The membranotropic regions of the endo and ecto domains of HIV gp41 envelope glycoprotein

We have identified the membranotropic regions of the full sequence of the HIV gp41 envelope glycoprotein by performing an exhaustive study of membrane rupture, phospholipid-mixing and fusion induced by two 15-mer gp41-derived peptide libraries from HIV strains HIV_MN and HIV_consensus_B on model membranes having different phospholipid compositions. The data obtained for the two strains and its comparison have led us to identify different gp41 membranotropic segments in both ecto- and endodomains which might be implicated in viral membrane fusion and/or membrane interaction. The membranotropic segments corresponding to the gp41 ectodomain were the fusion domain, a stretch located on the N-heptad repeat region adjacent to the fusion domain, part of the immunodominant loop, the pre-transmembrane domain and the transmembrane domain. The membranotropic segments corresponding to the gp41 endodomain were mainly located at some specific parts of the previously described lentivirus lytic sequences. Significantly, the C-heptad repeat region and the Kennedy sequence located in the ectodomain and in the endodomain, respectively, presented no membranotropic activity in any model membrane assayed. The identification of these gp41 segments as well as their membranotropic propensity sustain the notion that different segments of gp41 provide the driving force for the merging of the viral and target cell membranes as well as they help us to define those segments as attractive targets for further development of new anti-viral compounds.     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein.

Insertion of four amino acids into various locations within the amino-terminal halves of the human immunodeficiency virus type 1 gp120 or gp41 envelope glycoprotein disrupts the noncovalent association of these two envelope subunits (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. A. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987). To localize the determinants on the gp120 envelope glycoprotein important for subunit association, amino acids conserved among primate immunodeficiency viruses were changed. Substitution mutations affecting either of two highly conserved regions located at the amino (residues 36 to 45) and carboxyl (residues 491 to 501) ends of the mature gp120 molecule resulted in nearly complete dissociation of the envelope glycoprotein subunits. Partial dissociation phenotypes were observed for some changes affecting residues in the third and fourth conserved gp120 regions. These results suggest that hydrophobic regions at both ends of the gp120 glycoprotein contribute to noncovalent association with the gp41 transmembrane glycoprotein.     

Probing the HIV gp120 envelope glycoprotein conformation by NMR

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

The tryptophan-rich region of HIV gp41 and the promotion of cholesterol-rich domains

The peptide N-acetyl-KWASLWNWFNITNWLWYIK-amide has a sequence that corresponds to the juxtamembrane region of the HIV-1 gp41 fusion protein. We have studied how cholesterol modulates the interaction of this peptide with membranes containing cholesterol using differential scanning calorimetry, circular dichroism, fluorescence spectroscopy, and nuclear magnetic resonance. We find that this peptide is less able to sequester cholesterol into domains than is N-acetyl-LWYIK-amide. On the other hand, the peptide N-acetyl-LASWIK-amide, which corresponds to a segment of HIV-2 and SIV gp41 fusion proteins, has intermediate potency between N-acetyl-KWASLWNWFNITNWLWYIK-amide and N-acetyl-LWYIK-amide in forming areas enriched in cholesterol, even though it does not have a cholesterol recognition/interaction amino acid consensus sequence (CRAC). We suggest that the difference between HIV-1 and HIV-2 in their requirements for glycosphingolipids in determining their tropism is related to their difference in partitioning to cholesterol-rich domains in biological membranes.     

Peptide mimic of the HIV envelope gp120-gp41 interface

The human immunodeficiency virus envelope glycoprotein (Env) is composed of surface (gp120) and transmembrane (gp41) subunits, which are noncovalently associated on the viral surface. Human immunodeficiency virus Env mediates viral entry after undergoing a complex series of conformational changes induced by interaction with cellular CD4 and a chemokine coreceptor. These changes propagate from gp120 to gp41 via the gp120-gp41 interface, ultimately exposing gp41 and allowing it to form the trimer-of-hairpins structure that provides the driving force for membrane fusion. Key unresolved questions about the gp120-gp41 interface include the specific regions of gp41 and gp120 involved, the mechanism by which receptor and coreceptor-binding-induced conformational changes in gp120 are communicated to gp41, how trimer-of-hairpins formation is prevented in the prefusogenic gp120-gp41 complex, and, ultimately, the structure of the prefusion gp120-gp41 complex. Here, we develop a biochemical model system that mimics a key portion of the gp120-gp41 interface in the prefusogenic state. We find that a gp41 fragment containing the disulfide bond loop and C-peptide region binds primarily to the gp120 C5 region and that this interaction is incompatible with trimer-of-hairpins formation. Based on these data, we propose that in prefusogenic Env, gp120 sequesters the gp41 C-peptide region away from the N-trimer region, preventing trimer-of-hairpins formation until coreceptor binding disrupts this interface. This model system is a valuable tool for studying the gp120-gp41 complex, conformational changes induced by CD4 and coreceptor binding, and the mechanism of membrane fusion.     

Structure based sequence analysis & epitope prediction of gp41 HIV1 envelope glycoprotein isolated in Pakistan

ABSTRACT: BACKGROUND: Gp41 is an envelope glycoprotein of human immune deficiency virus (HIV). HIV viral glycoprotein gp41, present in complex with gp120, assists the viral entry into host cell. Over eighty thousands individuals are HIV infected in Pakistan which makes about 0.2% of 38.6 million infected patients worldwide. Hence, HIV gp41 protein sequences isolated in Pakistan were analyzed for the CD4 and CD8 T cells binding epitopes. RESULTS: Immunoinformatics tools were applied for the study of variant region of HIV gp41envelope protein. The protein nature was analyzed using freely accessible computational software. About 90 gp41 sequences of Pakistani origin were aligned and variable and conserved regions were found. Four segments were found to be conserved in gp41 viral protein. A method was developed, involving the secondary structure, surface accessibility, hydrophobicity, antigenicity and molecular docking for the prediction and location of epitopes in the viral glycoprotein. Some highly conserved CD4 and CD8 binding epitopes were also found using multiple parameters. The predicted continuous epitopes mostly fall in the conserved region of 1-12; 14-22 and 25-46 and can be used as effective vaccine candidates. CONCLUSION: The study revealed potential HIV subtype a derived cytotoxic T cell (CTL) epitopes from viral proteome of Pakistani origin. The conserved epitopes are very useful for the diagnosis of the HIV 1 subtype a. This study will also help scientists to promote research for vaccine development against HIV 1 subtype a, isolated in Pakistan.     

Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.     

HIV-1跨膜蛋白gp41螺旋结构的原核表达及功能研究

HIV-1跨膜蛋白gp41是HIV-1包膜与靶细胞膜的融合过程中的关键蛋白,是理想的HIV-1融合抑制剂靶点。为开展以gp41为靶点的抑制剂筛选,以HIV-1 B亚型病毒基因为模板,通过PCR、酶切、连接等方法构建得到gp41 5-helix与6-helix重组质粒,转入大肠杆菌BL21（DE3）进行表达,经变性和复性后亲和层析纯化蛋白。经SDS-PAGE鉴定,纯化后蛋白纯度较高。本研究还通过非变性凝胶电泳证明gp41 5-helix与C-多肽衍生物T-20存在相互作用,为下一步药物筛选模型的建立奠定了基础。     

Structural and immunological characterisation of heteroclitic peptide analogues corresponding to the 600-612 region of the HIV envelope gp41 glycoprotein

The conformational and immunological properties of different analogues corresponding to the 600-612 disulfide loop of the human immunodeficiency virus (HIV) gp41 glycoprotein envelope were studied. Fourteen analogues were designed and synthesised; namely, a series of seven analogues in which the disulfide bond was replaced by a lactam bridge and a series of seven analogues in which one residue of each analogue at a time, was replaced by its corresponding homologised alpha-amino acid (beta(3)-amino acid). In the case of the lactam analogues, the influence of the two possible CO-NH and NH-CO orientations of the lactam bridge as well as the size of the lactam ring was explored. The analogues were tested in ELISA with monoclonal antibodies raised against the 600-612 cyclic parent peptide as well as with sera from HIV-1 infected patients. A structural analysis of the parent and analogue peptides was carried out in dimethyl sulfoxide (DMSO-d(6)) using two-dimensional NMR techniques and molecular dynamics simulations. Comparison of the own conformation of the cyclic analogues with their either strong or weak reactivity with the antibodies reveals structural features that may be correlated with the antibody reactivity. Thus, a close structural similarity, particularly a characteristic orientation of the side-chains of residues Lys606, Leu607 and Ile608 in the loop, was found in certain beta(3)-analogues that were better recognised than the parent peptide by anti-peptide mouse monoclonal antibodies and patients' antibodies.     

Expression, purification, and characterization of recombinant HIV gp140. The gp41 ectodomain of HIV or simian immunodeficiency virus is sufficient to maintain the retroviral envelope glycoprotein as a trimer

Efforts to understand the molecular basis of human immunodeficiency virus (HIV) envelope glycoprotein function have been hampered by the inability to generate sufficient quantities of homogeneous material. We now report on the high level expression, purification, and characterization of soluble HIV gp140 ectodomain proteins in Chinese hamster ovary-Lec3.2.8.1 cells. Gel filtration and analytical ultracentrifugation show that the uncleaved ADA strain-derived gp140 proteins are trimeric without further modification required to maintain oligomers. These spike proteins are native as judged by soluble CD4 (sCD4) (K(D) = 1-2 nm) and monoclonal antibody binding studies using surface plasmon resonance. CD4 ligation induces conformational change in the trimer, exposing the chemokine receptor binding site as assessed by 17b monoclonal antibody reactivity. Lack of anti-cooperativity in sCD4-ADA trimer interaction distinct from that observed with sCD4-SIV mac32H implies quaternary structural differences in ground states of their respective spike proteins.     

Characterization of HIV-1 envelope gp41 genetic diversity and functional domains following perinatal transmission

Background

HIV-1 envelope gp41 is a transmembrane protein that promotes fusion of the virus with the plasma membrane of the host cells required for virus entry. In addition, gp41 is an important target for the immune response and development of antiviral and vaccine strategies, especially when targeting the highly variable envelope gp120 has not met with resounding success. Mutations in gp41 may affect HIV-1 entry, replication, pathogenesis, and transmission. We, therefore, characterized the molecular properties of gp41, including genetic diversity, functional motifs, and evolutionary dynamics from five mother-infant pairs following perinatal transmission.

Results

The gp41 open reading frame (ORF) was maintained with a frequency of 84.17% in five mother-infant pairs' sequences following perinatal transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in gp41 sequences. Both mother and infant gp41 sequences were under positive selection pressure, as determined by ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 157 mother-infant gp41 sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked sequences. The functional domains of gp41, including fusion peptide, heptad repeats, glycosylation sites and lentiviral lytic peptides were mostly conserved in gp41 sequences analyzed in this study. The CTL recognition epitopes and motifs recognized by fusion inhibitors were also conserved in the five mother-infant pairs.

Conclusion

The maintenance of an intact envelope gp41 ORF with conserved functional domains and a low degree of genetic variability as well as positive selection pressure for adaptive evolution following perinatal transmission is consistent with an indispensable role of envelope gp41 in HIV-1 replication and pathogenesis.     

Role of hydrophobic residues in the central ectodomain of gp41 in maintaining the association between human immunodeficiency virus type 1 envelope glycoprotein subunits gp120 and gp41

The interaction between the gp120 and gp41 subunits of the human immunodeficiency virus envelope glycoprotein serves to stabilize the virion form of the complex and to transmit receptor-induced conformational changes in gp120 to trigger the membrane fusion activity of gp41. In this study, we used site-directed mutagenesis to identify amino acid residues in the central ectodomain of gp41 that contribute to the stability of the gp120-gp41 association. We identified alanine mutations at six positions, including four tryptophan residues, which result in mutant envelope glycoprotein complexes that fail to retain gp120 on the cell surface. These envelope glycoproteins readily shed their gp120 and are unable to mediate cell-cell fusion. These findings suggest an important role for the conserved bulky hydrophobic residues in stabilizing the gp120-gp41 complex.     

Development of HIV entry inhibitors targeted to the coiled-coil regions of gp41

The discoveries that synthetic peptides corresponding to the N- and C-terminal heptad repeat (HR) regions of gp41 have potent anti-HIV activity opened a new avenue to identification of small molecule HIV entry inhibitors targeted to the HIV gp41 coiled-coil regions. Based on the structural information of the HIV gp41 core, three distinct approaches to develop small molecule anti-HIV agents have been reported. Each of these approaches has specific advantages, which will have complementary effects on the design of new strategies for identification of more potent HIV entry inhibitors. It is expected that novel antiviral drugs targeted to the HIV gp41 coiled-coil regions will be developed in the near future for the chemotherapy and/or prophylaxis of HIV infection and AIDS.     

gp120-independent fusion mediated by the human immunodeficiency virus type 1 gp41 envelope glycoprotein: a reassessment.

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.     

gp160, the envelope glycoprotein of human immunodeficiency virus type 1, is a dimer of 125-kilodalton subunits stabilized through interactions between their gp41 domains.

The molecular masses, carbohydrate contents, oligomeric status, and overall molecular structure of the env glycoproteins of human immunodeficiency virus type 1--gp120, gp160, and gp41--have been determined by quantitative electron microscopy. Using purified gp160s, a water-soluble form of env purified from a recombinant vaccinia virus expression system, we have measured the masses of several hundred individual molecules by dark-field scanning transmission electron microscopy. When combined with sequence-based information, these mass measurements establish that gp160s is a dimer of subunits with an average monomer mass of 123 kDa, of which approximately 32 kDa is carbohydrate and 91 kDa is protein. Similarly, gp120 was found to be a monomer of 89 kDa and to contain virtually all of env's glycosylation. gp41 is glycosylated only slightly, if at all, and is responsible for the interactions that stabilize the gp160s dimer. A molecular mass map of gp160s derived by image processing depicts an asymmetric dumbbell whose two domains have masses of approximately 173 and approximately 73 kDa, corresponding to a gp120 dimer and a gp41 dimer, respectively. We infer that the average monomer mass of native gp160 is 125 kDa and that in situ, env is either a dimer or a tetramer but is most unlikely to be a trimer.     

The membrane-spanning domain of gp41 plays a critical role in intracellular trafficking of the HIV envelope protein

Background

Each of the pathogenic human retroviruses (HIV-1/2 and HTLV-1) has a nonhuman primate counterpart, and the presence of these retroviruses in humans results from interspecies transmission. The passage of another simian retrovirus, simian foamy virus (SFV), from apes or monkeys to humans has been reported. Mandrillus sphinx, a monkey species living in central Africa, is naturally infected with SFV. We evaluated the natural history of the virus in a free-ranging colony of mandrills and investigated possible transmission of mandrill SFV to humans.

Results

We studied 84 semi-free-ranging captive mandrills at the Primate Centre of the Centre International de Recherches Médicales de Franceville (Gabon) and 15 wild mandrills caught in various areas of the country. The presence of SFV was also evaluated in 20 people who worked closely with mandrills and other nonhuman primates. SFV infection was determined by specific serological (Western blot) and molecular (nested PCR of the integrase region in the polymerase gene) assays. Seropositivity for SFV was found in 70/84 (83%) captive and 9/15 (60%) wild-caught mandrills and in 2/20 (10%) humans. The 425-bp SFV integrase fragment was detected in peripheral blood DNA from 53 captive and 8 wild-caught mandrills and in two personnel. Sequence and phylogenetic studies demonstrated the presence of two distinct strains of mandrill SFV, one clade including SFVs from mandrills living in the northern part of Gabon and the second consisting of SFV from animals living in the south. One man who had been bitten 10 years earlier by a mandrill and another bitten 22 years earlier by a macaque were found to be SFV infected, both at the Primate Centre. The second man had a sequence close to SFVmac sequences. Comparative sequence analysis of the virus from the first man and from the mandrill showed nearly identical sequences, indicating genetic stability of SFV over time.

Conclusion

Our results show a high prevalence of SFV infection in a semi-free-ranging colony of mandrills, with the presence of two different strains. We also showed transmission of SFV from a mandrill and a macaque to humans.     

The HIV-1 envelope glycoprotein gp120 features four heparan sulfate binding domains, including the co-receptor binding site

It is well established that the human immunodeficiency virus-1 envelope glycoprotein surface unit, gp120, binds to cell-associated heparan sulfate (HS). Virus infectivity is increased by such interaction, and a variety of soluble polyanions efficiently neutralize immunodeficiency virus-1 in vitro. This interaction has been mainly attributed to the gp120 V3 loop. However, although evidence suggested that this particular domain does not fully recapitulate the binding activity of the protein, the ability of HS to bind to other regions of gp120 has not been completely addressed, and the exact localizations of the polysaccharide binding sites are not known. To investigate in more detail the structural basis of the HS-gp120 interaction, we used a mapping strategy and compared the heparin binding activity of wild type and mutant gp120 using surface plasmon resonance-based binding assays. Four heparin binding domains (1-4) were identified in the V2 and V3 loops, in the C-terminal domain, and within the CD4-induced bridging sheet. Interestingly, three of them were found in domains of the protein that undergo structural changes upon binding to CD4 and are involved in co-receptor recognition. In particular, Arg(419), Lys(421), and Lys(432), which directly interact with the co-receptor, are targeted by heparin. This study provides a complete account of the gp120 residues involved in heparin binding and identified several binding surfaces that constitute potential target for viral entry inhibition.     

Biophysical characterization and membrane interaction of the most membranotropic region of the HIV-1 gp41 endodomain

The membrane fusion protein of HIV-1 is the envelope transmembrane gp41 glycoprotein, which is the responsible of the membrane fusion between the virus and the target cell. Gp41 has an unusual cytoplasmic tail, the endodomain, containing highly helicoidal segments with large hydrophobic moments, the so called lentivirus lytic peptides or LLPs. According to our previous work, one of the most membranotropic regions along the whole gp41 glycoprotein was located in the LLP3 region of the gp41. In order to get new insights into the viral membrane fusion mechanism, a peptide pertaining to the LLP3 domain has been studied by infrared, fluorescence and calorimetry regarding its structure, its ability to induce membrane rupture and aggregation, as well as its affinity towards specific phospholipids. Our results demonstrate that this peptide interacts with phospholipid-containing model membranes, affects the phase-behavior of membrane phospholipids and induces leakage and aggregation of liposomes. The membrane-perturbing properties of LLP3, together with the possibility that the Kennedy sequence could be part of an external loop, open the possibility that these domains might function in modulating viral membrane fusion or budding, synergistically with other membranotropic regions of the gp41 glycoprotein.