Transmembrane Glycoprotein Gp41 Research Articles

The TZM-bl Reporter Cell Line Expresses Kynureninase That Can Neutralize 2F5-like Antibodies in the HIV-1 Neutralization Assay.

Induction of broadly neutralizing antibodies targeting ectodomain of the transmembrane (TM) glycoprotein gp41 HIV-1 provides a basis for the development of a universal anti-viral vaccine. The HeLa cell-derived TZM-bl reporter cell line is widely used for the estimation of lentiviruses neutralization by immune sera. The cell line is highly permissive to infection by most strains of HIV, SIV, and SHIV. Here we demonstrated that TZM-bl cells express a 48 kDa non-glycosylated protein (p48) recognized by broadly neutralizing monoclonal antibody (mAb) 2F5 targeting the ELDKWA (aa 669–674) epitope of gp41TM of HIV-1. A significant amount of p48 was found in the cell supernatant. The protein was identified as human kynureninase (KYNU), which has the ELDKWA epitope. The protein is further called “p48 KYNU”. The HIV-1 neutralization by mAb 2F5 and 4E10 in the presence of p48KYNU was tested on Jurkat and TZM-bl cells. It was demonstrated that p48KYNU reduces neutralization by 2F5-like antibodies, but it has almost no effect on mAb 4E10. Therefore, p48KYNU can attenuate HIV-1 neutralization by 2F5-like antibodies and hence create false-negative results. Thus, previously tested immune sera that recognized the ELDKWA-epitope and demonstrated a “weak neutralization” of HIV-1 in TZM-bl assay should be reevaluated.

Small-Molecule HIV Entry Inhibitors Targeting gp120 and gp41.

Interrupting early events in the virus life cycle, such as those prior to the formation of provirus, could effectively prevent HIV-1 infection. HIV-1 entry into host cells is mediated by the envelope glycoprotein (Env) trimer, which is composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. Hence, the development of novel inhibitors targeting these two glycoproteins could hold the key to early inhibition of HIV-1 infection. Small-molecule entry inhibitors targeting early events in the virus life cycle comprise a well-established class of useful drugs. Many libraries of small-molecule inhibitors have been established to screen potential drug candidates for a variety of targets based on computer docking, FRET, or peptide-linked assay. This chapter reviews the mechanisms of some small-molecule inhibitors targeting HIV-1 gp120 and gp41 and corresponding high-efficiency screening strategies for potential small-molecule inhibitors.

Gp41-targeted antibodies restore infectivity of a fusion-deficient HIV-1 envelope glycoprotein.

The HIV-1 envelope glycoprotein (Env) mediates viral entry via conformational changes associated with binding the cell surface receptor (CD4) and coreceptor (CCR5/CXCR4), resulting in subsequent fusion of the viral and cellular membranes. While the gp120 Env surface subunit has been extensively studied for its role in viral entry and evasion of the host immune response, the gp41 transmembrane glycoprotein and its role in natural infection are less well characterized. Here, we identified a primary HIV-1 Env variant that consistently supports >300% increased viral infectivity in the presence of autologous or heterologous HIV-positive plasma. However, in the absence of HIV-positive plasma, viruses with this Env exhibited reduced infectivity that was not due to decreased CD4 binding. Using Env chimeras and sequence analysis, we mapped this phenotype to a change Q563R, in the gp41 heptad repeat 1 (HR1) region. We demonstrate that Q563R reduces viral infection by disrupting formation of the gp41 six-helix bundle required for virus-cell membrane fusion. Intriguingly, antibodies that bind cluster I epitopes on gp41 overcome this inhibitory effect, restoring infectivity to wild-type levels. We further demonstrate that the Q563R change increases HIV-1 sensitivity to broadly neutralizing antibodies (bNAbs) targeting the gp41 membrane-proximal external region (MPER). In summary, we identify an HIV-1 Env variant with impaired infectivity whose Env functionality is restored through the binding of host antibodies. These data contribute to our understanding of gp41 residues involved in membrane fusion and identify a mechanism by which host factors can alleviate a viral defect.

Strain-Dependent Activation and Inhibition of Human Immunodeficiency Virus Entry by a Specific PF-68742 Stereoisomer.

Human immunodeficiency virus (HIV-1) entry into cells is mediated by the viral envelope glycoprotein (Env) trimer, which consists of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. When gp120 binds sequentially to the receptors CD4 and CCR5 on the target cell, the metastable Env trimer is triggered to undergo entry-related conformational changes. PF-68742 is a small molecule that inhibits the infection of a subset of HIV-1 strains by interfering with an Env function other than receptor binding. Determinants of HIV-1 resistance to PF-68742 map to the disulfide loop and fusion peptide of gp41. Of the four possible PF-68742 stereoisomers, only one, MF275, inhibited the infection of CD4-positive CCR5-positive cells by some HIV-1 strains. MF275 inhibition of these HIV-1 strains occurred after CD4 binding but before the formation of the gp41 six-helix bundle. Unexpectedly, MF275 activated the infection of CD4-negative CCR5-positive cells by several HIV-1 strains resistant to the inhibitory effects of the compound in CD4-positive target cells. In contrast to CD4 complementation by CD4-mimetic compounds, activation of CD4-independent infection by MF275 did not depend upon the availability of the gp120 Phe 43 cavity. Sensitivity to inhibitors indicates that MF275-activated virus entry requires formation/exposure of the gp41 heptad repeat (HR1) as well as CCR5 binding. MF275 apparently activates a virus entry pathway parallel to that triggered by CD4 and CD4-mimetic compounds. Strain-dependent divergence in Env conformational transitions allows different outcomes, inhibition or activation, in response to MF275. Understanding the mechanisms of MF275 activity should assist efforts to optimize its utility.IMPORTANCE Envelope glycoprotein (Env) spikes on the surface of human immunodeficiency virus (HIV-1) bind target cell receptors, triggering changes in the shape of Env. We studied a small molecule, MF275, that also induced shape changes in Env. The consequences of MF275 interaction with Env depended on the HIV-1 strain, with infection by some viruses inhibited and infection by other viruses enhanced. These studies reveal the strain-dependent diversity of HIV-1 Envs as they undergo shape changes in proceeding down the entry pathway. Appreciation of this diversity will assist attempts to develop broadly active inhibitors of HIV-1 entry.

Recent Progress in the Development of HIV-1 Entry Inhibitors: From Small Molecules to Potent Anti-HIV Agents.

Viral entry, the first process in the reproduction of viruses, primarily involves attachment of the viral envelope proteins to membranes of the host cell. The crucial components that play an important role in viral entry include viral surface glycoprotein gp120, viral transmembrane glycoprotein gp41, host cell glycoprotein (CD4), and host cell chemokine receptors (CCR5 and CXCR4). Inhibition of the multiple molecular interactions of these components can restrain viruses, such as HIV-1, from fusion with the host cell, blocking them from reproducing. This review article specifically focuses on the recent progress in the development of small-molecule HIV-1 entry inhibitors and incorporates important aspects of their structural modification that lead to the discovery of new molecular scaffolds with more potency.

Optimized protocol for detection of native, full-length HIV-1 envelope on the surface of transfected cells.

AimsDesigning therapeutics against the HIV envelope glycoprotein (Env) is only as accurate as the structure of the Env they are targeting. Conserving the structure of the Env trimer is crucial for proper experimental assessment of antibody binding and neutralization. However, Env is notably difficult to express by transfection of a recombinant Env plasmid. To increase surface expression, researchers commonly utilize c‐tail mutants of the gp41 transmembrane glycoprotein of HIV‐1, but mutations and deletions in this region can impact the overall conformation and stability of the Env trimer. Multiple studies have shown that while tail mutants have higher Env surface expression, they are easier to neutralize and have altered trimer conformations compared with wild‐type Env found in vivo on infected cells. To assess and characterize native cell surface Env structures, we sought a protocol that could reliably detect wild‐type Env surface expression by flow cytometry.Methods and resultsBy avoiding fetal bovine serum–based buffers, significantly increasing the amounts of transfected plasmid and Env‐specific antibody and by selecting a bright, biotin + streptavidin‐PE detection system, we were able to increase the surface expression of transfected Env protein.ConclusionThis protocol will allow for more precise assessment of antibody binding, epitope exposure, and Env structure, all of which will contribute to designing more effective vaccines and immunotherapeutics.

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

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.

Activation and Inactivation of Primary Human Immunodeficiency Virus Envelope Glycoprotein Trimers by CD4-Mimetic Compounds.

The trimeric envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) mediate virus entry into host cells. Binding to the host cell receptors, CD4 and CCR5, triggers changes in the conformation of the HIV-1 envelope glycoprotein trimer important for virus entry. Small-molecule CD4-mimetic compounds inhibit HIV-1 infection by multiple mechanisms: (i) direct blockade of the interaction between the gp120 exterior envelope glycoprotein and CD4; (ii) premature triggering of conformational changes in the envelope glycoproteins, leading to irreversible inactivation; and (iii) exposure of cryptic epitopes to antibodies, allowing virus neutralization. The consequences of the binding of the CD4-mimetic compound to the HIV-1 envelope glycoproteins depends upon how many of the three subunits of the trimer are bound and upon the propensity of the envelope glycoproteins to undergo conformational changes. Understanding the mechanistic factors that influence the activity of CD4-mimetic compounds can help to improve their potency and coverage of diverse HIV-1 strains.

Characterization of Humoral Immune Responses against Capsid Protein p24 and Transmembrane Glycoprotein gp41 of Human Immunodeficiency Virus Type 1 in China.

The objective of this study was to extend our previous research and to further characterize the humoral immune responses against HIV-1 p24, gp41 and the specific peptides carrying the immunodominant epitopes (IDEs) that react with human serum samples from HIV-1-infected individuals in China. We found that the majority (90.45%, 180/199) of the samples did not react with any of the three HIV-1 p24 peptides carrying IDEs, but did react with the recombinant full-length p24, suggesting that these samples tested in China were primarily directed against the conformational epitopes of HIV-1 p24. In contrast, 84.54% (164/194) of the samples reacted with at least one HIV-1 linear gp41 peptide, in particular the gp41-p1 peptide (amino acids 560–616). Both recently and long-term HIV-1-infected individuals displayed similar humoral immune responses against the recombinant gp41. However, samples from long-term HIV-1-infected subjects but not from recently infected subjects, showed a very strong reaction against the gp41-p1 peptide. The different response patterns observed for the two groups against the gp41 and the peptide gp41-p1 were statistically significant (P<0.01, Chi-square test). These results have direct relevance and importance for design of improved HIV-1 p24 detection assays and the gp41- based immunoassay that can be used to reliably distinguish recent and long-term HIV-1 infection.

Structure-activity relationship studies of indole-based compounds as small molecule HIV-1 fusion inhibitors targeting glycoprotein 41.

We previously described indole-containing compounds with the potential to inhibit HIV-1 fusion by targeting the hydrophobic pocket of transmembrane glycoprotein gp41. Here we report optimization and structure–activity relationship studies on the basic scaffold, defining the role of shape, contact surface area, and molecular properties. Thirty new compounds were evaluated in binding, cell–cell fusion, and viral replication assays. Below a 1 μM threshold, correlation between binding and biological activity was diminished, indicating an amphipathic requirement for activity in cells. The most active inhibitor 6j exhibited 0.6 μM binding affinity and 0.2 μM EC50 against cell–cell fusion and live virus replication and was active against T20 resistant strains. Twenty-two compounds with the same connectivity displayed a consensus pose in docking calculations, with rank order matching the biological activity. The work provides insight into requirements for small molecule inhibition of HIV-1 fusion and demonstrates a potent low molecular weight fusion inhibitor.

The peptides and peptidomimetics: HIV-1 fusion inhibitors

The fusion of HIV and target cell membranes is mediated by the transmembrane glycoprotein gp41. HIV-1 fusion inhibitors target gp41 to prevent the virus-cell membrane fusion, thus interrupt the initial steps of viral replication. The first peptide fusion inhibitor (T-20) was approved by the U.S. FDA in 2003. However, the application of T-20 was limited by drawbacks such as rapid proteolysis, highly clinical dosage, poor patient adhesive and ineffectiveness to drug resistant HIV-1 isolates. In recent years, considerable progresses have been made in the understanding of the mechanism of fusion inhibitors and in the design of novel fusion inhibitors, to overcome the drawbacks of T-20. Various peptides and peptidomimetics used as fusion inhibitors have been developed, and these novel fusion inhibitors target different part of HIV-1 gp41 and show higher activity and better pharmacokinetic properties. In this paper, the recent progresses in the design and development of novel peptides and peptidominetics used as HIV-1 fusion inhibitors are reviewed, providing a reference for related drug development and basic research.

Reevaluation of the Requirement for TIP47 in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Incorporation

Incorporation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into assembling particles is crucial for virion infectivity. Genetic and biochemical data indicate that the matrix (MA) domain of Gag and the cytoplasmic tail of the transmembrane glycoprotein gp41 play an important role in coordinating Env incorporation; however, the molecular mechanism and possible role of host factors in this process remain to be defined. Recent studies suggested that Env incorporation is mediated by interactions between matrix and tail-interacting protein of 47 kDa (TIP47; also known as perilipin-3 and mannose-6-phosphate receptor-binding protein 1), a member of the perilipin, adipophilin, TIP47 (PAT) family of proteins implicated in protein sorting and lipid droplet biogenesis. We have confirmed by nuclear magnetic resonance spectroscopy titration experiments and surface plasmon resonance that MA binds TIP47. We also reevaluated the role of TIP47 in HIV-1 Env incorporation in HeLa cells and in the Jurkat T-cell line. In HeLa cells, TIP47 overexpression or RNA interference (RNAi)-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in Jurkat cells did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity.

Designing a Soluble Near Full-length HIV-1 gp41 Trimer

The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors, whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been extensively studied, the detailed mechanism is still poorly understood. Design of gp41 recombinants that mimic key intermediates is essential to elucidate the mechanism as well as to develop potent therapeutics and vaccines. Here, using molecular genetics and biochemical approaches, a series of hypotheses was tested to overcome the extreme hydrophobicity of HIV-1 gp41 and design a soluble near full-length gp41 trimer. The two long heptad repeat helices HR1 and HR2 of gp41 ectodomain were mutated to disrupt intramolecular HR1-HR2 interactions but not intermolecular HR1-HR1 interactions. This resulted in reduced aggregation and improved solubility. Attachment of a 27-amino acid foldon at the C terminus and slow refolding channeled gp41 into trimers. The trimers appear to be stabilized in a prehairpin-like structure, as evident from binding of a HR2 peptide to exposed HR1 grooves, lack of binding to hexa-helical bundle-specific NC-1 mAb, and inhibition of virus neutralization by broadly neutralizing antibodies 2F5 and 4E10. Fusion to T4 small outer capsid protein, Soc, allowed display of gp41 trimers on the phage nanoparticle. These approaches for the first time led to the design of a soluble gp41 trimer containing both the fusion peptide and the cytoplasmic domain, providing insights into the mechanism of entry and development of gp41-based HIV-1 vaccines.

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.

Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures.

The initial step in HIV-1 infection occurs with the binding of cell surface CD4 to trimeric HIV-1 envelope glycoproteins (Env), a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The design of soluble versions of trimeric Env that display structural and functional properties similar to those observed on intact viruses is highly desirable from the viewpoint of designing immunogens that could be effective as vaccines against HIV/AIDS. Using cryoelectron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env. We show that unliganded gp140 trimers adopt a quaternary arrangement similar to that displayed by native unliganded trimers on the surface of intact HIV-1 virions. When complexed with soluble CD4, Fab 17b, which binds to gp120 at its chemokine coreceptor binding site, or both soluble CD4 and 17b Fab, gp140 trimers display an open conformation in which there is an outward rotation and displacement of each gp120 protomer. We demonstrate that the molecular arrangements of gp120 trimers in the closed and open conformations of the soluble trimer are the same as those observed for the closed and open states, respectively, of trimeric gp120 on intact HIV-1 BaL virions, establishing that soluble gp140 trimers can be designed to mimic the quaternary structural transitions displayed by native trimeric Env.

Contribution of Intrinsic Reactivity of the HIV-1 Envelope Glycoproteins to CD4-Independent Infection and Global Inhibitor Sensitivity

Human immunodeficiency virus (HIV-1) enters cells following sequential activation of the high-potential-energy viral envelope glycoprotein trimer by target cell CD4 and coreceptor. HIV-1 variants differ in their requirements for CD4; viruses that can infect coreceptor-expressing cells that lack CD4 have been generated in the laboratory. These CD4-independent HIV-1 variants are sensitive to neutralization by multiple antibodies that recognize different envelope glycoprotein epitopes. The mechanisms underlying CD4 independence, global sensitivity to neutralization and the association between them are still unclear. By studying HIV-1 variants that differ in requirements for CD4, we investigated the contribution of CD4 binding to virus entry. CD4 engagement exposes the coreceptor-binding site and increases the “intrinsic reactivity” of the envelope glycoproteins; intrinsic reactivity describes the propensity of the envelope glycoproteins to negotiate transitions to lower-energy states upon stimulation. Coreceptor-binding site exposure and increased intrinsic reactivity promote formation/exposure of the HR1 coiled coil on the gp41 transmembrane glycoprotein and allow virus entry upon coreceptor binding. Intrinsic reactivity also dictates the global sensitivity of HIV-1 to perturbations such as exposure to cold and the binding of antibodies and small molecules. Accordingly, CD4 independence of HIV-1 was accompanied by increased susceptibility to inactivation by these factors. We investigated the role of intrinsic reactivity in determining the sensitivity of primary HIV-1 isolates to inhibition. Relative to the more common neutralization-resistant (“Tier 2-like”) viruses, globally sensitive (“Tier 1”) viruses exhibited increased intrinsic reactivity, i.e., were inactivated more efficiently by cold exposure or by a given level of antibody binding to the envelope glycoprotein trimer. Virus sensitivity to neutralization was dictated both by the efficiency of inhibitor/antibody binding to the envelope glycoprotein trimer and by envelope glycoprotein reactivity to the inhibitor/antibody binding event. Quantitative differences in intrinsic reactivity contribute to HIV-1 strain variability in global susceptibility to neutralization and explain the long-observed relationship between increased inhibitor sensitivity and decreased entry requirements for target cell CD4.

Molecular architectures of trimeric SIV and HIV-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure.

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.

Conformational characterization of aberrant disulfide-linked HIV-1 gp120 dimers secreted from overexpressing cells

The envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) mediate viral entry and are also the primary target of neutralizing antibodies. The gp160 envelope glycoprotein precursor undergoes proteolytic cleavage in the Golgi complex to produce the gp120 exterior glycoprotein and the gp41 transmembrane glycoprotein, which remain associated non-covalently in the trimeric Env complex. Monomeric soluble gp120 has been used extensively to investigate conformational states, structure, antigenicity and immunogenicity of the HIV-1 Env glycoproteins. Expression of gp120 alone (without gp41) leads to the accumulation not only of monomeric gp120 but also an aberrant dimeric form. The gp120 dimers were sensitive to reducing agents. The formation of gp120 dimers was disrupted by a single amino acid change in the inner domain, and was reduced by removal of the V1/V2 variable loops or the N and C termini. Epitopes on the gp120 inner domain and the chemokine receptor-binding surface were altered or occluded by gp120 dimerization. Awareness of the existence and properties of gp120 dimers should assist interpretation of studies of this key viral protein.

Relationship between Antibody 2F5 Neutralization of HIV-1 and Hydrophobicity of Its Heavy Chain Third Complementarity-Determining Region

The membrane-proximal external region (MPER) of the HIV-1 gp41 transmembrane glycoprotein is the target of the broadly neutralizing antibody 2F5. Prior studies have suggested a two-component mechanism for 2F5-mediated neutralization involving both structure-specific recognition of a gp41 protein epitope and nonspecific interaction with the viral lipid membrane. Here, we mutationally alter a hydrophobic patch on the third complementarity-determining region of the heavy chain (CDR H3) of the 2F5 antibody and assess the abilities of altered 2F5 variants to bind gp41 and to neutralize diverse strains of HIV-1. CDR H3 alterations had little effect on the affinity of 2F5 variants for a peptide corresponding to its gp41 epitope. In contrast, strong effects and a high degree of correlation (P < 0.0001) were found between virus neutralization and CDR H3 hydrophobicity, as defined by predicted free energies of transfer from water to a lipid bilayer interface or to octanol. The effect of CDR H3 hydrophobicity on neutralization was independent of isolate sensitivity to 2F5, and CDR H3 variants with tryptophan substitutions were able to neutralize HIV-1 approximately 10-fold more potently than unmodified 2F5. A threshold was observed for increased hydrophobicity of the 2F5 CDR H3 loop beyond which effects on 2F5-mediated neutralization leveled off. Together, the results provide a more complete understanding of the 2F5 mechanism of HIV-1 neutralization and indicate ways to enhance the potency of MPER-directed antibodies.

Role of a Putative gp41 Dimerization Domain in Human Immunodeficiency Virus Type 1 Membrane Fusion

The entry of human immunodeficiency virus type 1 (HIV-1) into a target cell entails a series of conformational changes in the gp41 transmembrane glycoprotein that mediates the fusion of the viral and target cell membranes. A trimer-of-hairpins structure formed by the association of two heptad repeat (HR) regions of the gp41 ectodomain has been implicated in a late step of the fusion pathway. Earlier native and intermediate states of the protein are postulated to mediate the antiviral activity of the fusion inhibitor enfuvirtide and of broadly neutralizing monoclonal antibodies (NAbs), but the details of these structures remain unknown. Here, we report the identification and crystal structure of a dimerization domain in the C-terminal ectodomain of gp41 (residues 630 to 683, or C54). Two C54 monomers associate to form an asymmetric, antiparallel coiled coil with two distinct C-terminal alpha-helical overhangs. This dimer structure is conferred largely by interactions within a central core that corresponds to the sequence of enfuvirtide. The mutagenic alteration of the dimer interface severely impairs the infectivity of Env-pseudotyped viruses. Moreover, the C54 structure binds tightly to both the 2F5 and 4E10 NAbs and likely represents a potential intermediate conformation of gp41. These results should enhance our understanding of the molecular basis of the gp41 fusogenic structural transitions and thereby guide rational, structure-based efforts to design new fusion inhibitors and vaccine candidates intended to induce broadly neutralizing antibodies.