Domain Of Gp41 Research Articles

D108 Synthetic HIV-1 Glycopeptides Enable Characterization of Fine Epitopes of Broadly Neutralizing Antibody PG9

Characterization of the precise epitopes for broadly neutralizing antibodies (bNAbs) is an important step for the design of an effective HIV-1 vaccine. Recent X-ray crystal structural studies indicate that the epitope of PG9 constitutes two N-glycans (at N160 and N156 positions) and a strand of peptide in the V1V2 domain of gp120. However, the precise nature of the neutralizing epitopes, particularly the fine structures of the N-glycans at N156 remains to be characterized. Further mapping of the epitopes is complicated by the complexity and heterogeneity of glycosylation of HIV-1 gp120. To address this problem, we launched a project aiming to identify the precise epitopes by synthesis and evaluation of a series of homogeneous cyclic glycopeptides corresponding to the V1/V2 domain. A chemoenzymatic method was developed to make such complex HIV-1 glycopeptides in which defined N-glycans were installed at the predetermined glycosylation sites. SPR binding studies with PG9 revealed that the fine structures of N-glycans at both the N156 and N160 sites were critical for the recognition of the synthetic antigens by PG9. In particular, a complex type sugar at the N156, rather than a Man5GlcNAc2 glycan, seems critical for the high affinity binding of PG9 to the antigen. The synthetic HIV-1 glycopeptides will be valuable to serve as coating antigens for detecting glycan-dependent neutralizing antibodies from sera of HIV-infected patients or those involved in HIV vaccine trials. These studies provide important insights for HIV-1 vaccine design.

Decoding distinct membrane interactions of HIV-1 fusion inhibitors using a combined atomic force and fluorescence microscopy approach

Enfuvirtide and T-1249 are two potent HIV-1 fusion inhibitor peptides. Recent studies indicate that lipids play an important role in the mode of action of those bioactive molecules. Using a combined tandem atomic force microscopy (AFM)–epifluorescence microscopy approach, we studied the interaction of both enfuvirtide and T-1249 with supported lipid bilayers. Fluid (ld)-gel (so) and ld-liquid ordered (lo) phase-separated membrane systems were tested. Results, especially for T-1249, show significant lipid membrane activity at a 15μM peptide concentration. T-1249, in opposition to enfuvirtide, induces an increase in membrane surface roughness, decrease in membrane fluidity, bilayer thinning at ld domains and disruption of the so domain borders. In terms of structural properties, both enfuvirtide and T-1249 possess distinct functional hydrophobic and amphipathic domains of HIV gp41. While enfuvirtide only yields the tryptophan-rich domain (TRD), T-1249 possesses both TRD and pocket-binding domain (PBD). TRD increases the hydrophobicity of the peptide while PBD enhances the amphipathic characteristics. As such, the enhanced membrane activity of T-1249 may be explained by a synergism between its amphipathic N-terminal segment and its hydrophophic C-terminal. Our findings provide valuable insights on the molecular-level mode of action of HIV-1 fusion inhibitors, unraveling the correlation between their structural properties and membrane interactions as a factor influencing their antiviral activity. Ultimately, this work validates the applicability of a combined AFM and fluorescence approach to evaluate the mechanic and structural properties of supported lipid bilayers upon interaction with membrane-active peptides.

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide 13CO to Lipid 31P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

Fusion of the human immunodeficiency virus (HIV) membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41, which is an integral membrane protein of HIV. The fusion peptide (FP) is the ∼25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of "HFP", i.e., a ∼25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was (13)CO backbone labeled. Samples were then prepared that each contained a singly (13)CO labeled HFP incorporated into membranes that lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric β sheet structure. Proximity between the HFP (13)CO nuclei and (31)P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct (13)CO shifts for the α helical and β sheet structures so that the proximities to (31)P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the (13)CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. "HFPmn" was a linear peptide that contained the 23 N-terminal residues of gp41. "HFPmn_V2E" contained the V2E mutation that for HIV leads to greatly reduced extent of fusion and infection. The present study shows that HFPmn_V2E induces much less vesicle fusion than HFPmn. "HFPtr" contained three strands with HFPmn sequence that were chemically cross-linked near their C-termini. HFPtr mimics the trimeric topology of gp41 and induces much more rapid and extensive vesicle fusion than HFPmn. For HFPmn and HFPtr, well-resolved α and β peaks were observed for A6-, L9-, and L12-labeled samples. For each of these samples, there were similar HFP (13)CO to lipid (31)P proximities in the α and β structures, which evidenced comparable membrane locations of the HFP in either structure including insertion into a single membrane leaflet. The data were also consistent with deeper insertion of HFPtr relative to HFPmn in both the α and β structures. The results supported a strong correlation between the membrane insertion depth of the HFP and its fusogenicity. More generally, the results supported membrane location of the HFP as an important determinant of its fusogenicity. The deep insertion of HFPtr in both the α and β structures provides the most relevant membrane location of the FP for HIV gp41-catalyzed membrane fusion because HIV gp41 is natively trimeric. Well-resolved α and β signals were observed in the HFPmn_V2E samples with L9- and L12- but not A6-labeling. The α signals were much more dominant for L9- and L12-labeled HFPmn_V2E than the corresponding HFPmn or HFPtr. The structural model for the less fusogenic HFPmn_V2E includes a shorter helix and less membrane insertion than either HFPmn or HFPtr. This greater helical population and different helical structure and membrane location could result in less membrane perturbation and lower fusogenicity of HFPmn_V2E and suggest that the β sheet fusion peptide is the most functionally relevant structure of HFPmn, HFPtr, and gp41.

Modulation of Cytokine Release and Gene Expression by the Immunosuppressive Domain of gp41 of HIV-1

The transmembrane envelope protein gp41 of the human immunodeficiency virus HIV-1 plays an important role during infection allowing fusion of the viral and cellular membrane. In addition, there is increasing evidence that gp41 may contribute to the immunodeficiency induced by HIV-1. Recombinant gp41 and a synthetic peptide corresponding to a highly conserved domain in gp41, the immunosuppressive (isu) domain, have been shown to inhibit mitogen-induced activation of human peripheral blood mononuclear cells (PBMCs) and to increase release of IL-6 and IL-10 from these cells. We recently reported that a single mutation in the isu domain of gp41 abrogated the immunosuppressive properties and that HIV-1 sequences containing such abrogating mutations had never been isolated from infected individuals. Here, we studied the influence of the isu peptide on the release of 66 cytokines and the expression of 27,000 genes in PBMCs. Incubation of PBMCs with isu peptide homopolymers increased the expression of 16 cytokines among them IL-6 and IL-10, and decreased that of IL-2 and CXCL9. Interestingly, the extend of cytokine modulation was donor-dependent. Among the genes up-regulated were IL-6, IL-8, IL-10 but also MMP-1, TREM-1 and IL-1beta. Most importantly, genes involved in innate immunity such as FCN1 and SEPP1 were found down-regulated. Many changes in cytokine expression demonstrated in our experiments were also found in HIV-1 infected individuals. These data indicate that the isu domain of gp41 has a broad impact on gene expression and cytokine release and therefore may be involved in HIV-1 induced immunopathogenesis.

Prime-boost immunization of rabbits with HIV-1 gp120 elicits potent neutralization activity against a primary viral isolate.

Development of a vaccine for HIV-1 requires a detailed understanding of the neutralizing antibody responses that can be experimentally elicited to difficult-to-neutralize primary isolates. Rabbits were immunized with the gp120 subunit of HIV-1 JR-CSF envelope (Env) using a DNA-prime protein-boost regimen. We analyzed five sera that showed potent autologous neutralizing activity (IC50s at ∼103 to 104 serum dilution) against pseudoviruses containing Env from the primary isolate JR-CSF but not from the related isolate JR-FL. Pseudoviruses were created by exchanging each variable and constant domain of JR-CSF gp120 with that of JR-FL or with mutations in putative N-glycosylation sites. The sera contained different neutralizing activities dependent on C3 and V5, C3 and V4, or V4 regions located on the glycan-rich outer domain of gp120. All sera showed enhanced neutralizing activity toward an Env variant that lacked a glycosylation site in V4. The JR-CSF gp120 epitopes recognized by the sera are generally distinct from those of several well characterized mAbs (targeting conserved sites on Env) or other type-specific responses (targeting V1, V2, or V3 variable regions). The activity of one serum requires specific glycans that are also important for 2G12 neutralization and this serum blocked the binding of 2G12 to gp120. Our findings show that different fine specificities can achieve potent neutralization of HIV-1, yet this strong activity does not result in improved breadth.

Effects of the Midspan Arginine on the Interactions between a Solvated Lipid Bilayer and the HIV-1 Gp41 Membrane Spanning Domain

The membrane spanning domain (MSD) of HIV-1 gp41 has been shown experimentally to anchor envelope protein gp41 in the viral membrane and to play a role in fusion/infection. One conserved structural motif of the MSD is a midspan arginine, probably charged, located in the hydrophobic lipid bilayer core. It has therefore been postulated that the positively-charged guanidino sidegroup of the midspan arginine (R694) snorkels to negatively-charged headgroups in the membrane. The configurational differences between the wild-type (WT) MSD and a fusion-impaired mutant (R694L) MSD are studied here using molecular dynamics (MD). The conformational distribution and PMF of the R694L MSD peptide were compared with that of the WT MSD peptide using the technique of metadynamics. The mutant and WT peptides both have stable, alpha-helical conformations. Equilibrium properties of these stable, alpha-helical conformations were studied by long (300ns) MD. The presence of greater water around the C-terminal and the greater tilt of the WT MSD indicates that perhaps a role of the charged midspan arginine is partial solvation of the C-terminal, which may function to hold the viral membrane in a metastable prefusion state.

193 Computer-aided design of novel HIV-1 entry inhibitors based on glycosphingolipids

Novel HIV-1 entry inhibitors targeting the envelope gp120 V3 loop were designed by computer modeling based on glycosphingolipid β-galactosylceramide (β-GalCer) forming on the surface of some susceptible host cells the primary receptor for HIV-1 alternative to CD4, which is used by the virus to enter macrophages and T-lymphocytes (e.g. Fantini et al., 1993). To achieve this goal, 3D structures of the twelve water-soluble analogs of β-GalCer containing different substitutes of its fatty acid residue were determined by quantum chemical calculations and evaluation of their potential anti-HIV-1 activity was carried out by molecular docking, molecular dynamics, and free energy simulations. Analysis of the structural complexes of these β-GalCer derivatives with the HIV-1 V3 loops from the five diverse viral strains makes it clear that, in all of the cases of interest, the third variable domain of gp120 forms two potential binding sites for glycolipids concerning the immunogenic tip and the base of V3. At the same time, nonconventional XH … π hydrogen bonds between XH sugar groups (X designates C or O) and overlapping π-orbitals of the conserved Phe-20, Tyr-21, and His-34 residues of the V3 loop were shown to play a key role in specific binding of the designed glycosphingolipids to the above conserved structural motifs of V3 that include residues critical for cell tropism (Andrianov et al., 2011; Andrianov, Kornoushenko, et al., 2012). These findings testifying to the ability of the simulated chemicals to specifically and effectively interact with the functionally important sites of V3 were confirmed by those on molecular dynamics and calculating the free energy of formation of the complexes for these β-GalCer analogs with the HIV-1 V3 loops from different viral modifications. Finally, the majority of the designed molecules were found to form much more stable complexes with V3, compared to that of the β-GalCer-based anti-HIV-1 agent developed previously (Andrianov, Anishchenko, et al., 2012) and used in the calculations as a control. In the light of the data obtained, these potential HIV-1 entry inhibitors present the promising basic compounds for the development of novel, potent, and broad antiviral drugs.

Conformational Dynamics of Single HIV Env Molecules on the Surface of Native Virions

Entry of HIV is accomplished by the trimeric envelope spike (Env), a conformational machine that avoids recognition by antibodies through a 2-receptor mechanism of activation, involving CD4 and a coreceptor. We introduced fluorophores into distinct domains of gp120, the receptor-binding subunit of the spike, and monitored the conformational changes though single-molecule fluorescence resonance energy transfer (smFRET) measurements on the surface of infectious HIV virions. smFRET trajectories revealed transitions between three conformations intrinsically accessible to the unliganded Env. Binding of CD4 stabilized one of the pre-existing states. Binding of CD4 and the coreceptor-surrogate antibody 17b stabilized a second pre-existing state. Markov modeling of the smFRET trajectories indicated a preferred order to the conformational changes with the CD4-stabilized state an intermediate critical to achieving the likely coreceptor-stabilized state. Distances estimated from the observed FRET states were integrated with known structures of HIV Env to produce a temporally accurate simulation of gp120, as it activates the underlying gp41-fusion machinery. Our work provides a framework for understanding the stepwise activation of HIV Env through successive binding of the receptor and coreceptor.

Specific VpU codon changes were significantly associated with gp120 V3 tropic signatures in HIV-1 B-subtype

After infection and integration steps, HIV-1 transcriptions increase sharply and singly-spliced mRNAs are produced. These encode Env (gp120 and gp41) and auxiliary proteins Vif, Vpr and VpU. The same localization within the unique structure of the mRNAs suggests that the VpU sequence prior to the Env could affect the Env polyprotein expression.The HIV-1 infection process begins when the gp120 subunit of the envelope glycoprotein complex interacts with its receptor(s) on the target cell. The V3 domain of gp120 is the major determinant of cellular co-receptor binding. According to phenotypic information of HIV-1 isolates, sequences from the VpU to V3 regions (119 in R5- and 120 X4-tropic viruses; one per patient) were analysed. The binomial correlation phi coefficient was used to assess covariation among VpU and gp120(V3) signatures. Subsequently, average linkage hierarchical agglomerative clustering was performed. Beyond the classical V3 signatures (R5-viruses: S11, E25D; X4-viruses: S11KR, E25KRQ), other specific V3 and novel VpU signatures were found to be statistically associated with co-receptor usage. Several statistically significant associations between V3 and VpU mutations were also observed. The dendrogram showed two distinct large clusters: one associated with R5-tropic sequences (bootstrap=0.94), involving: (a) H13NP(V3), E25D(V3), S11(V3), T22A(V3) and Q61H(VpU), (b) E25A(V3) and L12F(VpU), (c) D44E(VpU), R18Q(V3) and D80N(VpU); and another associated with X4-tropic sequences (bootstrap=0.97), involving: (i) E25I(V3) and V10A(VpU), (ii) 0-1insV(VpU), H13R(V3), I46L(VpU), I30M(V3) and 60-62del(VpU), (iii) S11KR(V3) and E25KRQ(V3). Some of these pairs of mutations were encoded always by one specific codon. These data indicate the possible VpU mutational patterns contributing to regulation of HIV-1 tropism.

The tale of the long tail: the cytoplasmic domain of HIV-1 gp41.

Envelope glycoproteins (Env) of lentiviruses typically possess unusually long cytoplasmic domains, often 150 amino acids or longer. It is becoming increasingly clear that these sequences contribute a diverse array of functional activities to the life cycle of their viruses. The cytoplasmic domain of gp41 (gp41CD) is required for replication of human immunodeficiency virus type 1 (HIV-1) in most but not all cell types, whereas it is largely dispensable for replication of simian immunodeficiency virus (SIV). Functionally, gp41CD has been shown to regulate rapid clathrin-mediated endocytosis of Env. The resultant low levels of Env expression at the cell surface likely serve as an immune avoidance mechanism to limit accessibility to the humoral immune response. Intracellular trafficking of Env is also regulated by gp41CD through interactions with a variety of cellular proteins. Furthermore, gp41CD has been implicated in the incorporation of Env into virions through an interaction with the virally encoded matrix protein. Most recently, the gp41CDs of HIV-1 and SIV were shown to activate the key cellular-transcription factor NF-κB via the serine/threonine kinase TAK1. Less well understood are the cytotoxicity- and apoptosis-inducing activities of gp41CD as well as potential roles in modulating the actin cytoskeleton and overcoming host cell restrictions. In this review, we summarize what is currently known about the cytoplasmic domains of HIV-1 and SIV and attempt to integrate the wealth of information in terms of defined functional activities.

Efficiency of bridging-sheet recruitment explains HIV-1 R5 envelope glycoprotein sensitivity to soluble CD4 and macrophage tropism.

HIV-1 R5 viruses vary extensively in their capacity to infect macrophages. R5 viruses that confer efficient infection of macrophages are able to exploit low levels of CD4 for infection and predominate in brain tissue, where macrophages are a major target for infection. HIV-1 R5 founder viruses that are transmitted were reported to be non-macrophage-tropic. Here, we investigated the sensitivities of macrophage-tropic and non-macrophage-tropic R5 envelopes to neutralizing antibodies. We observed striking differences in the sensitivities of Env(+) pseudovirions to soluble CD4 (sCD4) and to neutralizing monoclonal antibodies (MAbs) that target the CD4 binding site. Macrophage-tropic R5 Envs were sensitive to sCD4, while non-macrophage-tropic Envs were significantly more resistant. In contrast, all Envs were sensitive to VRC01 regardless of tropism, while MAb b12 conferred an intermediate neutralization pattern where all the macrophage-tropic and about half of the non-macrophage-tropic Envs were sensitive. CD4, b12, and VRC01 share binding specificities on the outer domain of gp120. However, these antibodies differ in their ability to induce conformational changes on the trimeric envelope and in specificity for residues on the V1V2 loop stem and β20-21 junction that are targets for CD4 in recruiting the bridging sheet. These distinct specificities of CD4, b12, and VRC01 likely explain the observed differences in Env sensitivity to inhibition by these reagents and provide an insight into the envelope mechanisms that control macrophage tropism. We present a model where the efficiency of bridging-sheet recruitment by CD4 is a major determinant of HIV-1 R5 envelope sensitivity to soluble CD4 and macrophage tropism.

Spontaneous Rearrangement of the β20/β21 Strands in Simulations of Unliganded HIV-1 Glycoprotein, gp120

Binding of the viral spike drives cell entry and infection by HIV-1 to the cellular CD4 and chemokine receptors with associated conformational change of the viral glycoprotein envelope, gp120. Crystal structures of the CD4-gp120-antibody ternary complex reveal a large internal gp120 cavity formed by three domains-the inner domain, outer domain, and bridging sheet domain-and are capped by CD4 residue Phe43. Several structures of gp120 envelope in complex with various antibodies indicated that the bridging sheet adopts varied conformations. Here, we examine bridging sheet dynamics using a crystal structure of gp120 bound to the F105 antibody exhibiting an open bridging sheet conformation and with an added V3 loop. The two strands of the bridging sheet β2/β3 and β20/β21 are dissociated from each other and are directed away from the inner and outer domains. Analysis of molecular dynamics (MD) trajectories indicates that the β2/β3 and β20/β21 strands rapidly rearrange to interact with the V3 loop and the inner and outer domains, respectively. Residue N425 on β20 leads the conformational rearrangement of the β20/β21 strands by interacting with W112 on the inner domain and F382 on the outer domain. An accompanying shift is observed in the inner domain as helix α1 exhibits a loss in helicity and pivots away from helix α5. The two simulations provide a framework for understanding the conformational diversity of the bridging sheet and the propensity of the β20/β21 strand to refold between the inner and outer domains of gp120, in the absence of a bound ligand.

The Conserved Residue Arg46 in the N-Terminal Heptad Repeat Domain of HIV-1 gp41 Is Critical for Viral Fusion and Entry

During the process of HIV-1 fusion with the target cell, the N-terminal heptad repeat (NHR) of gp41 interacts with the C-terminal heptad repeat (CHR) to form fusogenic six-helix bundle (6-HB) core. We previously identified a crucial residue for 6-HB formation and virus entry - Lys63 (K63) in the C-terminal region of NHR (aa 54–70), which forms a hydrophobic cavity. It can form an important salt bridge with Asp121 (D121) in gp41 CHR. Here, we found another important conserved residue for virus fusion and entry, Arg46 (R46), in the N-terminal region of NHR (aa 35–53), which forms a hydrogen bond with a polar residue, Asn43 (N43), in NHR, as a part of the hydrogen-bond network. R46 can also form a salt bridge with a negatively charged residue, Glu137 (E137), in gp41 CHR. Substitution of R46 with the hydrophobic residue Ala (R46A) or the negatively charged residue Glu (R46E) resulted in disruption of the hydrogen bond network, breakage of the salt bridge and reduction of 6-HB’s stability, leading to impairment of viral fusion and decreased inhibition of N36, an NHR peptide. Similarly, CHR peptide C34 with substitution of E137 for Ala (E137A) or Arg (E137R) also exhibited reduced inhibitory activity against HIV-1 infection and HIV-1-mediated cell-to-cell fusion. These results suggest that the positively charged residue R46 and its hydrogen bond network, together with the salt bridge between R46 and E137, are important for viral fusion and entry and may therefore serve as a target for designing novel HIV fusion/entry inhibitors.

Rationally designed HIV envelope glycoproteins delivered in a novel adjuvant elicited more broadly reactive antigen-specific antibody responses

Methods Here, we designed disulfide-stabilized recombinant HIV1 subtype B (SF162) envelope glycoproteins (Env), gp120 and gp140, by insertion of site-specific cysteine pairs between two layers (layer 1 and 2) in inner domain of gp120. In addition, we identified a novel adjuvant approach using Carbopol 971P, a cross-linked polyanionic carbomer, in combination with the Novartis proprietary oil-in water adjuvant, MF59, to augment humoral immune responses to the Env glycoprotein. We performed thorough in vitro analysis of the disulfide-stabilized Env glycoprotein followed by in vivo evaluations of the adjuvanted-Env glycoprotein boost in rabbits.

Single mutations in the transmembrane envelope protein abrogate the immunosuppressive property of HIV-1

BackgroundThe mechanism by which HIV-1 induces AIDS is still unknown. Previously, synthetic peptides corresponding to the conserved immunosuppressive (isu) domain in gp41 of HIV-1 had been shown to inhibit proliferation and to modulate cytokine expression of immune cells. The question is, whether the viral gp41 can do the same.ResultsWe show for the first time that two trimeric forms of glycosylated gp41 released from transfected human cells modulated expression of cytokines and other genes in human PBMCs in the same manner, but at least seven hundred-fold stronger compared to that induced by the isu peptide. Single amino acid substitutions in the isu domain of gp41 introduced by site-directed mutagenesis abrogated this property. Furthermore, replication-competent HIV-1 with a mutation in the isu domain of gp41 did not modulate the cytokine expression, while wild-type virus did. Interestingly, most of the abrogating mutations were not reported in viral sequences derived from infected individuals, suggesting that mutated non-immunosuppressive viruses were eliminated by immune responses. Finally, immunisation of rats with gp41 mutated in the isu domain resulted in increased antibody responses compared with the non-mutated gp41. These results show that non-mutated gp41 is immunosuppressive in immunisation experiments, i.e. in vivo, and this has implications for the vaccine development.ConclusionsThese findings indicate that the isu domain of gp41 modulates cytokine expression in vitro and suppresses antibody response in vivo and therefore may contribute to the virus induced immunodeficiency.

Subunit organization of the membrane-bound HIV-1 envelope glycoprotein trimer

The trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike is a molecular machine that mediates virus entry into host cells and is the sole target for virus-neutralizing antibodies. The mature Env spike results from cleavage of a trimeric gp160 precursor into three gp120 and three gp41 subunits. Here we describe an ~11-Å cryo-EM structure of the trimeric HIV-1 Env precursor in its unliganded state. The three gp120 and three gp41 subunits form a cage-like structure with an interior void surrounding the trimer axis. Interprotomer contacts are limited to the gp41 transmembrane region, the torus-like gp41 ectodomain, and a gp120 trimer association domain composed of the V1/V2 and V3 variable regions. The cage-like architecture, which is unique among characterized viral envelope proteins, restricts antibody access, reflecting requirements imposed by HIV-1 persistence in the host.

Structural elements of primary CCR5-using HIV-1 gp120 proteins influencing sensitivity and resistance to the broadly neutralizing monoclonal antibody b12

Structure-guided approaches to HIV-1 vaccine design depend on knowledge of the presentation of neutralizing epitopes on gp120, such as the epitope for the broadly neutralizing mAb b12. Here, we characterized predicted three-dimensional structures of functionally diverse gp120 proteins in their b12-bound conformation, to better understand the gp120 determinants that expose or occlude the b12 epitope. Mapping the gp120–b12 binding interface identified amino acid polymorphisms within the C2, C3, C4 and V5 regions of gp120 associated with augmented b12 binding, and importantly, identified residues in the b12-exclusive binding domain of gp120 that are important for b12 neutralization resistance. Structural studies suggest that these b12 resistance variants promote reduced conformational flexibility in the b12 recognition site, which we show involves structural alterations within the gp120 CD4 binding loop and the V4 loop. Together, our studies provide new mechanistic insights into the gp120 determinants influencing sensitivity and resistance to HIV-1 neutralization by b12.

Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance

A rare pathway of HIV-1 resistance to small molecule CCR5 inhibitors such as Vicriviroc (VCV) involves changes solely in the gp41 fusion peptide (FP). Here, we show that the G516V change is critical to VCV resistance in PBMC and TZM-bl cells, although it must be accompanied by either M518V or F519I to have a substantial impact. Modeling VCV inhibition data from the two cell types indicated that G516V allows both double mutants to use VCV-CCR5 complexes for entry. The model further identified F519I as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5. From inhibitor-free reversion cultures, we also identified a substitution in the inner domain of gp120, T244A, which appears to counter the resistance phenotype created by the FP substitutions. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.

Vicriviroc Resistance Decay and Relative Replicative Fitness in HIV-1 Clinical Isolates under Sequential Drug Selection Pressures

We previously described an HIV-1-infected individual who developed resistance to vicriviroc (VCV), an investigational CCR5 antagonist, during 28 weeks of therapy (Tsibris AM et al., J. Virol. 82:8210-8214, 2008). To investigate the decay of VCV resistance mutations, a standard clonal analysis of full-length env (gp160) was performed on plasma HIV-1 samples obtained at week 28 (the time of VCV discontinuation) and at three subsequent time points (weeks 30, 42, and 48). During 132 days, VCV-resistant HIV-1 was replaced by VCV-sensitive viruses whose V3 loop sequences differed from the dominant pretreatment forms. A deep-sequencing analysis showed that the week 48 VCV-sensitive V3 loop form emerged from a preexisting viral variant. Enfuvirtide was added to the antiretroviral regimen at week 30; by week 48, enfuvirtide treatment selected for either the G36D or N43D HR-1 mutation. Growth competition experiments demonstrated that viruses incorporating the dominant week 28 VCV-resistant env were less fit than week 0 viruses in the absence of VCV but more fit than week 48 viruses. This week 48 fitness deficit persisted when G36D was corrected by either site-directed mutagenesis or week 48 gp41 domain swapping. The correction of N43D, in contrast, restored fitness relative to that of week 28, but not week 0, viruses. Virus entry kinetics correlated with observed fitness differences; the slower entry of enfuvirtide-resistant viruses corrected to wild-type rates in the presence of enfuvirtide. These findings suggest that while VCV and enfuvirtide select for resistance mutations in only one env subunit, gp120 and gp41 coevolve to maximize viral fitness under sequential drug selection pressures.

A10 The Potential Role of Antibodies to the C5 Domain of gp120 in Mitigating Generalized Immune Activation Associated With HIV-1 Infection

A10 The Potential Role of Antibodies to the C5 Domain of gp120 in Mitigating Generalized Immune Activation Associated With HIV-1 Infection