectively, these results show that targeting and activation of immune cells using engineered cytokine domains within the protein can improve the immunogenicity of Env subunit vaccines.

A new paradigm in molecular recognition? specific antibody binding to membrane-inserted HIV-1 epitopes

The conserved membrane proximal external region (MPER), adjacent to the transmembrane domain (TMD) of human immunodeficiency virus type-1 (HIV-1) gp41 glycoprotein subunit, is accessible to the broadly neutralizing 4E10 and 2F5 monoclonal antibodies (mAbs) and, therefore, constitutes a potential target for vaccine design. This gp41 domain is postulated to be functional during the Env glycoprotein-mediated fusion reaction by destabilizing the highly rigid viral envelope. To perform this task, the aromatic-rich MPER is believed to insert into the interfacial region of the viral membrane external monolayer, thereby inducing the restructuring of the lipid bilayer required for fusion-pore opening. This model predicts that: (i) 2F5 and 4E10 mAbs are capable of binding epitopes inserted into the membrane interface; (ii) in-membrane binding will result in effective blocking of MPER membrane activity; and (iii) both processes, in-membrane recognition and blocking of membrane activity, can be modulated by altering both the lipid composition and the MPER amino acid sequence. We review here recently reported experimental data consistent with those predictions, and further speculate on their relevance for prospective anti-HIV vaccine development.

Mutagenesis of tyrosine and di-leucine motifs in the HIV-1 envelope cytoplasmic domain results in a loss of Env-mediated fusion and infectivity

BackgroundThe gp41 component of the Human Immunodeficiency Virus (HIV) envelope glycoprotein (Env) contains a long cytoplasmic domain (CD) with multiple highly conserved tyrosine (Y) and dileucine (LL) motifs. Studies suggest that the motifs distal to major endocytosis motif (Y712HRL), located at residues 712-715 of Env, may contribute to Env functionality in the viral life cycle. In order to examine the biological contribution of these motifs in the biosynthesis, transport, and function of Env, we constructed two panels of mutants in which the conserved Y- and LL-motifs were sequentially substituted by alternative residues, either in the presence or absence of Y712. Additional mutants targeting individual motifs were then constructed.ResultsAll mutant Envs, when expressed in the absence of other viral proteins, maintained at least WT levels of Env surface staining by multiple antibodies. The Y712 mutation (Y712C) contributed to at least a 4-fold increase in surface expression for all mutants containing this change. Sequential mutagenesis of the Y- and LL-motifs resulted in a generally progressive decrease in Env fusogenicity. However, additive mutation of dileucine and tyrosine motifs beyond the tyrosine at residue 768 resulted in the most dramatic effects on Env incorporation into virions, viral infectivity, and virus fusion with target cells.ConclusionsFrom the studies reported here, we show that mutations of the Y- and LL-motifs, which effectively eliminate the amphipathic nature of the lytic peptide 2 (LLP2) domain or disrupt YW and LL motifs in a region spanning residues 795-803 (YWWNLLQYW), just C-terminal of LLP2, can dramatically interfere with biological functions of HIV-1 Env and abrogate virus replication. Because these mutant proteins are expressed at the cell surface, we conclude that tyrosine and di-leucine residues within the cytoplasmic domain of gp41 play critical roles in HIV-1 replication that are distinct from that of targeting the plasma membrane.

Conserved arginine residue in the membrane-spanning domain of HIV-1 gp41 is required for efficient membrane fusion

Despite the high mutation rate of HIV-1, the amino acid sequences of the membrane-spanning domain (MSD) of HIV-1 gp41 are well conserved. Arginine residues are rarely found in single membrane-spanning domains, yet an arginine residue, R(696) (the numbering is based on that of HXB2), is highly conserved in HIV-1 gp41. To examine the role of R(696), it was mutated to K, A, I, L, D, E, N, and Q. Most of these substitutions did not affect the expression, processing or surface distribution of the envelope protein (Env). However, a syncytia formation assay showed that the substitution of R(696) with amino acid residues other than K, a naturally observed mutation in the gp41 MSD, decreased fusion activity. Substitution with hydrophobic amino acid residues (A, I, and L) resulted in a modest decrease, while substitution with D or E, potentially negatively-charged residues, almost abolished the syncytia formation. All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein (DSP) assay that scores the degree of membrane fusion based on pore formation between fusing cells. Interestingly, the D and E substitutions did show some fusion activity in the DSP assays, suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability. However, nascent pores failed to develop, due probably to impaired activity in the pore enlargement process. Our data show the importance of this conserved arginine residue for efficient membrane fusion.

HIV-1 gp41 Core with Exposed Membrane-Proximal External Region Inducing Broad HIV-1 Neutralizing Antibodies

The membrane-proximal external region (MPER) of the HIV-1 gp41 consists of epitopes for the broadly cross-neutralizing monoclonal antibodies 2F5 and 4E10. However, antigens containing the linear sequence of these epitopes are unable to elicit potent and broad neutralizing antibody responses in vaccinated hosts, possibly because of inappropriate conformation of these epitopes. Here we designed a recombinant antigen, designated NCM, which comprises the N- and C-terminal heptad repeats that can form a six-helix bundle (6HB) core and the MPER domain of gp41. Two mutations (T569A and I675V) previously reported to expose the neutralization epitopes were introduced into NCM to generate mutants named NCM(TA), NCM(IV), and NCM(TAIV). Our results showed that NCM and its mutants could react with antibodies specific for 6HB and MPER of gp41, suggesting that these antigens are in the form of a trimer of heterodimer (i.e., 6HB) with three exposed MPER tails. Antigen with double mutations, NCM(TAIV), elicited much stronger antibody response in rabbits than immunogens with single mutation, NCM(TA) and NCM(IV), or no mutation, NCM. The purified MPER-specific antibodies induced by NCM(TAIV) exhibited broad neutralizing activity, while the purified 6HB-specific antibodies showed no detectable neutralizing activity. Our recombinant antigen design supported by an investigation of its underlying molecular mechanisms provides a strong scientific platform for the discovery of a gp41 MPER-based AIDS vaccine.

Theoretical Studies on the Interactions and Interferences of HIV-1 Glycoprotein gp120 and Its Coreceptor CCR5

The interaction between the HIV gp120 protein and coreceptor CCR5 or CXCR4 of the host cell is critical in mediating the HIV entry process. A model for the CCR5-gp120 complex has been developed. In the model, the N-terminus of CCR5 binds to three discontinuous domains of gp120, including the fourth conserved (C4) region, β19/β20 connecting loop, and V3 loop. The second extra-cellular loop (ECL2) of CCR5 also interacts with the crown part of the gp120 V3 loop. The bindings of the three CCR5 antagonists, maraviroc, aplaviroc, and vicriviroc, to the trans-membrane domain of CCR5 have been modeled. The bindings are found to affect the conformation of the ECL2 domain, which in turn drives the N-terminus of CCR5 to an altered state. Aplaviroc is more hydrophilic than maraviroc and vicriviroc, and its binding is more interfered by solvent, resulting in a quite different effect to the structure of CCR5 compared with those of the other two molecules. The above results are in accord with experimental observations and provide a structural basis for further design of CCR5 antagonists.

Trans-Membrane Domain of HIV gp41 Interacts with the Externally Added gp41 Fusion Peptide: TMD-FP Complex Inhibits Model Membrane Fusion

We report the effects of a complex between fusion peptide (FP) and trans-membrane domain (TMD) of gp41, that separately promote fusion, on the kinetics of PEG-mediated fusion and on the structure of the bilayer. Circular dichroism (CD) measurements showed that FP in the membrane exists mostly in a beta sheet at L/P ratios 1200/1 to 200/1, while the gp41 TMD displayed ∼32% helix, 23% beta, 21% turn and 24% other secondary structure content at L/P of 300/1. Adding FP to membranes containing TMD resulted in a TMD-FP complex with reduced helical content and increased beta and turn content. DPH and TMA-DPH anisotropy measurements revealed that the FP-TMD complex reduced membrane order in both the interface and interior regions of the bilayer, whereas FP alone increased order in both regions. The complex also increased water penetration into the bilayer (lifetime ratio of TMA-DPH in H2O and D2O) much more than for the individual peptides. While the FP decreased membrane free volume (from partitioning of C6NBDPC from micellar to bilayer phase), the presence of TMD enhanced this effect enormously. The time courses of lipid mixing (LM), content mixing (CM) and content leakage (L) were fitted globally to 3-state or 4-state sequential models (Biophys. J., 2007, 92; 4012), providing estimates of rate constants for inter-conversion between states as well as probabilities of the occurrence of LM, CM or L in each state. The FP-TMD complex inhibited the rates of initial intermediate but especially pore formation. The effect on pore formation was predominantly due to a large increase in the activation enthalpy not matched by as large an increase activation entropy. Supported by NIGMS grant 32707 to BRL.