Rational site-directed mutations of the LLP-1 and LLP-2 lentivirus lytic peptide domains in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41 indicate common functions in cell-cell fusion but distinct roles in virion envelope incorporation

Two highly conserved cationic amphipathic alpha-helical motifs, designated lentivirus lytic peptides 1 and 2 (LLP-1 and LLP-2), have been characterized in the carboxyl terminus of the transmembrane (TM) envelope glycoprotein (Env) of lentiviruses. Although various properties have been attributed to these domains, their structural and functional significance is not clearly understood. To determine the specific contributions of the Env LLP domains to Env expression, processing, and incorporation and to viral replication and syncytium induction, site-directed LLP mutants of a primary dualtropic infectious human immunodeficiency virus type 1 (HIV-1) isolate (ME46) were examined. Substitutions were made for highly conserved arginine residues in either the LLP-1 or LLP-2 domain (MX1 or MX2, respectively) or in both domains (MX4). The HIV-1 mutants with altered LLP domains demonstrated distinct phenotypes. The LLP-1 mutants (MX1 and MX4) were replication defective and showed an average of 85% decrease in infectivity, which was associated with an evident decrease in gp41 incorporation into virions without a significant decrease in Env expression or processing in transfected 293T cells. In contrast, MX2 virus was replication competent and incorporated a full complement of Env into its virions, indicating a differential role for the LLP-1 domain in Env incorporation. Interestingly, the replication-competent MX2 virus was impaired in its ability to induce syncytia in T-cell lines. This defect in cell-cell fusion did not correlate with apparent defects in the levels of cell surface Env expression, oligomerization, or conformation. The lack of syncytium formation, however, correlated with a decrease of about 90% in MX2 Env fusogenicity compared to that of wild-type Env in quantitative luciferase-based cell-cell fusion assays. The LLP-1 mutant MX1 and MX4 Envs also exhibited an average of 80% decrease in fusogenicity. Altogether, these results demonstrate for the first time that the highly conserved LLP domains perform critical but distinct functions in Env incorporation and fusogenicity.     

Surface exposure of the HIV-1 env cytoplasmic tail LLP2 domain during the membrane fusion process: interaction with gp41 fusion core

HIV-1 gp41 cytoplasmic tail (CT) is highly conserved among HIV-1 isolates, particularly the region designated lentivirus lytic peptide (LLP1-2), which includes two alpha-helical domains LLP1 and LLP2. Although the gp41 CT is recognized as a modulator of viral fusogenicity, little is known about the regulatory mechanism of this region in the viral fusion process. Here we report that anti-LLP1-2 and anti-LLP2 antibodies (IgG) inhibited HIV-1 Env-mediated cell fusion and bound to the interface between effector and target cells at a suboptimal temperature (31.5 degrees C), which slows down the fusion process and prolongs the fusion intermediate state. This suggests that LLP1-2, especially the LLP2 region located inside the viral membrane, is transiently exposed on the membrane surface during the fusion process. Synthetic LLP2 peptide could bind to the gp41 six-helix bundle core with high binding affinity. These results suggest that the gp41 CT may interact with the gp41 core, via the surface-exposed LLP2 domain, to regulate Env-mediated membrane fusion.     

Theta-defensins prevent HIV-1 Env-mediated fusion by binding gp41 and blocking 6-helix bundle formation

Retrocyclin-1, a -defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 microm) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.     

Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41

A previous study from our laboratory reported a preferential conservation of arginine relative to lysine in the C-terminal tail (CTT) of HIV-1 envelope (Env). Despite substantial overall sequence variation in the CTT, specific arginines are highly conserved in the lentivirus lytic peptide (LLP) motifs and are scarcely substituted by lysines, in contrast to gp120 and the ectodomain of gp41. However, to date, no explanation has been provided to explain the selective incorporation and conservation of arginines over lysines in these motifs. Herein, we address the functions in virus replication of the most conserved arginines by performing conservative mutations of arginine to lysine in the LLP1 and LLP2 motifs. The presence of lysine in place of arginine in the LLP1 motif resulted in significant impairment of Env expression and consequently virus replication kinetics, Env fusogenicity, and incorporation. By contrast, lysine exchanges in LLP2 only affected the level of Env incorporation and fusogenicity. Our findings demonstrate that the conservative lysine substitutions significantly affect Env functional properties indicating a unique functional role for the highly conserved arginines in the LLP motifs. These results provide for the first time a functional explanation to the preferred incorporation of arginine, relative to lysine, in the CTT of HIV-1 Env. We propose that these arginines may provide unique functions for Env interaction with viral or cellular cofactors that then influence overall Env functional properties.     

人类免疫缺陷病毒1型gp41结构与功能的研究进展

人类免疫缺陷病毒1型(HIV-1)通过其包膜糖蛋白(Env)介导侵入靶细胞.Env由受体特异性结合单位gp120和膜融合单位gp41组成.HIV-1的gp41分为3个功能区:膜外区、跨膜区和膜内区.膜外区是病毒感染时膜融合的主要结构基础;跨膜区通过疏水残基使Env锚定在脂质膜上;膜内区则表现多重功能,参与病毒的感染、复...     

Mutations in the membrane-spanning domain of the human immunodeficiency virus envelope glycoprotein that affect fusion activity.

A chimeric protein consisting of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) ectodomain joined to the transmembrane and cytoplasmic-tail domains of vesicular stomatitis virus G protein lost the ability to fuse CD4+ HeLa cells yet was transported to the cell surface and cleaved normally. These results suggested some critical role of the HIV gp41 transmembrane or cytoplasmic domain in fusion. Subsequent mutagenic analysis of the HIV-1 Env transmembrane domain revealed that the sequence of amino acid residues from positions 696 to 707 of the transmembrane domain was important for fusion function but was not required for anchoring of the Env protein in the lipid bilayer or for transport to the cell surface. Further analysis indicated that the basic residues at positions 696 and 707 were critical for membrane fusion activity, as was the spacing between these residues. These results demonstrate that in addition to providing an anchoring function, the specific amino acid sequence in the transmembrane domain plays a crucial role in the membrane fusion process.     

Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS

Background

CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively.

Results

Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure.

Conclusion

Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.     

The crystal structure of the SIV gp41 ectodomain at 1.47 A resolution.

Cell membrane fusion by human (HIV) and simian (SIV) immunodeficiency viruses is mediated by the envelope glycoproteins gp120 and gp41. Although the precise mechanism of the fusion process is unknown, the ectodomain of gp41 is thought to undergo dramatic rearrangement from its prefusogenic state. To elucidate this process further, the crystal structure of the SIV gp41 ectodomain (residues 27-149) was determined at 1.47 A resolution and is reported herein. It is the most accurate and complete structure of a retroviral gp41 ectodomain determined to date. The rod-like trimeric structure of SIV gp41 comprises three parallel N-terminal alpha-helices assembled as a coiled coil in the center with three antiparallel C-terminal alpha-helices packed on the outside connected by highly flexible loops. Portions of the loops in all three monomers are crystallographically disordered and could not be accurately modeled. The core of the structure is similar (but not identical) to those of smaller HIV/SIV gp41 segments previously determined by X-ray crystallography with root mean square deviations in main chain atoms of less than 1.0 A. The crystal structure differs more substantially from the reported NMR solution structure of the identical SIV construct. The mechanisms of viral fusion and the inhibition by peptides are discussed in the context of the three-dimensional structure.     

Antigenic properties of the human immunodeficiency virus transmembrane glycoprotein during cell-cell fusion

Human immunodeficiency virus (HIV) entry is triggered by interactions between a pair of heptad repeats in the gp41 ectodomain, which convert a prehairpin gp41 trimer into a fusogenic three-hairpin bundle. Here we examined the disposition and antigenic nature of these structures during the HIV-mediated fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various lengths of time and then arrested. Fusion intermediates were then examined for reactivity with various monoclonal antibodies (MAbs) against immunogenic cluster I and cluster II epitopes in the gp41 ectodomain. All of these MAbs produced similar staining patterns indicative of reactivity with prehairpin gp41 intermediates or related structures. MAb staining was seen on Env cells only upon exposure to soluble CD4, CD4-positive, coreceptor-negative cells, or stromal cell-derived factor-treated target cells. In the fusion system, the MAbs reacted with the interfaces of attached Env and target cells within 10 min of coculture. MAb reactivity colocalized with the formation of gp120-CD4-coreceptor tricomplexes after longer periods of coculture, although reactivity was absent on cells exhibiting cytoplasmic dye transfer. Notably, the MAbs were unable to inhibit fusion even when allowed to react with soluble-CD4-triggered or temperature-arrested antigens prior to initiation of the fusion process. In comparison, a broadly neutralizing antibody, 2F5, which recognizes gp41 antigens in the HIV envelope spike, was immunoreactive with free Env cells and Env-target cell clusters but not with fused cells. Notably, exposure of the 2F5 epitope required temperature-dependent elements of the HIV envelope structure, as MAb binding occurred only above 19 degrees C. Overall, these results demonstrate that immunogenic epitopes, both neutralizing and nonneutralizing, are accessible on gp41 antigens prior to membrane fusion. The 2F5 epitope appears to depend on temperature-dependent elements on prefusion antigens, whereas cluster I and cluster II epitopes are displayed by transient gp41 structures. Such findings have important implications for HIV vaccine approaches based on gp41 intermediates.     

Promoting trimerization of soluble human immunodeficiency virus type 1 (HIV-1) Env through the use of HIV-1/simian immunodeficiency virus chimeras

The envelope proteins (Env) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) form homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained, but is less stable, after cleavage in a Golgi compartment and transport to the surface of infected cells. Functional, virion-associated HIV-1 and SIV Env have an almost exclusively trimeric structure. In addition, a soluble form of SIV Env (gp140) forms a nearly homogeneous population of trimers. Here, we describe the oligomeric structure of soluble, uncleaved HIV-1 gp140 and modifications that promote a stable trimeric structure. Biochemical and biophysical analyses, including sedimentation equilibrium and scanning transmission electron microscopy, revealed that unmodified HIV-1 gp140 purified as a heterogeneous range of oligomeric species, including dimers and aggregates. Deletion of the V2 domain alone or, especially, both the V1 and V2 domains reduced dimer formation but promoted aggregation rather than trimerization. Expressing gp140 with mannose-only oligosaccharides did not eliminate heterogeneity. Replacement of the entire gp41 segment of HIV-1 gp140 or just the N-terminal half (85 amino acids) of this segment with the corresponding region of SIV was sufficient to confer efficient trimerization for gp140 derived from clade B and C isolates. Importantly, the relatively small segment of the HIV Env replaced by SIV sequences contains no known targets of neutralizing antibody. The soluble trimeric form of HIV-1 Env should prove useful for assessment of antigenic structure and immunogenicity.     

Model for the structure of the HIV gp41 ectodomain: insight into the intermolecular interactions of the gp41 loop

In human immunodeficiency virus (HIV) the viral envelope proteins gp41 and gp120 form a non-covalent complex, which is a potential target for AIDS therapies. In addition gp41 plays a possible role in HIV infection of B cells via the complement system. In an effort to better understand the molecular interactions of gp41, the structure of the HIV gp41 ectodomain has been modeled using the NMR restraints of the simian immunodeficiency virus (SIV) gp41 ectodomain (M. Caffrey, M. Cai, J. Kaufman, S.J. Stahl, P.T. Wingfield, A.M. Gronenborn, G.M. Clore, Solution structure of the 44 kDa ectodomain of SIV gp41, EMBO J. 17 (1998) 4572--4584). The resulting model presents the first structural information for the HIV gp41 loop, which has been implicated to play a direct role in binding to gp120 and C1q of the complement system.     

Highly conserved structural properties of the C-terminal tail of HIV-1 gp41 protein despite substantial sequence variation among diverse clades: implications for functions in viral replication

Although the HIV-1 Env gp120 and gp41 ectodomain have been extensively characterized in terms of structure and function, similar characterizations of the C-terminal tail (CTT) of HIV gp41 remain relatively limited and contradictory. The current study was designed to examine in detail CTT sequence conservation relative to gp120 and the gp41 ectodomain and to examine the conservation of predicted physicochemical and structural properties across a number of divergent HIV clades and groups. Results demonstrate that CTT sequences display intermediate levels of sequence evolution and diversity in comparison to the more diverse gp120 and the more conserved gp41 ectodomain. Despite the relatively high level of CTT sequence variation, the physicochemical properties of the lentivirus lytic peptide domains (LLPs) within the CTT are evidently highly conserved across clades/groups. Additionally, predictions using PEP-FOLD indicate a high level of structural similarity in the LLP regions that was confirmed by circular dichroism measurements of secondary structure of LLP peptides from clades B, C, and group O. Results demonstrate that LLP peptides adopt helical structure in the presence of SDS or trifluoroethanol but are predominantly unstructured in aqueous buffer. Thus, these data for the first time demonstrate strong conservations of characteristic CTT physicochemical and structural properties despite substantial sequence diversity, apparently indicating a delicate balance between evolutionary pressures and the conservation of CTT structure and associated functional roles in virus replication.     

Evaluation of the cytopathicity (fusion/hemifusion) of patient-derived HIV-1 envelope glycoproteins comparing two effector cell lines

HIV-1 envelope glycoprotein (Env) is a major determinant of viral pathogenicity. The evaluation of the biological properties of patient-derived envelopes by comparing two effector cell lines (293T and HeLa) is reported. A standard cell-to-cell fusion assay was used to evaluate fusogenicity, whereas a coculture with CD4(+) cells was used to evaluate absolute cell loss, single cell death, and hemifusion events. Fusion and absolute cell loss assays showed that Env-expressing 293T and HeLa cells had different fusion efficiencies; fusion was magnified in 293T cells despite a significantly lower cell-surface Env expression. Conversely, gp41-mediated single cell death and hemifusion induced in CD4(+) cells by 293T-Env-positive cells were significantly lower than that induced by HeLa-Env-positive cells. These data showed that the effector cell line used in the in vitro assays is crucial, and a combination of assays is recommended to evaluate the biological properties of patient-derived envelope glycoproteins: preferentially, 293T-Env-positive cells for the evaluation of fusogenicity and HeLa-Env-positive cells for the evaluation of cell death parameters. The combination of assays described in our work could be a valuable tool for dual screenings of large collections of primary Envs or Env mutants and drugs acting on these Envs.     

Role of the fusion peptide and membrane-proximal domain in HIV-1 envelope glycoprotein-mediated membrane fusion

The N-terminal fusion peptide and the interfacial sequence preceding the transmembrane anchor of HIV-1 gp41 are required for viral fusion. Studies with synthetic peptides indicated that these regions function by destabilizing membranes, which is regarded as a crucial step in the membrane fusion reaction. However, it is not clear whether membrane destabilization is induced by these sequences in the intact gp41. We address this question by examining fusion and destabilization of membranes expressing HIV-1(IIIB) wild-type Env and two mutant Envs. (1) A Glu residue at position 2 of the gp41 fusion peptide is substituted for Val (V2E) to produce one mutant. (2) Residues 665-682 in the membrane-proximal domain are deleted to form the other. The process of membrane destabilization was monitored by the influx of Sytox, an impermeant fluorescent dye, into the Env-expressing cells following the interaction with CD4-CXCR4 complexes, and fusion was monitored by observing dye transfer between Env-expressing cells and appropriate target cells. We also monitored the conformational changes in the Envs following their interactions with CD4 and CXCR4 by immunofluorescence using an anti-gp41 mAb that reacts with the six-helix bundle. In contrast to the wild type, both Env mutants did not mediate cell fusion. The V2E Env did not mediate membrane destabilization. However, the Env with an unmodified fusion peptide but with a deletion of residues 665-682 in the membrane-proximal domain did mediate membrane destabilization. The wild type and both mutant Envs undergo conformational changes detected by the anti-gp41 six-helix bundle mAbs. Our results suggest that in intact HIV-1 Env the membrane-proximal domain is not required for membrane perturbations, but rather enables the bending of gp41 that is required for viral and target membranes to come together. Moreover, the observation that the Delta665-683 Env self-inserts its fusion peptide but does not cause fusion suggests that self-insertion of the fusion peptide is not sufficient for HIV-1 Env-mediated fusion.     

Replication-competent variants of human immunodeficiency virus type 2 lacking the V3 loop exhibit resistance to chemokine receptor antagonists

Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.     

Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ

The envelope glycoprotein (Env) complexes of the human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate viral entry and are a target for neutralizing antibodies. The receptor binding surfaces of Env are in large part sterically occluded or conformationally masked prior to receptor binding. Knowledge of the unliganded, trimeric Env structure is key for an understanding of viral entry and immune escape, and for the design of vaccines to elicit neutralizing antibodies. We have used cryo-electron tomography and averaging to obtain the structure of the SIV Env complex prior to fusion. Our result reveals novel details of Env organisation, including tight interaction between monomers in the gp41 trimer, associated with a three-lobed, membrane-distal gp120 trimer. A cavity exists at the gp41-gp120 trimer interface. Our model for the spike structure agrees with previously predicted interactions between gp41 monomers, and furthers our understanding of gp120 interactions within an intact spike.     

The simian immunodeficiency virus envelope glycoprotein contains multiple signals that regulate its cell surface expression and endocytosis

The cell surface expression of the envelope glycoproteins (Envs) of primate immunodeficiency viruses is, at least in part, regulated by endocytosis signal(s) located in the Env cytoplasmic domain. Here, we show that a membrane proximal signal that directs the simian immunodeficiency virus (SIV) Env to clathrin‐coated pits, and is conserved in all SIV and human immunodeficiency virus Envs, conforms to a Yxxø motif (where x can be any amino acid and Ø represents a large hydrophobic residue). This motif is similar to that described for a number of cellular membrane proteins. By surface plasmon resonance we detected a high affinity interaction between peptides containing this membrane proximal signal and both AP1 and AP2 clathrin adaptor complexes. Mutation of the tyrosine in this membrane proximal motif in a SIV Env with a prematurely truncated cytoplasmic domain leads to a ≥25‐fold increase in Env expression on infected cells. By contrast, the same mutation in an Env with a full‐length cytoplasmic domain increases cell surface expression only 4‐fold. We show that this effect results from the presence of additional endocytosis signals in the full‐length cytoplasmic domain. Chimeras containing CD4 ecto‐ and membrane spanning domains and a full‐length SIV Env cytoplasmic domain showed rapid endocytosis even when the membrane proximal tyrosine‐based signal was disrupted. Mapping experiments indicated that at least some of the additional endocytosis information is located between residues 743 and 812 of Env from the SIV_mac239 molecular clone. Together, our findings indicate that the cytoplasmic domain of SIV Env contains multiple endocytosis and/or trafficking signals that modulate its surface expression on infected cells, and suggest an important role for this function in pathogenesis.     

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

The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P) in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1_BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1_BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1_BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1_BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env spikes and I559P Envs.     

Conserved structures exposed in HIV-1 envelope glycoproteins stabilized by flexible linkers as potent entry inhibitors and potential immunogens

The HIV-1 envelope glycoprotein (Env) undergoes conformational changes while driving entry. We hypothesized that some of the intermediate Env conformations could be represented in tethered constructs where gp120 and the ectodomain of gp41 are joined by flexible linkers. Tethered Envs with long linkers (gp140-14 with 15 aa and gp140-24 with 26 aa) were stable and recognized by conformationally dependent anti-gp120 and anti-gp41 monoclonal antibodies (mAbs). Surprisingly, these proteins potently inhibited membrane fusion mediated by R5, X4, and R5X4 Envs with 5-100-fold lower IC50 than a tethered Env with short linker (gp140-4 with 4 aa), gp120, gp140, soluble CD4, or DP178 (T20). Compared to gp140, gp140-14,24 exhibited increased binding to anti-gp41 cluster II mAbs but not to cluster I mAbs. Cluster II mAbs but not cluster I, IV, or V mAbs reversed the inhibitory effect of gp140-14,24 suggesting a role of exposed conserved gp41 structures for the mechanism of inhibition. These findings suggest the existence of conserved gp41 structures that are important for HIV-1 entry and can be stably exposed in the native environment of the Env even in the absence of receptor-mediated activation. Thus, tethered Envs with long linkers may not only be important as HIV-1 inhibitors but also for elucidation of viral entry mechanisms and development of novel vaccine immunogens.