Transitions to and from the CD4-Bound Conformation Are Modulated by a Single-Residue Change in the Human Immunodeficiency Virus Type 1 gp120 Inner Domain

Binding to the primary receptor CD4 induces conformational changes in the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein that allow binding to the coreceptor (CCR5 or CXCR4) and ultimately trigger viral membrane-cell membrane fusion mediated by the gp41 transmembrane envelope glycoprotein. Here we report the derivation of an HIV-1 gp120 variant, H66N, that confers envelope glycoprotein resistance to temperature extremes. The H66N change decreases the spontaneous sampling of the CD4-bound conformation by the HIV-1 envelope glycoproteins, thus diminishing CD4-independent infection. The H66N change also stabilizes the HIV-1 envelope glycoprotein complex once the CD4-bound state is achieved, decreasing the probability of CD4-induced inactivation and revealing the enhancing effects of soluble CD4 binding on HIV-1 infection. In the CD4-bound conformation, the highly conserved histidine 66 is located between the receptor-binding and gp41-interactive surfaces of gp120. Thus, a single amino acid change in this strategically positioned gp120 inner domain residue influences the propensity of the HIV-1 envelope glycoproteins to negotiate conformational transitions to and from the CD4-bound state.Human immunodeficiency virus type 1 (HIV-1), the cause of AIDS (6, 29, 66), infects target cells by direct fusion of the viral and target cell membranes. The viral fusion complex is composed of gp120 and gp41 envelope glycoproteins, which are organized into trimeric spikes on the surface of the virus (10, 51, 89). Membrane fusion is initiated by direct binding of gp120 to the CD4 receptor on target cells (17, 41, 53). CD4 binding creates a second binding site on gp120 for the chemokine receptors CCR5 and CXCR4, which serve as coreceptors (3, 12, 19, 23, 25). Coreceptor binding is thought to lead to further conformational changes in the HIV-1 envelope glycoproteins that facilitate the fusion of viral and cell membranes. The formation of an energetically stable six-helix bundle by the gp41 ectodomain contributes to the membrane fusion event (9, 10, 79, 89, 90).The energy required for viral membrane-cell membrane fusion derives from the sequential transitions that the HIV-1 envelope glycoproteins undergo, from the high-energy unliganded state to the low-energy six-helix bundle. The graded transitions down this energetic slope are initially triggered by CD4 binding (17). The interaction of HIV-1 gp120 with CD4 is accompanied by an unusually large change in entropy, which is thought to indicate the introduction of order into the conformationally flexible unliganded gp120 glycoprotein (61). In the CD4-bound state, gp120 is capable of binding CCR5 with high affinity; moreover, CD4 binding alters the quaternary structure of the envelope glycoprotein complex, resulting in the exposure of gp41 ectodomain segments (27, 45, 77, 92). The stability of the intermediate state induced by CD4 binding depends upon several variables, including the virus (HIV-1 versus HIV-2/simian immunodeficiency virus [SIV]), the temperature, and the nature of the CD4 ligand (CD4 on a target cell membrane versus soluble forms of CD4 [sCD4]) (30, 73). For HIV-1 exposed to sCD4, if CCR5 binding occurs within a given period of time, progression along the entry pathway continues. If CCR5 binding is impeded or delayed, the CD4-bound envelope glycoprotein complex decays into inactive states (30). In extreme cases, the binding of sCD4 to the HIV-1 envelope glycoproteins induces the shedding of gp120 from the envelope glycoprotein trimer (31, 56, 58). Thus, sCD4 generally inhibits HIV-1 infection by triggering inactivation events, in addition to competing with CD4 anchored in the target cell membrane (63).HIV-1 isolates vary in sensitivity to sCD4, due in some cases to a low affinity of the envelope glycoprotein trimer for CD4 and in other cases to differences in propensity to undergo inactivating conformational transitions following CD4 binding (30). HIV-1 isolates that have been passaged extensively in T-cell lines (the tissue culture laboratory-adapted [TCLA] isolates) exhibit lower requirements for CD4 than primary HIV-1 isolates (16, 63, 82). TCLA viruses bind sCD4 efficiently and are generally sensitive to neutralization compared with primary HIV-1 isolates. Differences in sCD4 sensitivity between primary and TCLA HIV-1 strains have been mapped to the major variable loops (V1/V2 and V3) of the gp120 glycoprotein (34, 42, 62, 81). Sensitivity to sCD4 has been shown to be independent of envelope glycoprotein spike density or the intrinsic stability of the envelope glycoprotein complex (30, 35).In general, HIV-1 isolates are more sensitive to sCD4 neutralization than HIV-2 or SIV isolates (4, 14, 73). The relative resistance of SIV to sCD4 neutralization can in some cases be explained by a reduced affinity of the envelope glycoprotein trimer for sCD4 (57); however, at least some SIV isolates exhibit sCD4-induced activation of entry into CD4-negative, CCR5-expressing target cells that lasts for several hours after exposure to sCD4 (73). Thus, for some primate immunodeficiency virus envelope glycoproteins, activated intermediates in the CD4-bound conformation can be quite stable.The HIV-1 envelope glycoprotein elements important for receptor binding, subunit interaction, and membrane fusion are well conserved among different viral strains (71, 91). Thus, these elements represent potential targets for inhibitors of HIV-1 entry. Understanding the structure and longevity of the envelope glycoprotein intermediates along the virus entry pathway is relevant to attempts at inhibition. For example, peptides that target the heptad repeat 1 region of gp41 exhibit major differences in potency against HIV-1 strains related to efficiency of chemokine receptor binding (20, 21), which is thought to promote the conformational transition to the next step in the virus entry cascade. The determinants of the duration of exposure of targetable HIV-1 envelope glycoprotein elements during the entry process are undefined.To study envelope glycoprotein determinants of the movement among the distinct conformational states along the HIV-1 entry pathway, we attempted to generate HIV-1 variants that exhibit improved stability. Historically, labile viral elements have been stabilized by selecting virus to replicate under conditions, such as high temperature, that typically weaken protein-protein interactions (38, 39, 76, 102). Thus, we subjected HIV-1 to repeated incubations at temperatures between 42°C and 56°C, followed by expansion and analysis of the remaining replication-competent virus fraction. In this manner, we identified an envelope glycoprotein variant, H66N, in which histidine 66 in the gp120 N-terminal segment was altered to asparagine. The resistance of HIV-1 bearing the H66N envelope glycoproteins to changes in temperature has been reported elsewhere (37). Here, we examine the effect of the H66N change on the ability of the HIV-1 envelope glycoproteins to negotiate conformational transitions, either spontaneously or in the presence of sCD4. The H66N phenotype was studied in the context of both CD4-dependent and CD4-independent HIV-1 variants.     

Matrix and Envelope Coevolution Revealed in a Patient Monitored since Primary Infection with Human Immunodeficiency Virus Type 1

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode envelope glycoproteins (Env) with long cytoplasmic tails (CTs). The strong conservation of CT length in primary isolates of HIV-1 suggests that this factor plays a key role in viral replication and persistence in infected patients. However, we report here the emergence and dominance of a primary HIV-1 variant carrying a natural 20-amino-acid truncation of the CT in vivo. We demonstrated that this truncation was deleterious for viral replication in cell culture. We then identified a compensatory amino acid substitution in the matrix protein that reversed the negative effects of CT truncation. The loss or rescue of infectivity depended on the level of Env incorporation into virus particles. Interestingly, we found that a virus mutant with defective Env incorporation was able to spread by cell-to-cell transfer. The effects on viral infectivity of compensation between the CT and the matrix protein have been suggested by in vitro studies based on T-cell laboratory-adapted virus mutants, but we provide here the first demonstration of the natural occurrence of similar mechanisms in an infected patient. Our findings provide insight into the potential of HIV-1 to evolve in vivo and its ability to overcome major structural alterations.The envelope glycoprotein complex of the human immunodeficiency virus type 1 (HIV-1) is involved principally in virion attachment to target cell surfaces and in the entry process (15, 18, 27, 29, 52). Envelope glycoproteins (Env) are initially translated as a gp160 precursor glycoprotein, which is then processed during its trafficking through the secretory pathway, to yield a surface subunit gp120 noncovalently attached to a transmembrane subunit gp41. During HIV-1 assembly, Env proteins are incorporated at the surface of the viral particle as a trimeric structure consisting of three gp120/gp41 dimers (59, 62).The gp41 consists of an ectodomain, a hydrophobic transmembrane anchor, and a cytoplasmic tail (CT). Lentiviruses, including HIV-1 and simian immunodeficiency virus (SIV), are unusual in having a transmembrane subunit with much longer CTs (∼150 amino acids) than most other retroviruses (20 to 50 amino acids) (27). Early studies with T-cell laboratory-adapted HIV-1 mutants showed that the gp41 CT region played an important role in regulating Env functions, the incorporation of Env into virus particles and, consequently, viral replication (16, 21, 35, 63). The integrity of the gp41 CT thus appears to be crucial for replication in primary T cells, macrophages, and in many transformed T-cell lines (1, 44). Viral variants with truncated gp41 are rarely isolated from infected patients. One study reported the isolation of a CD4-independent variant harboring a sharply truncated CT (64). However, this atypical isolate existed as a minority variant in the original quasispecies of the patient (54). SIV variants with truncated CTs obtained in cell culture in vitro have also been shown to revert rapidly (to full-length CT) when introduced into macaques (39). These observations indicate that the long CTs of lentiviruses, such as HIV-1 and SIV, have functions specific to viral replication and persistence in vivo.Two groups of conserved sequence motifs have been identified in the gp41 CT that are likely to be involved in its functions. The first group, involved in regulating the intracellular trafficking of Env, includes a membrane-proximal tyrosine-based endocytic motif, Y₇₁₂SPL, (9, 47); a diaromatic motif, Y₈₀₂W₈₀₃, implicated in the retrograde transport of Env to the trans-Golgi network (8), and a C-terminal dileucine motif recently identified as a second endocytic motif (7, 10, 60). We have also provided evidence for the existence of additional as-yet-unidentified signals in studies of primary HIV-1 (34). The second group of motifs consists of three structurally conserved amphipathic α-helical domains: lentivirus lytic peptides 1, 2, and 3 (LLP-1, LLP-2, and LLP-3) (11, 17, 33). LLP domains have been implicated in various functions, including Env fusogenicity and the incorporation of Env into HIV-1 particles (28, 32, 43, 45, 50, 61).Several lines of evidence suggest that Env incorporation requires direct or indirect interactions between the matrix domain of the structural protein precursor Pr55^Gag (matrix) and the gp41 CT during HIV-1 assembly. This possibility was first suggested by the observation that HIV-1 Env drives the basolateral budding of Gag in polarized cells (37, 48). A direct interaction between the matrix and a glutathione S-transferase fusion protein containing Env CT was subsequently observed in vitro (13). Synthetic peptides corresponding to various domains of the gp41 CT have also been shown to interact directly with Pr55^Gag molecules (26). Furthermore, effects on viral infectivity of compensation between the CT and the matrix protein have been suggested by studies based on T-cell laboratory-adapted virus mutants (19, 40, 43). Finally, the cellular protein TIP47 was recently implicated in Env incorporation, based on its ability to bind both the matrix protein and the gp41 CT (38).In a previous study describing the evolutionary dynamics of the glycan shield of HIV-1 Env, we identified a patient (patient 153) for whom the 15 env clones obtained during primary infection (early stage) encoded full-length Env, whereas the 15 env sequences from the HIV-1 present 6 years later (late stage) encoded truncated gp41 CTs (14). These late-stage sequences contained a deletion introducing an in-frame stop codon, resulting in a 20-amino-acid truncation of the Env. Note that, unlike a point mutation, this deletion cannot easily revert to the full-length form. Such a deletion affecting various known motifs of the gp41 CT would be expected to impair viral replication. However, the plasma viral load measured in patient 153 demonstrated that the virus had retained its ability to replicate.In the present study, we explored the molecular mechanisms by which a primary HIV-1 maintained its capacity to replicate efficiently in this patient and demonstrated for the first time the occurrence of matrix and Env coevolution in vivo, providing insight into the ability of HIV-1 to overcome major structural alterations.     

The Heptad Repeat 2 Domain Is a Major Determinant for Enhanced Human Immunodeficiency Virus Type 1 (HIV-1) Fusion and Pathogenicity of a Highly Pathogenic HIV-1 Env

Human immunodeficiency virus type 1 (HIV-1)-mediated depletion of CD4⁺ lymphocytes in an infected individual is the hallmark of progression to AIDS. However, the mechanism for this depletion remains unclear. To identify mechanisms of HIV-1-mediated CD4 T-cell death, two similar viral isolates obtained from a rapid progressor patient with significantly different pathogenic phenotypes were studied. One isolate (R3A) demonstrates enhanced pathogenesis in both in vivo models and relevant ex vivo lymphoid organ model systems compared to another isolate, R3B. The pathogenic determinants were previously mapped to the V5-gp41 envelope region, correlating functionally with enhanced fusion activity and elevated CXCR4 binding affinity. To further elucidate specific differences between R3A and R3B within the V5-gp41 domains that enhance CD4 depletion, R3A-R3B chimeras to study the V5-gp41 region were developed. Our data demonstrate that six residues in the ectodomain of R3A provide the major determinant for both enhanced Env-cell fusion and pathogenicity. Furthermore, three amino acid differences in the heptad repeat 2 (HR-2) domain of R3A determined its fusion activity and significantly elevated its pathogenic activity. The chimeric viruses with enhanced fusion activity, but not elevated CXCR4 affinity, correlated with high pathogenicity in the thymus organ. We conclude that the functional domain of a highly pathogenic HIV-1 Env is determined by mutations in the HR-2 region that contribute to enhanced fusion and CD4 T-cell depletion.Human immunodeficiency virus type 1 (HIV-1) is the causative agent for AIDS, which is characterized by a dramatic loss of CD4⁺ lymphocytes and impairment of the immune system against invading pathogens (13, 21, 22). Though much has been determined regarding interactions between HIV-1 virus and CD4⁺ target cells, the mechanisms by which the HIV-1 virus depletes CD4⁺ lymphocytes remain incompletely understood. Various studies have demonstrated that in an HIV-infected host, both infected and uninfected cells are prone to destruction, albeit by different pathways (15, 18, 29). Recently, our group and others have shown that while binding of CD4 and chemokine receptors contribute to syncytium formation in vitro, viral membrane fusion by the envelope glycoprotein plays an important role in depletion of both uninfected and infected cells by HIV-1 and simian-human immunodeficiency virus in vivo (1, 11, 12, 26, 29).HIV-1 entry into a cell is mediated by a multistep process that begins with high-affinity binding between viral envelope (gp120) and the cellular CD4 receptor (9, 14, 16). This binding causes a conformational change in the viral envelope, allowing for subsequent coreceptor binding (mainly CCR5 or CXCR4). Upon coreceptor binding, another conformational change is thought to take place that allows gp41 to engage the cell to form a fusion complex. Envelope proteins have been demonstrated to exist as a trimer, allowing for three gp41s to form a fusion assembly through noncovalent interactions. This fusion assembly is determined to exist in a six-helix bundle formation as the fusion event takes place, allowing for the virion to fuse to the host cell (5, 24).The envelope glycoprotein (Env) of HIV plays a significant role in viral pathogenesis, as seen in several in vitro and in vivo models of infection. The Env functions to mediate virus entry of cells and is also a major target for immune responses (31, 39). While the envelope initially forms as a precursor protein (gp160), subsequent cleavage by a cellular protease yields the surface subunit gp120 and the transmembrane gp41 although the gp120 and gp41 interact noncovalently (36). The gp120 protein is comprised of five variable (V1 to V5) and five conserved constant (C1 to C5) domains and binds CD4 and the coreceptors. The gp41 protein is comprised of an amino-terminal fusion domain and two heptad repeats (HR-1 and HR-2) in the ectodomain (extracellular domain), a single transmembrane domain, and a cytoplasmic tail (intracellular domain) (8, 10, 36, 37). Due to the discovery of fusion inhibitor peptides such as C34 (23, 24) and T20 (38), much is now known about the fusion complex formed by the HIV-1 fusion domain. Similar to other viral envelopes that carry a type 1 fusion complex (such as influenza and corona viruses), the ectodomain of HIV-1 Env carries two HRs that form a coiled-coiled structure. In order for HIV-cell fusion to occur, the HR-1 domains of the trimeric Env protein must interact with the cell surface. Following this initial interaction, HR-2 domains are thought to intertwine over the HR-1 coils to form a stable six-helix bundle, which represents the gp41 core structure. X-ray crystallographic studies show that the six-helix bundle core consists of the HR-1 and HR-2 peptides bound in an antiparallel manner. This structure brings the fusion peptide to the target cell membrane, allowing for the formation of a fusion pore and the entry of virions into the cell.HIV-1 Env expressed on the surface of infected cells can induce cell-cell fusion with adjacent uninfected cells to form multinucleated syncytia and single cell lysis in cell culture and apoptosis in primary cells. Various models (both ex vivo and in vivo) have been utilized to study HIV-1-induced depletion of CD4⁺ lymphocytes. Models such as SCID-human thymus-liver (SCID-hu thy/liv), tonsil histoculture, and human fetal thymus organ culture (HFTOC) have demonstrated significant use in the study of acute infection and pathogenesis in the appropriate lymphoid organ microenvironment as they retain the organ structure and do not require exogenous stimulation for productive viral infection to occur (2, 20, 28, 32). More importantly, tissue culture-adapted HIV-1 isolates such as HXB2 fail to replicate in the SCID-hu thy/liv or HFTOC models (30, 33). Organ models such as the SCID-hu thy/liv and HFTOC thus more accurately demonstrate infection, replication, and pathogenicity of primary HIV-1 strains.Here, HFTOC is used to investigate mechanisms by which an HIV-1 virus with a highly pathogenic viral Env is able to deplete CD4⁺ lymphocytes. Two viral isolates obtained from rapid progressor patient 3 of the ALIVE cohort (40) show significant sequence homology, particularly in the Env region, while they carry stark differences in pathogenic ability (26, 27). One isolate (denoted R3A) was found to demonstrate enhanced fusion in cell-cell fusion assays as well as enhanced pathogenesis in relevant ex-vivo/in vivo organ model systems compared to another isolate, R3B. To define the pathogenic determinants that differentiate R3A from R3B, this study demonstrates that the enhanced fusogenicity of R3A (governed by the ectodomain of the gp41), but not the elevated CXCR4 binding affinity, confers the pathogenic phenotype in HFTOC. We further demonstrate that three amino acid differences in the HR-2 domain allow for this enhanced fusion for R3A Env, defining a possible mechanism for a pathogenic HIV-1 envelope.     

A V3 Loop-Dependent gp120 Element Disrupted by CD4 Binding Stabilizes the Human Immunodeficiency Virus Envelope Glycoprotein Trimer

Human immunodeficiency virus (HIV-1) entry into cells is mediated by a trimeric complex consisting of noncovalently associated gp120 (exterior) and gp41 (transmembrane) envelope glycoproteins. The binding of gp120 to receptors on the target cell alters the gp120-gp41 relationship and activates the membrane-fusing capacity of gp41. Interaction of gp120 with the primary receptor, CD4, results in the exposure of the gp120 third variable (V3) loop, which contributes to binding the CCR5 or CXCR4 chemokine receptors. We show here that insertions in the V3 stem or polar substitutions in a conserved hydrophobic patch near the V3 tip result in decreased gp120-gp41 association (in the unliganded state) and decreased chemokine receptor binding (in the CD4-bound state). Subunit association and syncytium-forming ability of the envelope glycoproteins from primary HIV-1 isolates were disrupted more by V3 changes than those of laboratory-adapted HIV-1 envelope glycoproteins. Changes in the gp120 β2, β19, β20, and β21 strands, which evidence suggests are proximal to the V3 loop in unliganded gp120, also resulted in decreased gp120-gp41 association. Thus, a gp120 element composed of the V3 loop and adjacent beta strands contributes to quaternary interactions that stabilize the unliganded trimer. CD4 binding dismantles this element, altering the gp120-gp41 relationship and rendering the hydrophobic patch in the V3 tip available for chemokine receptor binding.The entry of human immunodeficiency virus type 1 (HIV-1) is mediated by the viral envelope glycoproteins (9, 79). The HIV-1 envelope glycoproteins are synthesized as an ∼850-amino acid precursor, which trimerizes and is posttranslationally modified by carbohydrates to create a 160-kDa glycoprotein (gp160). The gp160 envelope glycoprotein precursor is proteolytically processed in the Golgi apparatus, resulting in a gp120 exterior envelope glycoprotein and a gp41 transmembrane envelope glycoprotein (16, 17, 66, 76). In the mature HIV-1 envelope glycoprotein trimer, the three gp120 subunits are noncovalently bound to three membrane-anchored gp41 subunits (32).HIV-1 entry involves the binding of gp120 in a sequential fashion to CD4 and one of the chemokine receptors, CCR5 or CXCR4 (1, 8, 15, 18, 25, 36). CD4 binding triggers the formation of an activated intermediate that is competent for binding to CCR5 or CXCR4 (29, 69, 73, 78). These chemokine receptors are G protein-coupled, 7-transmembrane segment receptors with relatively short N termini. The choice of chemokine receptors is dictated primarily by the sequence of a gp120 region, the third variable (V3) loop, that exhibits variability among HIV-1 strains and becomes exposed upon CD4 binding (4, 8, 10, 33, 37, 38, 49, 59, 75). X-ray crystal structures of CD4-bound HIV-1 gp120 have revealed that the gp120 “core” consists of a gp41-interactive inner domain, a surface-exposed and heavily glycosylated outer domain, and a conformationally flexible bridging sheet (38, 43, 79). In the CD4-bound state, the V3 loop projects 30 Å from the gp120 core, toward the chemokine receptor (38). The V3 loop in these structures consists of three elements: (i) conserved antiparallel β strands that contain a disulfide bond at the base of the loop; (ii) a conformationally flexible stem; and (iii) a conserved tip (37, 38). During the virus entry process, the base of the gp120 V3 loop and elements of the bridging sheet interact with the CCR5 N terminus, which is acidic and contains sulfotyrosine residues (12-14, 23, 24). Sulfotyrosine 14 of CCR5 is thought to insert into a highly conserved pocket near the V3 base, driving further conformational rearrangements that result in the rigidification of the V3 stem (37). The conserved β-turn at the tip of the V3 loop, along with some residues in the V3 stem, is believed to bind the “body” of CCR5, i.e., the extracellular loops and membrane-spanning helices. CCR5 binding is thought to induce further conformational changes in the HIV-1 envelope glycoproteins, leading to the fusion of the viral and target cell membranes by the gp41 transmembrane envelope glycoproteins.CCR5 binding involves two points of contact with the gp120 V3 loop: (i) the CCR5 N terminus with the V3 base and (ii) the CCR5 body with the V3 tip and distal stem (12-14, 23, 24, 37, 38). The intervening V3 stem can tolerate greater conformational and sequence variation, features that might decrease HIV-1 susceptibility to host antibodies (30). Despite amino acid variation, the length of the V3 loop is well conserved among naturally occurring group M (major group) HIV-1 strains (30, 42). This conserved length may be important for aligning the two CCR5-binding elements of the V3 loop. In addition to allowing optimal CCR5 binding, the conserved V3 length and orientation may be important for CCR5 binding to exert effects on the conformation of the HIV-1 envelope glycoproteins. We examine here the consequences of introducing extra amino acid residues into the V3 stem. The residues were introduced either into both strands of the V3 loop, attempting to preserve the symmetry of the structure, or into one of the strands, thereby kinking the loop. The effects of these changes on assembly, stability, receptor binding, and the membrane-fusing capacity of the HIV-1 envelope glycoproteins were assessed. In addition to effects on chemokine receptor binding, unexpected disruption of gp120-gp41 association was observed. Further investigation revealed a conserved patch in the tip of the V3 loop that is important for the association of gp120 with the trimeric envelope glycoprotein complex, as well as for chemokine receptor binding. Apparently, the V3 loop and adjacent gp120 structures contribute to the stability of the trimer in the unliganded HIV-1 envelope glycoproteins. These structures are known to undergo rearrangement upon CD4 binding, suggesting their involvement in receptor-induced changes in the virus entry process.     

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 α-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.The entry of human immunodeficiency virus type 1 (HIV-1) into its target cell to establish an infection requires the fusion of viral and cellular membranes, a process that is mediated by the viral envelope glycoprotein (Env) through interactions with receptors on the target cell membrane (CD4 and a coreceptor, such as CCR-5 or CXCR-4) (14). HIV-1 Env is synthesized as the glycoprotein precursor gp160, which oligomerizes in the endoplasmic reticulum and subsequently is cleaved by the cellular furin endoprotease to create a metastable state that is primed for the induction of membrane fusion activity (19). The resulting Env complex is a trimeric structure comprising three gp120 surface glycoproteins, each associated noncovalently with one of three subunits of the gp41 transmembrane glycoprotein (24, 27, 47, 48). This native (prefusion) Env spike protrudes from the virus surface and is the target for neutralizing antibodies (NAbs) (reviewed in reference 3). It is generally accepted that HIV-1 membrane fusion is promoted by a series of receptor binding-triggered conformational changes in the Env complex, culminating in the formation of an energetically stable trimer of α-helical hairpins in gp41 (10, 14).The core structure of the trimer-of-hairpins is an antiparallel six-helix bundle: a central, three-stranded coiled coil formed by the first heptad repeat (HR_N) region of gp41 is sheathed by three α-helices derived from the second HR (HR_C) (5, 27, 42, 44). HR_N is immediately C terminal to the fusion peptide, while HR_C is adjacent to the transmembrane helix anchored in the viral membrane. The interaction of gp120 with CD4 and a chemokine receptor is thought to alter intersubunit interactions in the native Env complex, leading to gp41 reorganization into a postulated prehairpin intermediate (reviewed in references 10 and 14). At this point, the N-terminal HR_N coiled-coil trimer is formed, relocating the fusion peptides to allow them to insert into the cellular membrane. The HR_C region then is thought to jackknife so as to pack against the inner coiled-coil core and form the postfusion trimer-of-hairpin structure that brings the attached target cell and viral membranes together. Evidence for the existence of these different gp41 conformational states in the fusion pathway is indirect, being inferred from the antiviral activity of peptides derived from the two HR regions of gp41 (20, 45). These peptide inhibitors likely act in a dominant-negative manner by binding to the prehairpin intermediate, preventing the formation of the trimer-of-hairpins (6, 13, 27, 31). This intermediate is relatively stable, with a half-life of many minutes, as detected by the capacity of such peptides to inhibit fusion once prefusion gp41 has undergone a conformational transition (21, 31). Although mounting evidence indicates that the prefusogenic and intermediate states are important targets for drug- and vaccine-elicited NAbs (reviewed in references 3 and 10), little is known about their structures and how they modulate gp41 fusogenicity or serve as targets for inhibition.The C-terminal part of the gp41 ectodomain consists of HR_C (or C34) and the membrane-proximal external region (MPER) (Fig. ​(Fig.1).1). The C34 peptide is intrinsically disordered in isolation and forms an outer-layer α-helix only in the six-helix bundle (27, 29). Structural studies of the trimeric coiled-coil state of the MPER and of its bent helix conformation after binding to lipid membranes have begun to provide clues regarding the function of this unusual and important NAb-associated segment (25, 41). The MPER is the established target for two very rare but broadly reactive NAbs, 2F5 and 4E10/z13, which are elicited during natural human infection (50). These neutralizing epitopes seem to be poorly exposed on the surface of both HIV-1-infected cells and virions (reviewed in reference 3). Their exposure is enhanced or triggered by receptor binding but diminishes on the formation of the trimer-of-hairpins, suggesting that both of the NAbs target a more extended intermediate conformation rather than the native gp41 structure (8, 12). Despite extensive efforts, how structural aspects of the MPER explain its antigenicity and immunogenicity remains unclear. Here, we report the identification of the C-terminal dimerization domain of gp41 and present the 1.65-Å crystal structure of this domain. We characterize the role of this antiparallel two-stranded coiled-coil structure in NAb reactivity and viral function. Our study provides a potential structure for the fusion-intermediate state of gp41 and for the future design of new HIV-1 immunogens that may elicit broad and potent NAbs.Open in a separate windowFIG. 1.Structural and functional domains of HIV-1 gp41. (Upper) Schematic view of gp41 showing the location of the fusion peptide (FP), the two HR regions, the MPER, the transmembrane segment (TM), and the cytoplasmic region (CP). HR_C and MPER are depicted in blue and green, respectively. (Lower) Sequences of the C56, C54, C54_N656L, and C39 peptides employed in the study. The Asn-656→Leu mutation in C54_N656L is shown in red. The sequences of T-20 and core epitopes recognized by the human 2F5 and 4E10 MAbs are indicated.     

Truncation of the Membrane-Spanning Domain of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Defines Elements Required for Fusion,Incorporation, and Infectivity

The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a “snorkeling” model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.Human immunodeficiency virus type 1 (HIV-1) infection is initiated by fusion of the viral membrane with that of the target cell and is mediated by the viral envelope glycoprotein (Env). HIV-1 Env, a type 1 membrane-spanning glycoprotein, is a trimeric complex composed of three noncovalently linked heterodimers of gp120, the receptor-binding surface (SU) component, and gp41, the membrane-spanning, transmembrane (TM) component (12, 26, 44, 45). The gp120 and gp41 glycoproteins are synthesized as a precursor gp160 glycoprotein, which is encoded by the env gene. The gp160 precursor is cotranslationally glycosylated and, following transport to the trans-Golgi network, is cleaved into the mature products by a member of the furin family of endoproteases (45). Mature Env proteins are transported to the plasma membrane, where they are rapidly endocytosed or incorporated into virions (5, 33, 43). Recent evidence suggests that endocytosis and intracellular trafficking of Env is required for its interaction with Gag precursors and for efficient assembly into virions (20).HIV-1 Env molecules function as quasistable “spring-loaded” fusion machines. Recent studies have suggested that several regions of gp120 are reoriented following CD4 binding so that a planar “bridging sheet,” which forms the binding site for the coreceptor (CCR5 or CXCR4), can form (6, 7). Coreceptor binding is necessary for additional conformational changes in gp41 and for complete fusion (3). The gp41 monomer has three subdomains, an ectodomain, a membrane-spanning domain (MSD), and a cytoplasmic domain (39). The ectodomain of gp41, which mediates membrane fusion, is composed of a fusion peptide, two heptad repeats, and a tryptophan-rich membrane-proximal external region. Following the binding of gp120 to the CD4 receptor and the CCR5/CXCR4 coreceptor, conformational changes are induced in Env that result in the exposure of the gp41 fusion peptide (32). This peptide inserts into the target cell membrane, allowing gp41 to form a bridge between the viral and cellular membranes. Interaction of the heptad repeats to form a six-helix bundle then brings the target and viral membranes together, allowing membrane fusion to occur (24).While heptad repeat regions 1 and 2 in the N-terminal ectodomain play key roles in Env-mediated fusion by bringing the viral and cell membranes into close proximity, an important function of gp41 is to anchor the glycoprotein complex within the host-derived viral membrane (18). The precise boundaries of the HIV-1 MSD have not been clearly defined; however, the MSD is one of the most conserved regions in the gp41 sequence. Based on the initial functional studies of HIV-1, the MSD of Env was defined as a stretch of 25 predominantly hydrophobic amino acids that span residues K681 to R705 in the NL4-3 sequence (14, 16, 18). These residues were suggested to cross the viral membrane in the form of an alpha helix, the length of which is approximately equal to the theoretical depth of a membrane bilayer. A major caveat of this model is that it places a basic amino acid residue (R694) into the hydrophobic center of the lipid bilayer. While some transmembrane proteins do contain charged amino acid residues in their MSDs, it is normally considered to be energetically unfavorable without some mechanism to neutralize the charge (8, 13). Point mutation studies have yielded varying results, but in general, substitution of K681 is detrimental to fusion and infectivity while mutation of R694 or R705 has only a limited effect on these activities (16, 29). On the other hand, accumulating data argue for a different intramembrane structure of the HIV-1 MSD. Serial small deletions (3 amino acid residues) in the region between R694 and R705 showed normal cell-cell fusion, although larger deletions were detrimental (29), suggesting that, with respect to the biological functions of the Env glycoprotein, the length of this region is more important than its amino acid conservation.Previous C-terminal-truncation studies of simian immunodeficiency virus (SIV) Env (19, 41) suggested that the entire 27-amino-acid region is not required for the biological function of the protein. In the case of SIV, only the 15 apolar amino acids flanked by K689 and R705 (equivalent to K681 and R694 in HIV) and 6 additional amino acids (for a total of 23 amino acids) were required for near-wild-type (WT) fusion (19, 41). Two subsequent residues were required (total, 25 amino acids) for virus-cell entry and infectivity, while a length of 21 amino acid residues was sufficient for SIV Env to be incorporated into viral particles. These results led to a basic amino acid “snorkeling” model for the SIV MSD (41). In this model, the lysine and arginine (NL4-3 equivalents of K681 and R694) are buried in the lipid bilayer, while their long side chains are proposed to extend outward to the membrane surface and present the positively charged amino groups to the negatively charged head groups of the lipid bilayers. Applied to HIV-1 MSD, this model predicts a hydrophobic intramembrane core of only 12 amino acid residues (compared to 15 amino acid residues in the SIV MSD) between K681 and R694. The hydrophobic region C-terminal to K681 is not sufficient to effectively anchor the protein, since mutation of R694 to a stop codon yielded a nonfunctional protein that appeared to be retained in the endoplasmic reticulum (11). This contrasts with truncation experiments with the vesicular stomatitis virus (VSV) G glycoprotein, which have shown that a region of 12 hydrophobic amino acids flanked by basic residues is sufficient to anchor the protein in the membrane (1).In order to understand if the “snorkeling” model is applicable to the HIV-1 MSD, we constructed a series of nonsense mutants with HIV-1 gp41 truncated in single-amino-acid steps at the C terminus from residue R707 to residue R694. For each mutant Env, we determined the membrane stability, fusogenicity, and ability to mediate infectivity. The results of these studies suggest that the 12-residue “core” (36) plus three subsequent hydrophobic amino acids is the minimal anchor domain for HIV-1 Env, as well as the minimal sequence to mediate cell-cell fusion. In contrast to SIV Env, HIV-1 Env requires the entire 25-amino-acid region from K681 to R707 to mediate near-WT incorporation and infectivity.     

Effect of Mutations in the Human Immunodeficiency Virus Type 1 Protease on Cleavage of the gp41 Cytoplasmic Tail

We previously reported that human immunodeficiency virus type 1 (HIV-1) develops resistance to the cholesterol-binding compound amphotericin B methyl ester (AME) by acquiring mutations (P203L and S205L) in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 that create cleavage sites for the viral protease (PR). In the present study, we observed that a PR inhibitor-resistant (PIR) HIV-1 mutant is unable to efficiently cleave the gp41 cytoplasmic tail in P203L and S205L virions, resulting in loss of AME resistance. To define the pathway to AME resistance in the context of the PIR PR, we selected for resistance with an HIV-1 isolate expressing the mutant enzyme. We identified a new gp41 mutation, R236L, that results in cleavage of the gp41 tail by the PIR PR. These results highlight the central role of gp41 cleavage as the primary mechanism of AME resistance.Cholesterol-enriched membrane microdomains, often referred to as lipid rafts (4, 18, 24), play an important role in the replication of many enveloped viruses, including human immunodeficiency virus type 1 (HIV-1) (22, 30). Lipid rafts are involved in both HIV-1 entry and egress (reviewed in references 6, 22, and 30), and the lipid bilayer of HIV-1 virions is significantly enriched in cholesterol and highly saturated lipids characteristic of lipid rafts (3, 5, 8). We recently demonstrated that the cholesterol-binding polyene fungal antibiotic amphotericin B methyl ester (AME) potently inhibits HIV-1 replication. The antiviral activity of AME is due to a profound inhibition of viral entry (27, 28) and impairment of virus particle production (29).In our previous studies, we showed that the propagation of HIV-1 in the presence of AME leads to viral escape from this compound. The mutations that confer resistance map to the cytoplasmic tail (CT) of the gp41 transmembrane envelope (Env) glycoprotein (27, 28). AME-resistant mutants (P203L and S205L) overcome the defect in viral entry imposed by AME by a novel mechanism of resistance whereby the gp41 CT is cleaved by the viral protease (PR) after incorporation of Env into virions (28). The introduction of stop codons into the gp41-coding region that prematurely truncate the CT also renders virions AME resistant. In the present study, we evaluated the interplay between protease inhibitor resistance (PIR) mutations and AME resistance.     

gp340 Promotes Transcytosis of Human Immunodeficiency Virus Type 1 in Genital Tract-Derived Cell Lines and Primary Endocervical Tissue

The human scavenger receptor gp340 has been identified as a binding protein for the human immunodeficiency virus type 1 (HIV-1) envelope that is expressed on the cell surface of female genital tract epithelial cells. This interaction allows such epithelial cells to efficiently transmit infective virus to susceptible targets and maintain viral infectivity for several days. Within the context of vaginal transmission, HIV must first traverse a normally protective mucosa containing a cell barrier to reach the underlying T cells and dendritic cells, which propagate and spread the infection. The mechanism by which HIV-1 can bypass an otherwise healthy cellular barrier remains an important area of study. Here, we demonstrate that genital tract-derived cell lines and primary human endocervical tissue can support direct transcytosis of cell-free virus from the apical to basolateral surfaces. Further, this transport of virus can be blocked through the addition of antibodies or peptides that directly block the interaction of gp340 with the HIV-1 envelope, if added prior to viral pulsing on the apical side of the cell or tissue barrier. Our data support a role for the previously described heparan sulfate moieties in mediating this transcytosis but add gp340 as an important facilitator of HIV-1 transcytosis across genital tract tissue. This study demonstrates that HIV-1 actively traverses the protective barriers of the human genital tract and presents a second mechanism whereby gp340 can promote heterosexual transmission.Through correlative studies with macaques challenged with simian immunodeficiency virus (SIV), the initial targets of infection in nontraumatic vaginal exposure to human immunodeficiency virus type 1 (HIV-1) have been identified as subepithelial T cells and dendritic cells (DCs) (18, 23, 31, 36-38). While human transmission may differ from macaque transmission, the existing models of human transmission remain controversial. For the virus to successfully reach its CD4⁺ targets, HIV must first traverse the columnar mucosal epithelial cell barrier of the endocervix or uterus or the stratified squamous barrier of the vagina or ectocervix, whose normal functions include protection of underlying tissue from pathogens. This portion of the human innate immune defense system represents a significant impediment to transmission. Studies have placed the natural transmission rate of HIV per sexual act between 0.005 and 0.3% (17, 45). Breaks in the epithelial barrier caused by secondary infection with other sexual transmitted diseases or the normal physical trauma often associated with vaginal intercourse represent one potential means for viral exposure to submucosal cells and have been shown to significantly increase transmission (reviewed in reference 11). However, studies of nontraumatic exposure to SIV in macaques demonstrate that these disruptions are not necessary for successful transmission to healthy females. This disparity indicates that multiple mechanisms by which HIV-1 can pass through mucosal epithelium might exist in vivo. Identifying these mechanisms represents an important obstacle to understanding and ultimately preventing HIV transmission.Several host cellular receptors, including DC-specific intercellular adhesion molecule-grabbing integrin, galactosyl ceramide, mannose receptor, langerin, heparan sulfate proteoglycans (HSPGs), and chondroitin sulfate proteoglycans, have been identified that facilitate disease progression through binding of HIV virions without being required for fusion and infection (2, 3, 12, 14, 16, 25, 29, 30, 43, 46, 50). These host accessory proteins act predominately through glycosylation-based interactions between HIV envelope (Env) and the host cellular receptors. These different host accessory factors can lead to increased infectivity in cis and trans or can serve to concentrate and expose virus at sites relevant to furthering its spread within the body. The direct transcytosis of cell-free virus through primary genital epithelial cells and the human endometrial carcinoma cell line HEC1A has been described (7, 9); this is, in part, mediated by HSPGs (7). Within the HSPG family, the syndecans have been previously shown to facilitate trans infection of HIV in vitro through binding of a specific region of Env that is moderately conserved (7, 8). This report also demonstrates that while HSPGs mediate a portion of the viral transcytosis that occurs in these two cell types, a significant portion of the observed transport occurs through an HSPG-independent mechanism. Other host cell factors likely provide alternatives to HSPGs for HIV-1 to use in subverting the mucosal epithelial barrier.gp340 is a member of the scavenger receptor cysteine-rich (SRCR) family of innate immune receptors. Its numerous splice variants can be found as a secreted component of human saliva (34, 41, 42) and as a membrane-associated receptor in a large number of epithelial cell lineages (22, 32, 40). Its normal cellular function includes immune surveillance of bacteria (4-6, 44), interaction with influenza A virus (19, 20, 32, 51) and surfactant proteins in the lung (20, 22, 33), and facilitating epithelial cell regeneration at sites of cellular inflammation and damage (27, 32). The secreted form of gp340, salivary agglutinin (SAG), was identified as a component of saliva that inhibits HIV-1 transmission in the oral pharynx through a specific interaction with the viral envelope protein that serves to agglutinate the virus and target it for degradation (34, 35, 41). Interestingly, SAG was demonstrated to form a direct protein-protein interaction with HIV Env (53, 54). Later, a cell surface-associated variant of SAG called gp340 was characterized as a binding partner for HIV-1 in the female genital tract that could facilitate virus transmission to susceptible targets of infection (47) and as a macrophage-expressed enhancer of infection (10).     

Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.Fc receptors (FcRs) are differentially expressed on a variety of cells of hematopoietic lineage, where they bind the constant region of antibody (Ab) and provide a link between humoral and cellular immunity. Humans possess two classes of FcRs for the constant region of IgG (FcγRs) that, when cross-linked, are distinguished by their ability to either activate or inhibit cell signaling (69, 77, 79). The activating receptors FcγRI (CD64), FcγRIIa (CD32), and FcγRIII (CD16) signal through an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb (CD32) contains an inhibitory motif (ITIM) that counters ITAM signals and B-cell receptor signals. It has been suggested that a balance between activating and inhibitory FcγRs coexpressed on the same cells plays an important role in regulating adaptive immunity (23, 68). Moreover, the inhibitory FcγRIIb, being the sole FcγR on B cells, appears to play an important role in regulating self-tolerance (23, 68). The biologic role of FcγRs may be further influenced by differences in their affinity for immunoglobulin G (IgG); thus, FcγRI is a high-affinity receptor that binds monomeric IgG (mIgG) and IgG immune complexes (IC), whereas FcγRIIa, FcγRIIb, and FcγRIIIa are medium- to low-affinity receptors that preferentially bind IgG IC (10, 49, 78). FcγRs also exhibit differences in their relative affinity for the four IgG subclasses (10), which has been suggested to influence the balance between activating and inhibitory FcγRs (67).In addition to their participation in acquired immunity, FcγRs can mediate several innate immune functions, including phagocytosis of opsonized pathogens, Ab-dependent cell cytotoxicity (ADCC), antigen uptake by professional antigen-presenting cells, and the production of inflammatory cytokines and chemokines (26, 35, 41, 48, 69). In some cases, interaction of Ab-coated viruses with FcγRs may be exploited by viruses as a means to facilitate entry into FcγR-expressing cells (2, 33, 47, 84). Several groups have reported FcγR-mediated Ab-dependent enhancement (ADE) of HIV-1 infection in vitro (47, 51, 58, 63, 94, 96), whereas other reports have implicated FcγRs in efficient inhibition of the virus in vitro (19, 21, 29, 44-46, 62, 98) and possibly as having beneficial effects against HIV-1 in vivo (5, 27, 28, 42). These conflicting results are further complicated by the fact that HIV-1-susceptible cells, such as monocytes and macrophages, can coexpress more than one FcγR (66, 77, 79).HIV-1 entry requires sequential interactions between the viral surface glycoprotein, gp120, and its cellular receptor (CD4) and coreceptor (usually CCR5 or CXCR4), followed by membrane fusion that is mediated by the viral transmembrane glycoprotein gp41 (17, 106). Abs neutralize the virus by binding either gp120 or gp41 and blocking entry into cells. Several human monoclonal Abs that neutralize a broad spectrum of HIV-1 variants have attracted considerable interest for vaccine design. Epitopes for these monoclonal Abs include the receptor binding domain of gp120 in the case of b12 (71, 86), a glycan-specific epitope on gp120 in the case of 2G12 (13, 85, 86), and two adjacent epitopes in the membrane-proximal external region (MPER) of g41 in the cases of 2F5 and 4E10 (3, 11, 38, 93). At least three of these monoclonal Abs have been shown to interact with FcRs and to mediate ADCC (42, 43).A highly standardized and validated assay for neutralizing Abs against HIV-1 that quantifies reductions in luciferase (Luc) reporter gene expression after a single round of virus infection in TZM-bl cells has been developed (60, 104). TZM-bl (also called JC53BL-13) is a CXCR4-positive HeLa cell line that was engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly Luc and Escherichia coli β-galactosidase under the control of the HIV-1 Tat-regulated promoter in the long terminal repeat terminal repeat sequence (74, 103). TZM-bl cells are permissive to infection by a wide variety of HIV-1, simian immunodeficiency virus, and human-simian immunodeficiency virus strains, including molecularly cloned Env-pseudotyped viruses. Here we report the creation and characterization of four new TZM-bl cell lines, each expressing one of the major human FcγRs. These new cell lines were used to gain a better understanding of the individual roles that FcγRs play in determining the fate of HIV-1 IC. Two FcγRs that potentiated the neutralizing activity of gp41 MPER-specific Abs were identified.     

Antagonism to and Intracellular Sequestration of Human Tetherin by the Human Immunodeficiency Virus Type 2 Envelope Glycoprotein

Tetherin (CD317/BST-2), an interferon-induced membrane protein, restricts the release of nascent retroviral particles from infected cell surfaces. While human immunodeficiency virus type 1 (HIV-1) encodes the accessory gene vpu to overcome the action of tetherin, the lineage of primate lentiviruses that gave rise to HIV-2 does not. It has been previously reported that the HIV-2 envelope glycoprotein has a Vpu-like function in promoting virus release. Here we demonstrate that the HIV-2 Rod envelope glycoprotein (HIV-2 Rod Env) is a tetherin antagonist. Expression of HIV-2 Rod Env, but not that of HIV-1 or the closely related simian immunodeficiency virus (SIV) SIVmac1A11, counteracts tetherin-mediated restriction of Vpu-defective HIV-1 in a cell-type-specific manner. This correlates with the ability of the HIV-2 Rod Env to mediate cell surface downregulation of tetherin. Antagonism requires an endocytic motif conserved across HIV/SIV lineages in the gp41 cytoplasmic tail, but specificity for tetherin is governed by extracellular determinants in the mature Env protein. Coimmunoprecipitation studies suggest an interaction between HIV-2 Rod Env and tetherin, but unlike studies with Vpu, we found no evidence of tetherin degradation. In the presence of HIV-2 Rod Env, tetherin localization is restricted to the trans-Golgi network, suggesting Env-mediated effects on tetherin trafficking sequester it from virus assembly sites on the plasma membrane. Finally, we recapitulated these observations in HIV-2-infected CD4⁺ T-cell lines, demonstrating that tetherin antagonism and sequestration occur at physiological levels of Env expression during virus replication.Various stages of the replication cycle of primate lentiviruses can be targeted by host antiviral restriction factors (reviewed in reference 49). In addition to the well-characterized antiviral effects of members of the APOBEC3 family of cytidine deaminases, particularly APOBEC3G and -3F, and species-specific variants of tripartite motif family 5α, the release of nascent retroviral particles has recently been shown to be a target for a novel restriction factor, tetherin (CD317/bone marrow stromal cell antigen 2 [BST-2]) (31, 46). Tetherin is an interferon-inducible gene that was originally shown to impart a restriction on the release of mutants of human immunodeficiency virus type 1 (HIV-1) that lack a vpu gene (31, 46). In tetherin-positive cells, mature Vpu-defective HIV-1 particles are retained on the cell surface, linked to the plasma membrane (PM) and each other via protease-sensitive tethers, and can be subsequently endocytosed and accumulate in late endosomes (30, 31). Tetherin is not HIV specific and restricts the release of virus-like particles derived from all retroviruses tested (18), as well as those of filoviruses and arenaviruses (18, 19, 39).Tetherin is a small (181-amino-acid) type II membrane protein with an unusual topology that exists mainly as a disulfide-linked dimer (34). It consists of an N-terminal cytoplasmic tail, a transmembrane anchor, an extracellular domain that includes three cysteine residues important for dimerization, a putative coiled-coil, and finally a glycophosphatidyinosityl-linked lipid anchor (22) that is essential for restriction (31). Tetherin localizes to retroviral assembly sites on the PM (18, 31), and this unusual structure is highly suggestive that tetherin restricts virion release by incorporation into the viral membrane and cross-linking virions to cells. Such a mechanism would make tetherin a powerful antiviral effector that can target an obligate part of most, if not all, enveloped virus assembly strategies. Moreover, since tetherin restriction has no specific requirement for virus protein sequences, to avoid its action, mammalian viruses have evolved to encode several distinct countermeasures that specifically inhibit tetherin''s antiviral function.The Vpu accessory protein antagonizes tetherin-mediated restriction of HIV-1 (31, 46). In the presence of Vpu, tetherin is downregulated from the cell surface (2, 46) and is targeted for degradation (10, 13, 14), although whether these processes are required for antagonism of tetherin function is unclear (27). HIV-1 Vpu displays a distinct species specificity in that it is unable to target tetherin orthologues from rhesus macaques or African green monkeys (14, 25). This differential sensitivity maps to the tetherin transmembrane domain, particularly residues that are predicted to have been under high positive selection pressure during primate evolution (14, 16, 25). This suggests that tetherin evolution may have been driven in part by viral countermeasures like Vpu. Vpu, however, is only encoded by HIV-1 and its direct simian immunodeficiency virus (SIV) lineage precursors. The majority of SIVs, including the SIVsm, the progenitor of both HIV-2 and SIVmac, do not encode a Vpu protein (21). In some of these SIVs, tetherin antagonism was recently shown to map to the nef gene (16, 51). SIV Nef proteins, however, are generally ineffective against human tetherin because they target a (G/D)DIWK motif that was deleted from the human tetherin cytoplasmic tail sometime after the divergence of humans and chimpanzees (51). This raises the question of how HIV-2 is able to overcome human tetherin, as recent data show chronically HIV-2-infected CEM T cells have reduced tetherin levels on their surface (10).Interestingly, it has long been known that the envelope glycoprotein of certain HIV-2 isolates can stimulate the release of Vpu-defective HIV-1 virions from cells we now know to be tetherin positive (5, 6, 43). HIV and SIV Envs form trimeric spikes of dimers of the surface subunit (SU-gp105 in HIV-2/SIVmac and gp120 in HIV-1) that bind CD4 and the chemokine coreceptor and gp41 (the transmembrane [TM] subunit that facilitates fusion with and entry into the target cell). Envelope precursors (gp140 or gp160) are synthesized in the endoplasmic reticulum, where they become glycosylated and are exported to the surface via the secretory pathway (8). During transit through the Golgi apparatus and possibly in endosomal compartments, the immature precursors are cleaved by furin-like proteases to form mature spikes (15, 29). Multiple endocytosis motifs in the gp41 cytoplasmic tail lead to only minor quantities of Env being exposed at the cell surface at any given time (7, 40). Recent data demonstrated that the conserved GYxxθ motif, a binding site for the clathrin adaptor protein AP-2 (3), in the membrane-proximal region of HIV-2 gp41 is required to promote Vpu-defective HIV-1 release from HeLa cells (1, 32). Based on experiments with HIV-1/HIV-2 chimeric envelopes, an additional requirement in the extracellular component was suggested (1). In this study we set out to examine the Vpu-like activity of HIV-2 envelope in light of the discovery of tetherin. We demonstrate that the HIV-2 Env is a tetherin antagonist, and we provide mechanistic insight into the basis of this antagonism.     

Crystallographic Definition of the Epitope Promiscuity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5: Vaccine Design Implications

The quest to create a human immunodeficiency virus type 1 (HIV-1) vaccine capable of eliciting broadly neutralizing antibodies against Env has been challenging. Among other problems, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, Z13, and 4E10. In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnMAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab′. A variety of amino acid substitutions outside the ⁶⁶⁴DKW⁶⁶⁶ core epitope are tolerated. However, changes at the ⁶⁶⁴DKW⁶⁶⁶ motif itself are restricted to those residues that preserve the aspartate''s negative charge, the hydrophobic alkyl-π stacking arrangement between the β-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnMAb 2F5, which could guide future attempts at designing small-molecule MPER-like vaccines capable of eliciting 2F5-like antibodies.Eliciting broadly neutralizing antibodies (bnAbs) against primary isolates of human immunodeficiency virus type I (HIV-1) has been identified as a major milestone to attain in the quest for a vaccine in the fight against AIDS (12, 28). These antibodies would need to interact with HIV-1 envelope glycoproteins gp41 and/or gp120 (Env), target conserved regions and functional conformations of gp41/gp120 trimeric complexes, and prevent new HIV-1 fusion events with target cells (21, 57, 70, 71). Although a humoral response generating neutralizing antibodies against HIV-1 can be detected in HIV-1-positive individuals, the titers are often very low, and virus control is seldom achieved by these neutralizing antibodies (22, 51, 52, 66, 67). The difficulty in eliciting a broad and potent neutralizing antibody response against HIV-1 is thought to reside in the high degree of genetic diversity of the virus, in the heterogeneity of Env on the surface of HIV-1, and in the masking of functional regions by conformational covering, by an extensive glycan shield, or by the ability of some conserved domains to partition to the viral membrane (24, 25, 29, 30, 38, 39, 56, 68, 69). So far, vaccine trials using as immunogens mimics of Env in different conformations have primarily elicited antibodies with only limited neutralization potency across different HIV-1 clades although recent work has demonstrated more encouraging results (4, 12, 61).The use of conserved regions on gp41 and gp120 Env as targets for vaccine design has been mostly characterized by the very few anti-HIV-1 broadly neutralizing monoclonal antibodies (bnMAbs) that recognize them: the CD4 binding-site on gp120 (bnMAb b12), a CD4-induced gp120 coreceptor binding site (bnMAbs 17b and X5), a mannose cluster on the outer face of gp120 (bnMAb 2G12), and the membrane proximal external region (MPER) of gp41 (bnMAbs 2F5, Z13 and 4E10) (13, 29, 44, 58, 73). The gp41 MPER region is a particularly conserved part of Env that spans residues 659 to 683 (HXB2 numbering) (37, 75). Substitution and deletion studies have linked this unusually tryptophan-rich region to the fusion process of HIV-1, possibly involving a series of conformational changes (5, 37, 41, 49, 54, 74). Additionally, the gp41 MPER has been implicated in gp41 oligomerization, membrane leakage ability facilitating pore formation, and binding to the galactosyl ceramide receptor on epithelial cells for initial mucosal infection mediated by transcytosis (2, 3, 40, 53, 63, 64, 72). This wide array of roles for the gp41 MPER will put considerable pressure on sequence conservation, and any change will certainly lead to a high cost in viral fitness.Monoclonal antibody 2F5 is a broadly neutralizing monoclonal anti-HIV-1 antibody isolated from a panel of sera from naturally infected asymptomatic individuals. It reacts with a core gp41 MPER epitope spanning residues 662 to 668 with the linear sequence ELDKWAS (6, 11, 42, 62, 75). 2F5 immunoglobulin G binding studies and screening of phage display libraries demonstrated that the DKW core is essential for 2F5 recognition and binding (15, 36, 50). Crystal structures of 2F5 with peptides representing its core gp41 epitope reveal a β-turn conformation involving the central DKW residues, flanked by an extended conformation and a canonical α-helical turn for residues located at the N terminus and C terminus of the core, respectively (9, 27, 45, 47). In addition to binding to its primary epitope, evidence is accumulating that 2F5 also undergoes secondary interactions: multiple reports have demonstrated affinity of 2F5 for membrane components, possibly through its partly hydrophobic flexible elongated complementarity-determining region (CDR) H3 loop, and it has also been suggested that 2F5 might interact in a secondary manner with other regions of gp41 (1, 10, 23, 32, 33, 55). Altogether, even though the characteristics of 2F5 interaction with its linear MPER consensus epitope have been described extensively, a number of questions persist about the exact mechanism of 2F5 neutralization at a molecular level.One such ambiguous area of the neutralization mechanism of 2F5 is investigated in this study. Indeed, compared to bnMAb 4E10, 2F5 is the more potent neutralizing antibody although its breadth across different HIV-1 isolates is more limited (6, 35). In an attempt to shed light on the exact molecular requirements for 2F5 recognition of its primary gp41 MPER epitope, we performed structural studies of 2F5 Fab′ with a variety of peptides. The remarkable breadth of possible 2F5 interactions reveals a somewhat surprising promiscuity of the 2F5 binding site. Furthermore, we link our structural observations with the natural variation observed within the gp41 MPER and discuss possible routes of 2F5 escape from a molecular standpoint. Finally, our discovery of 2F5''s ability to tolerate a rather broad spectrum of amino acids in its binding, a spectrum that even includes nonnatural amino acids, opens the door to new ways to design small-molecule immunogens potentially capable of eliciting 2F5-like neutralizing antibodies.     

Potent and Broadly Reactive HIV-2 Neutralizing Antibodies Elicited by a Vaccinia Virus Vector Prime-C2V3C3 Polypeptide Boost Immunization Strategy

Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism.Human immunodeficiency virus type 2 (HIV-2) infection affects 1 to 2 million individuals, most of whom live in India, West Africa, and Europe (17). HIV-2 has diversified into eight genetic groups named A to H, of which group A is by far the most prevalent worldwide. Nucleotide sequences of Env can differ up to 21% within a particular group and by over 35% between groups.The mortality rate in HIV-2-infected patients is at least twice that of uninfected individuals (26). Nonetheless, the majority of HIV-2-infected individuals survive as elite controllers (17). In the absence of antiretroviral therapy, the numbers of infected cells (39) and viral loads (36) are much lower among HIV-2-infected individuals than among those who are HIV-1 infected. This may be related to a more effective immune response produced against HIV-2. In fact, most HIV-2-infected individuals have proliferative T-cell responses and strong cytotoxic responses to Env and Gag proteins (17, 31). Moreover, autologous and heterologous neutralizing antibodies (NAbs) are raised in most HIV-2-infected individuals (8, 32, 48, 52), and the virus seems unable to escape from these antibodies (52). As for HIV-1, the antibody specificities that mediate HIV-2 neutralization and control are still elusive. The V3 region in the envelope gp125 has been identified as a neutralizing target by some but not by all investigators (3, 6, 7, 11, 40, 47, 54). Other weakly neutralizing epitopes were identified in the V1, V2, V4, and C5 regions in gp125 and in the COOH-terminal region of the gp41 ectodomain (6, 7, 41). A better understanding of the neutralizing determinants in the HIV-2 Env will provide crucial information regarding the most relevant targets for vaccine design.The development of immunogens that elicit the production of broadly reactive NAbs is considered the number one priority for the HIV-1 vaccine field (4, 42). Most current HIV-1 vaccine candidates intended to elicit such broadly reactive NAbs are based on purified envelope constructs that mimic the structure of the most conserved neutralizing epitopes in the native trimeric Env complex and/or on the expression of wild-type or modified envelope glycoproteins by different types of expression vectors (4, 5, 29, 49, 58). With respect to HIV-2, purified gp125 glycoprotein or synthetic peptides representing selected V3 regions from HIV-2 strain SBL6669 induced autologous and heterologous NAbs in mice or guinea pigs (6, 7, 22). However, immunization of cynomolgus monkeys with a subunit vaccine consisting of gp130 (HIV-2BEN) micelles offered little protection against autologous or heterologous challenge (34). Immunization of rhesus (19, 44, 45) and cynomolgus (1) monkeys with canarypox or attenuated vaccinia virus expressing several HIV-2 SBL6669 proteins, including the envelope glycoproteins, in combination with booster immunizations with gp160, gp125, or V3 synthetic peptides, elicited a weak neutralizing response and partial protection against autologous HIV-2 challenge. Likewise, vaccination of rhesus monkeys with immunogens derived from the historic HIV-2ROD strain failed to generate neutralizing antibodies and to protect against heterologous challenge (55). Finally, baboons inoculated with a DNA vaccine expressing the tat, nef, gag, and env genes of the HIV-2UC2 group B isolate were partially protected against autologous challenge without the production of neutralizing antibodies (33). These studies illustrate the urgent need for new vaccine immunogens and/or vaccination strategies that elicit the production of broadly reactive NAbs against HIV-2. The present study was designed to investigate in the mouse model the immunogenicity and neutralizing response elicited by novel recombinant envelope proteins derived from the reference primary HIV-2ALI isolate, when administered alone or in different prime-boost combinations.     

Virological Synapse-Mediated Spread of Human Immunodeficiency Virus Type 1 between T Cells Is Sensitive to Entry Inhibition

Human immunodeficiency virus type 1 (HIV-1) can disseminate between CD4⁺ T cells via diffusion-limited cell-free viral spread or by directed cell-cell transfer using virally induced structures termed virological synapses. Although T-cell virological synapses have been well characterized, it is unclear whether this mode of viral spread is susceptible to inhibition by neutralizing antibodies and entry inhibitors. We show here that both cell-cell and cell-free viral spread are equivalently sensitive to entry inhibition. Fluorescence imaging analysis measuring virological synapse lifetimes and inhibitor time-of-addition studies implied that inhibitors can access preformed virological synapses and interfere with HIV-1 cell-cell infection. This concept was supported by electron tomography that revealed the T-cell virological synapse to be a relatively permeable structure. Virological synapse-mediated HIV-1 spread is thus efficient but is not an immune or entry inhibitor evasion mechanism, a result that is encouraging for vaccine and drug design.As with enveloped viruses from several viral families, the human immunodeficiency virus type 1 (HIV-1) can disseminate both by fluid-phase diffusion of viral particles and by directed cell-cell transfer (39). The primary target cell for HIV-1 replication in vivo is the CD4⁺ T-cell (13), which is infectible by CCR5-tropic (R5) and CXCR4-tropic (X4) viral variants (29). R5 HIV-1 is the major transmitted viral phenotype and dominates the global pandemic, whereas X4 virus is found later in infection in ca. 50% of infected individuals, and its presence indicates a poor disease progression prognosis (23). Cell-cell HIV-1 transfer between T cells is more efficient than diffusion-limited spread (8, 16, 32, 38), although recent estimates for the differential range from approximately 1 (42) to 4 (6) orders of magnitude. Two structures have been proposed to support contact-mediated intercellular movement of HIV-1 between T cells: membrane nanotubes (33, 43) and macromolecular adhesive contacts termed virological synapses (VS) (15, 17, 33). VS appear to be the dominant structure involved in T-cell-T-cell spread (33), and both X4 (17) and R5 HIV-1 (6, 15, 42) can spread between T cells via this mechanism.VS assembly and function are dependent on HIV-1 envelope glycoprotein (Env) engaging its primary cellular receptor CD4 (2, 6, 17). This interaction recruits more CD4 and coreceptor to the site of cell-cell contact in an actin-dependent manner (17). Adhesion molecules cluster at the intercellular junction and are thought to stabilize the VS (18). In parallel, viral Env and Gag are recruited to the interface by a microtubule-dependent mechanism (19), where polarized viral budding may release virions into the synaptic space across which the target cell is infected (17). The precise mechanism by which HIV-1 subsequently enters the target T-cell cytoplasm remains unclear: by fusion directly at the plasma membrane, fusion from within an endosomal compartment, or both (4, 6, 15, 25, 34).Viruses from diverse families including herpesviruses (9), poxviruses (22) and hepatitis C virus (44) evade neutralizing antibody attack by direct cell-cell spread, since the tight junctions across which the these viruses move are antibody impermeable. It has been speculated that transfer of HIV-1 across VS may promote evasion from immune or therapeutic intervention with the inference that the junctions formed in retroviral VS may be nonpermissive to antibody entry (39). However, available evidence regarding whether neutralizing antibodies (NAb) and other entry inhibitors can inhibit HIV-1 cell-cell spread is inconsistent (25). An early analysis suggested that HIV-1 T-cell-T-cell spread is relatively resistant to neutralizing monoclonal antibodies (NMAb) (12). A later study agreed with this conclusion by demonstrating a lack of permissivity of HIV-1 T-cell-T-cell spread, measured by transfer of viral Gag, to interference with viral fusion using a gp41-specific NMAb and a peptidic fusion inhibitor (6). In contrast, another analysis reported that anti-gp41-specific NMAb interfered effectively with HIV-1 spread between T cells (26). Inhibitors of the HIV-1 surface glycoprotein (gp120)-CD4 or gp120-CXCR4 interaction reduced X4 HIV-1 VS assembly and viral transfer if applied prior to mixing of infected and receptor-expressing target cells (17, 19), but the effect of these inhibitors has not been tested on preformed VS. Thus, the field is currently unclear on whether direct T-cell-T-cell infectious HIV-1 spread is susceptible or not to antibody and entry inhibitor-mediated disruption of VS assembly, and the related question, whether the VS is permeable to viral entry inhibitors, including NAb. Addressing these questions is of central importance to understanding HIV-1 pathogenesis and informing future drug and vaccine design.Since estimates reported in the literature of the relative efficiency of direct HIV-1 T-cell-T-cell spread compared to cell-free spread vary by approximately 3 orders of magnitude (6, 38, 42), and the evidence for the activity of viral entry inhibitors on cell-cell spread is conflicting, we set out to quantify the efficiency of infection across the T-cell VS and analyze the susceptibility of this structure to NAb and viral entry inhibitors. Assays reporting on events proximal to productive infection show that the R5 HIV-1 T-cell VS is approximately 1 order of magnitude more efficient than cell-free virus infection, and imaging analyses reveal that the VS assembled by HIV-1 is most likely permeable to inhibitors both during, and subsequent to, VS assembly. Thus, we conclude that the T-cell VS does not provide a privileged environment allowing HIV-1 escape from entry inhibition.     

Combinations of the First and Next Generations of Human Immunodeficiency Virus (HIV) Fusion Inhibitors Exhibit a Highly Potent Synergistic Effect against Enfuvirtide- Sensitive and -Resistant HIV Type 1 Strains

T20 (generic name, enfuvirtide; brand name, Fuzeon) is a first-generation human immunodeficiency virus (HIV) fusion inhibitor approved for salvage therapy of HIV-infected patients refractory to current antiretroviral drugs. However, its clinical use is limited because of rapid emergence of T20-resistant viruses in T20-treated patients. Therefore, T1249 and T1144 are being developed as the second- and third-generation HIV fusion inhibitors, respectively, with improved efficacy and drug resistance profiles. Here, we found that combinations of T20 with T1249 and/or T1144 resulted in exceptionally potent synergism (combination index, <0.01) against HIV-1-mediated membrane fusion by 2 to 3 orders of magnitude in dose reduction. Highly potent synergistic antiviral efficacy was also achieved against infection by laboratory-adapted and primary HIV-1 strains, including T20-resistant variants. The mechanism underlying the synergistic effect could be attributed to the fact that T20, T1249, and T1144 all contain different functional domains and have different primary binding sites in gp41. As such, they may work cooperatively to inhibit gp41 six-helix bundle core formation, thereby suppressing virus-cell fusion. Therefore, these findings strongly imply that, rather than replacing T20, combining it with HIV fusion inhibitors of different generations might produce synergistic activity against both T20-sensitive and -resistant HIV-1 strains, suggesting a new therapeutic strategy for the treatment of HIV-1 infection/AIDS.In the early 1990s, a number of highly potent anti-human immunodeficiency virus type 1 (HIV-1) peptides derived from the C-heptad repeat (CHR) domain of the HIV-1 envelope glycoprotein (Env) transmembrane subunit gp41 were discovered (21, 22, 35, 59, 61). Biophysical and biochemical analyses suggest that the CHR peptides inhibit HIV-1 Env-mediated membrane fusion by interacting with the viral gp41 N-heptad repeat (NHR) domain to form heterologous trimer-of-heterodimer complexes, thus blocking gp41 six-helix bundle (6-HB) core formation, a critical step in virus-cell fusion (4, 5, 31, 52, 57).T20 (generic name, enfuvirtide; brand name, Fuzeon), a 36-mer CHR peptide (amino acids [aa] 638 to 673) containing a heptad repeat (HR) sequence-binding domain (HBD) and a tryptophan-rich domain (TRD) (Fig. ​(Fig.1)1) (30, 61), was licensed by the U.S. FDA as a first-generation HIV fusion inhibitor. T20 is very effective in inhibiting infection by HIV-1, especially the strains resistant to current antiretroviral therapies (24). However, many patients are now failing to respond to T20 because the viruses have developed T20 resistance (34, 51, 56, 62).Open in a separate windowFIG. 1.Functional domains of HIV fusion inhibitors and the interaction model. (A) Schematic view of the HIV-1_HXB2 gp41 molecule and sequences of the first-, second-, and third-generation HIV fusion inhibitors. FP, fusion peptide; TM, transmembrane domain; CP, cytoplasmic domain. (B) Interaction between the NHR and CHR peptides. The dashed lines between the NHR and CHR domains indicate the interaction between the residues located at the e and g and a and d positions in the NHR and CHR, respectively. The PBD, HBD, and TRD in the CHR peptides are shown in blue, light blue, and orange, respectively. The HR sequence, the region of aa 36 to 45 (determinant for T20 resistance and the primary binding site for T20), and the pocket-forming sequence in the NHR are shown in red, purple, and green, respectively. The interaction between the PBD and pocket-forming sequence is critical for stabilization of the 6-HB (3).T1249, a second-generation HIV fusion inhibitor, is a 39-mer peptide consisting of a pocket-binding domain (PBD), an HBD, and a TRD (Fig. ​(Fig.1).1). T1249 was shown to have a longer half-life than T20 in primates (7) and greater anti-HIV-1 potency than T20 in clinical studies and to be active against some T20-resistant HIV-1 variants (7, 14, 27, 38). However, the clinical development of T1249 was discontinued due to formulation difficulties (37).T1144, a third-generation HIV fusion inhibitor, is a 38-mer peptide containing a PBD and an HBD (Fig. ​(Fig.1).1). T1144 was designed by modifying the amino acid sequence of T651 (peptide C38; aa 626 to 673) to increase α-helicity and 6-HB stability and to improve pharmacokinetic properties (10). T1144 and its analog peptides are effective against viruses that are resistant to T20 (11).Sifuvirtide, a new generation of HIV fusion inhibitor, is a 34-mer peptide analogue of C34 containing a PBD and an HBD. Our previous studies have shown that sifuvirtide is more effective than T20 against both primary and laboratory-adapted HIV-1 strains. Pharmacokinetic studies of sifuvirtide demonstrated longer decay half-lives than T20 (19). Sifuvirtide is under phase II clinical trial (www.fusogen.com). Most recently, we found that the combination of sifuvirtide with T20 resulted in potent synergistic effect against T20-sensitive and -resistant HIV-1 strains (43). These findings encouraged us to test whether combining T20 with T1249 and/or T1144 would also have synergistic anti-HIV-1 activity since next-generation HIV fusion inhibitors, like C34 and sifuvirtide, also contain a PBD that can interact with pocket-forming sequence in the gp41 NHR. In this study, we were also motivated to address the mechanism(s) underlying a synergic effect. Once this effect is confirmed, a novel combination therapy could be designed for the treatment of HIV/AIDS patients who have failed to respond to T20 or other antiretroviral drugs.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Mutations in gp120 Contribute to the Resistance of Human Immunodeficiency Virus Type 1 to Membrane-Anchored C-Peptide maC46

Binding of the human immunodeficiency virus (HIV) envelope glycoprotein (Env) to the cellular CD4 receptor and a chemokine coreceptor initiates a series of conformational changes in the Env subunits gp120 and gp41. Eventually, the trimeric gp41 folds into a six-helix bundle, thereby inducing fusion of the viral and cellular membranes. C peptides derived from the C-terminal heptad repeat (CHR) of gp41 are efficient entry inhibitors as they block the six-helix bundle formation. Previously, we developed a membrane-anchored C peptide (maC46) expressed from a retroviral vector that also shows high activity against virus strains resistant to enfuvirtide (T-20), an antiviral C peptide approved for clinical use. Here, we present a systematic analysis of mutations in Env that confer resistance of HIV type 1 (HIV-1) to maC46. We selected an HIV-1 BaL strain with 10-fold reduced sensitivity to maC46 (BaL_C46) by passaging virus for nearly 200 days in the presence of gradually increasing concentrations of maC46. In comparison to wild-type BaL, BaL_C46 had five mutations at highly conserved positions in Env, three in gp120, one in the N-terminal heptad-repeat (NHR), and one in the CHR of gp41. No mutations were found in the NHR domain around the GIV motif that are known to cause resistance to enfuvirtide. Instead, maC46 resistance was found to depend on complementary mutations in the NHR and CHR that considerably favor binding of the mutated NHR to the mutated CHR over binding to maC46. In addition, resistance was highly dependent on mutations in gp120 that accelerated entry. Taken together, resistance to maC46 did not develop readily and required multiple cooperating mutations at conserved positions of the viral envelope glycoproteins gp120 and gp41.The entry process of the human immunodeficiency virus type 1 (HIV-1) has become a major target for new antiviral drugs. Viral entry is initiated by binding of the HIV-1 envelope glycoprotein subunit gp120 to the CD4 receptor and a chemokine coreceptor, generally CCR5 or CXCR4. Upon coreceptor binding, the viral transmembrane subunit gp41 undergoes conformational changes that eventually lead to the formation of the six-helix bundle (6HB) and membrane fusion. The 6HB is composed of a central trimeric coiled-coil structure formed by the N-terminal heptad repeat (NHR) domains of three gp41 molecules and the corresponding C-terminal heptad repeats (CHRs) that pack into the longitudinal grooves on the surface of the NHR coiled-coil in an antiparallel orientation (23). C-peptide fusion inhibitors (CFI) derived from the CHR of gp41 compete with the viral CHR for binding to the NHR trimer, thus blocking 6HB formation and viral entry (18).T-20 (enfuvirtide) is the first clinically approved CFI with high antiviral activity and a low-toxicity profile. However, as with many anti-HIV-1 drugs, resistance can emerge rapidly (13). The majority of the resistance mutations are found in the NHR of gp41 among the amino acids 544 to 553 (32, 35) (numbering refers to gp160 of the HIV-1 HXB2 strain throughout the article). Most of these mutations cause resistance by reducing the affinity of the NHR target region to inhibitory C peptides (13). Additionally, viral entry kinetics were found to correlate with the baseline susceptibility of different HIV strains to CFI. Determinants for viral entry kinetics are found in gp41 as well as in gp120 (1, 14, 35). Here, the influence of coreceptor affinity on virus entry kinetics and CFI susceptibility has been studied extensively (28, 30, 31). Recently, a statistical approach was used that highlighted positions in gp120 that underwent mutations in patients under enfuvirtide treatment (38). However, to our knowledge, selected CFI resistance mutations outside of gp41 have never been confirmed experimentally.Previously, we developed a retroviral vector expressing a membrane-anchored antiviral C peptide (maC46) that efficiently inhibits a broad range of different HIV-1 isolates. Enfuvirtide-resistant HIV-1 strains with mutations in the GIV motif of NHR were fully susceptible to maC46 (10). In the present study, we selected an HIV-1 variant with reduced sensitivity to maC46 by passaging an enfuvirtide-resistant BaL strain of HIV-1 on cells expressing increasing concentrations of maC46. Mutations in gp120 and gp41 were found to contribute to maC46 resistance.     

Analysis of Human Immunodeficiency Virus Type 1 Matrix Binding to Membranes and Nucleic Acids

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein targets HIV-1 precursor Gag (PrGag) proteins to assembly sites at plasma membrane (PM) sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P₂ through headgroup and 2′ acyl chain contacts. MA also binds nucleic acids, although the significance of this association with regard to the viral life cycle is unclear. We have devised a novel MA binding assay and used it to examine MA interactions with membranes and nucleic acids. Our results indicate that cholesterol increases the selectivity of MA for PI(4,5)P₂-containing membranes, that PI(4,5)P₂ binding tolerates 2′ acyl chain variation, and that the MA myristate enhances membrane binding efficiency but not selectivity. We also observed that soluble PI(4,5)P₂ analogues do not compete effectively with PI(4,5)P₂-containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P₂-containing liposomes successfully outcompete nucleic acids for MA binding, whereas other liposomes do not. These results support a model in which RNA binding protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites.The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein serves several functions in the viral replication cycle. One essential function is to target PrGag proteins to their assembly sites at the plasma membranes (PMs) of infected cells (4, 5, 11, 16, 25, 29, 30, 33, 35, 39, 43-45, 47, 50, 54, 56, 57). A second function is the recruitment of the viral surface/transmembrane (SU/TM; also referred to as gp120/gp41) envelope (Env) protein complex into virions (14, 15, 18, 19, 27, 51-53). In addition to these activities, numerous reports have attributed nucleic acid binding properties to retroviral MAs (24, 38, 47), and with some viruses MA appears to serve in an encapsidation capacity (24). While no encapsidation role has been assigned for HIV-1 MA, experiments have shown that MA can substitute for the HIV-1 nucleocapsid (NC) protein assembly function (38) under some circumstances, presumably by virtue of its facility to concentrate PrGag proteins by binding them to RNAs (38).A number of structural studies have been conducted on HIV-1 MA (1, 22, 41, 42, 49). The protein is N terminally myristoylated and composed of six α helices, capped by a three-strand β sheet (7, 22, 41, 42, 49). The protein trimerizes in solution and in crystals (22, 28, 49) and recently has been shown to organize as hexamers of trimers on lipid membranes (1). The membrane binding face of HIV-1 MA is basic, fostering its ability to associate with negatively charged phospholipid headgroups (1, 22, 30, 41, 42, 49). The importance of such an interaction has been underscored in molecular genetic experiments which demonstrated that depletion of PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] reduced the assembly efficiency of HIV-1 (9, 36). Consistent with these observations, HIV-1 MA preferentially binds to soluble PI(4,5)P₂ mimics through contacts with the headgroup and 2′ acyl chain, and binding promotes exposure of the MA myristate group and protein oligomerization (17, 21, 40-43, 46). However, PI(4,5)P₂ is not the only lipid to demonstrate an association with HIV-1. In particular, HIV-1 appears to assemble at cholesterol-rich PM sites, cholesterol is highly enriched in HIV-1 virions, and cholesterol depletion reduces viral infectivity (2, 6, 8, 20, 23, 26, 31, 34, 37). The HIV-1 lipidome shows additional differences from the PM lipids of infected cells (2, 5, 8), suggesting that other lipids could affect PrGag-membrane binding or virus assembly site selection.To gain a better understanding of the functions and interactions of HIV-1 MA, we have examined the liposome and nucleic acid binding properties of purified myristoylated MA. Using liposome flotation assays and a novel liposome bead binding assay, we have demonstrated that the PI(4,5)P₂ binding specificity of MA is enhanced by cholesterol, that protein myristoylation increases membrane binding efficiency but not specificity, and that 2′ acyl chain variation is compatible with PI(4,5)P₂ binding. We also examined whether soluble PI(4,5)P₂ mimics could compete with liposomes for MA binding. Surprisingly, we found that soluble mimics not only failed to compete with PI(4,5)P₂ liposomes but also increased MA binding to membranes that do not contain acidic phospholipids. Finally, we have observed that while MA does bind nucleic acids, nucleic acid binding is outcompeted by PI(4,5)P₂-containing liposomes. Our results suggest models for PrGag-membrane and RNA association and the HIV-1 assembly pathway.     

A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies

The membrane-proximal external region (MPER) of the human immunodeficiency virus (HIV) envelope glycoprotein (gp41) is critical for viral fusion and infectivity and is the target of three of the five known broadly neutralizing HIV type 1 (HIV-1) antibodies, 2F5, Z13, and 4E10. Here, we report the crystal structure of the Fab fragment of Z13e1, an affinity-enhanced variant of monoclonal antibody Z13, in complex with a 12-residue peptide corresponding to the core epitope (W⁶⁷⁰NWFDITN⁶⁷⁷) at 1.8-Å resolution. The bound peptide adopts an S-shaped conformation composed of two tandem, perpendicular helical turns. This conformation differs strikingly from the α-helical structure adopted by an overlapping MPER peptide bound to 4E10. Z13e1 binds to an elbow in the MPER at the membrane interface, making relatively few interactions with conserved aromatics (Trp672 and Phe673) that are critical for 4E10 recognition. The comparison of the Z13e1 and 4E10 epitope structures reveals a conformational switch such that neutralization can occur by the recognition of the different conformations and faces of the largely amphipathic MPER. The Z13e1 structure provides significant new insights into the dynamic nature of the MPER, which likely is critical for membrane fusion, and it has significant implications for mechanisms of HIV-1 neutralization by MPER antibodies and for the design of HIV-1 immunogens.The continued spread of human immunodeficiency virus (HIV) worldwide and, in particular, in sub-Saharan Africa, where an estimated 22 million people currently are living with HIV/AIDS, underscores the urgent need for a preventative vaccine. However, despite nearly 25 years of intense international research, a vaccine is not yet available. Passive immunization with broadly neutralizing antibodies can confer sterilizing protection against infection in animal models (4, 12, 39-41, 51, 64), providing encouragement for the development of an antibody-inducing component of an HIV type 1 (HIV-1) vaccine. Such a vaccine should elicit neutralizing antibodies with activity against the broadest range of primary circulating isolates. However, a lack of understanding of how to raise potent, cross-reactive antibodies by immunization, the so-called neutralizing antibody problem, is a major hurdle in this effort (6, 24, 72). Thus, an understanding of the structure and presentation of neutralizing epitopes on the virus and the antibodies that recognize them is vital for vaccine development.The targets of antibody neutralization are the surface envelope (Env) glycoprotein trimers (gp120/gp41) that mediate the fusion of the viral membrane with that of the host. The majority of antibodies elicited during natural infection or immunization show limited or no cross-reactivity against diverse isolates. However, a few rare, broadly neutralizing, monoclonal antibodies have been isolated from HIV-1-infected individuals and exhibit activity against a wide range of isolates by binding to functionally conserved epitopes exposed on native gp120/gp41 trimers. These epitopes include the CD4 binding site, recognized by antibody b12, and a relatively well-conserved cluster of N-linked glycans, located on the outer domain of gp120, that is recognized by antibody 2G12 (12, 13, 71, 76). V3-directed antibodies, which are common in natural infection, also are able to sporadically neutralize across clades, as exemplified by 447-52D and F425-B4e8 (7, 16, 49, 66). The identification of three broadly neutralizing antibodies, 2F5, Z13, and 4E10, that target the conserved tryptophan-rich membrane-proximal external region (MPER) of gp41 has implicated this region as a highly promising vaccine target and has, therefore, spurred interest in its structural characterization (15, 35, 45, 47, 48, 50, 80).The MPER plays a critical, but not fully understood, role in membrane fusion and is situated between the C-terminal heptad repeat (CHR) and the transmembrane domain (TM) of gp41 (Fig. ​(Fig.1).1). Following the binding of gp120 to the cell surface receptors CD4 and CXCR4/CCR5, the gp41 glycoprotein undergoes a series of conformational changes that trigger the membrane fusion activity. Notably, a relatively long-lived prehairpin intermediate of gp41 is formed, in which the coiled-coil of the N-terminal heptad repeats (NHR) extends so as to enable the fusion peptides to embed into the target membrane. In the postfusion or fusogenic state, the CHR and NHR reassemble into an antiparallel 6-helix bundle in a process that drives membrane fusion (18). The MPER contains several functionally conserved tryptophan residues that are critical for membrane fusion and viral entry, although the structural basis for their specific role has not been firmly established (22, 44, 58). Their mutation to alanine leads to the attenuation of viral infectivity, which is most pronounced for Trp666 and Trp672 (numbered according to the HXB2 isolate) (46, 58, 78). In addition, peptides based on the MPER can induce membrane leakage (68). Such membrane-disrupting properties of the MPER have been suggested to be functionally important in the expansion of the fusion pore created after receptor engagement (42, 44, 58, 68, 77).Open in a separate windowFIG. 1.Major features of gp41 include the fusion peptide (FP), NHR, CHR, TM, and cytoplasmic domain (CD). The MPER is located between the CHR and TM regions of gp41. The core epitopes of 2F5 (green), Z13e1 (yellow), and 4E10 (orange) are indicated. The epitope of Z13e1 is located between those of 2F5 and 4E10, but it overlaps more closely with 4E10.From initial explorations using solution nuclear magnetic resonance, the structure of a 19-residue MPER peptide (residues 665 to 683) was found to be helical in dodecylphosphocholine micelles, with the hydrophobic and hydrophilic residues distributed evenly around the helix axis (62). Another study found that an MPER peptide comprising residues 659 to 671 adopts a 3₁₀-helix in water (10). More recently, the structure of an MPER peptide (residues 662 to 683) in liposomes was elucidated by a combination of nuclear magnetic resonance and spin-label electron paramagnetic resonance (69), and it was found to adopt a kinked, amphipathic structure composed of two helices connected by a short hinge (Phe673 and Asn674). Crystal structures of Fab 2F5 in complex with a 7-mer (E⁶⁶²LDKWAS⁶⁶⁸) and 17-mer encompassing residues 654 to 670 previously had revealed a mostly extended conformation characterized by a central β-turn involving Asp664, Lys665, and Trp666 (47, 48). This motif is the key recognition determinant for 2F5 and becomes deeply buried in the antibody combining site, suggesting that it is exposed at some stage in viral entry (45, 47, 78). The crystal structure of Fab 4E10 in complex with peptide-spanning residues W⁶⁷⁰NWFDITNW⁶⁷⁸ revealed an amphipathic α-helical structure with a narrow hydrophilic face (15). The N terminus of the 4E10 epitope forms a 3₁₀-helix that transitions into a regular α-helix at residue Asp674 and continues to Lys683, which constitutes the end of the gp41 ectodomain (14). Thus, while the structure of the MPER within functional, membrane-embedded Env trimers is not known, the observation that unconstrained peptides are able to adopt more than one defined structure suggests an inherent degree of flexibility.Like 4E10, Z13 was identified from an HIV-1-infected individual, the former being isolated from an immortalized B-cell line and the latter from a bone marrow RNA phage display library (80). The epitope of MAb Z13 spans residues S⁶⁶⁸LWNWFDITN⁶⁷⁷, as determined by peptide mapping, scanning mutagenesis, and antibody competition studies (46, 80). This region lies between the 2F5 and 4E10 epitopes but overlaps more closely with 4E10 (Fig. ​(Fig.1).1). 4E10 and Z13 are both able to neutralize primary as well as laboratory-adapted isolates; nevertheless, Z13 is not as broadly neutralizing as 4E10, which has the greatest breadth of any HIV-1 antibody described to date (9). Z13e1 is an affinity-enhanced variant of Z13 and was evolved by randomizing the complementarity determining region (CDR) L3 loop sequence to identify tighter-binding mutants using phage display (46). Z13e1 displays higher affinity for both peptide and recombinant gp41 substrates, as well as increased neutralization potency, suggesting that the L3 mutations optimize binding to the linear MPER epitope. The neutralization breadth of Z13e1 is limited by the requirement for Asn671 and Asp674 in the MPER, which are approximately 71 and 58% conserved, respectively, among sequences in the Los Alamos HIV sequence database (80). Based on the clear relationship between Env trimer binding and neutralization, the neutralizing activity of Z13e1 derives from binding to a functional trimer (8, 20, 25, 43, 52, 55, 60, 73, 74). While Z13e1 and 4E10 have identical affinities for optimized linear peptides, Z13e1 is still about an order of magnitude less potent than 4E10 against a variety of primary isolates. Although the occlusion of the Z13e1 epitope on virion-associated trimers is thought to be the major limitation (46), the structural basis for the lower potency of Z13e1 relative to those of 2F5 and 4E10 is unclear.Whereas neutralization by 4E10 depends critically on Trp672 and Phe673, Z13e1 instead requires the flanking Asn671 and Asp674 residues (46). Based on a helical model of the MPER, it was predicted that Z13e1 binds the narrow hydrophilic face that displays Asn671, Asp674, and Asn677 that is opposite that recognized by 4E10. As Z13e1 and 4E10 bind to functional trimers, both epitopes must be exposed at some stage before membrane fusion (20). To examine how Z13e1 recognizes its MPER epitope, we determined the crystal structure of Fab Z13e1 in complex with a 12-residue peptide corresponding to the core epitope with C-terminal flanking lysines to aid peptide solubility (W⁶⁷⁰NWFDITN⁶⁷⁷KKKK). The crystal structure at 1.8-Å resolution uncovers a conformation of the MPER that is distinct from that visualized in complex with 4E10. Our findings show that Z13e1 and 4E10 recognize different conformers of the MPER and reveal a novel conformational switch that is relevant for HIV-1 neutralization and membrane fusion.