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.     

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.     

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

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

HIV-1 envelope accessible surface and polarity: clade, blood, and brain

The human immunodeficiency virus type-1 (HIV-1) gp160 (gp120-gp41 complex) trimer envelope (ENV) protein is a potential vaccine candidate for HIV/AIDS. HIV-1 vaccine development has been problematic and charge polarity as well as sequence variation across clades may relate to the difficulties. Further obstacles are caused by sequence variation between blood and brain-derived sequences, since the brain is a separate compartment for HIV-1 infection. We utilize a threedimensional residue measure of solvent exposure, accessible surface area (ASA), which shows that major segments of gp120 and gp41 known structures are solvent exposed across clades. We demonstrate a large percent sequence polarity for solvent exposed residues in gp120 and gp41. The range of sequence polarity varies across clades, blood, and brain from different geographical locations. Regression analysis shows that blood and brain gp120 and gp41 percent sequence polarity range correlate with mean Shannon entropy. These results point to the use of protein modifications to enhance HIV-1 ENV vaccines across multiple clades, blood, and brain. It should be noted that we do not address the issue of protein glycosylation here; however, this is an important issue for vaccine design and development. ABBREVIATIONS: HIV-1 - human immunodeficiency virus type 1, AIDS - acquired immunodeficiency syndrome, ENV - envelope, gp160 - 160,000d glycoprotein, gp120 - 120,000d glycoprotein, gp41 - 41,000d glycoprotein, LANL - Los Alamos National Laboratories, PDB - Protein Data Bank, HVTN - STEP HIV vaccine trial, AA - amino acids, MSA - multiple sequence alignment, ASA - accessible surface area, SNPs- single nucleotide polymorphisms, HAART - Highly Active Antiretroviral Therapy, CCR5 - C-C chemokine receptor type 5, CNS - central nervous system, HIVE - HIV encephalitis, P - polarity, NP - non-polarity, CTL - cytotoxic T lymphocyte, NIAID - National Institute of Allergy and Infectious Diseases.     

Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines

Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.     

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.     

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.     

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.     

Permanent inhibition of viral entry by covalent entrapment of HIV gp41 on the virus surface

HIV entry occurs by concerted conformational changes in the envelope protein complex on the surface of the virus. This complex is made up of a trimer of heterodimers of two subunits: surface subunit, gp120, and transmembrane subunit, gp41. Conformational changes in the envelope complex allow gp41 to mediate membrane fusion leading to exposure of two gp41 regions: N-heptad repeat (NHR) and C-heptad repeat (CHR). Peptides from the NHR or the CHR have been found to inhibit HIV entry. Herein we show that we can covalently inhibit HIV viral entry by permanently trapping the gp41 intermediate on the virus surface using a covalently reactive group on inhibitory peptides. This is evidence showing that vulnerable conformational intermediates exist transiently during HIV viral entry, and the details presented herein will facilitate development of envelope as a target for therapeutics and potential chemopreventive agents that could disable the virus before contact with the host cell.     

The helical hairpin structure of the influenza fusion peptide can be seen on a hydrophobic moment map

An assignment of the helical hairpin of the influenza fusion peptide has been made based on the hydrophobic moments, represented in a form of two-dimensional map. Such assignment holds for all serotypes, even for the cases of mutations altering the amino acid character. Similar results are obtained for the experimentally developed hydrophobicity scales, whose values reflect the transfer energies between aqueous and membrane environments. A distinct, however still structure-related hydrophobic map corresponds to a helical and contiguous HIV gp41 fp. The method may be used as a simple tool for sequence-based prediction of structures adopted by viral fusion peptides.     

Membrane interactions of synthetic peptides corresponding to amphipathic helical segments of the human immunodeficiency virus type-1 envelope glycoprotein.

The human and simian immunodeficiency virus envelope glycoproteins, which mediate virus-induced cell fusion, contain two putative amphipathic helical segments with large helical hydrophobic moments near their carboxyl-terminal ends. In an attempt to elucidate the biological role of these amphipathic helical segments, we have synthesized peptides corresponding to residues 768-788 and 826-854 of HIV-1/WMJ-22 gp160. Circular dichroism studies of the peptides showed that the alpha helicity of the peptides increased with the addition of dimyristoyl phosphatidylcholine (DMPC) indicating that the peptides form lipid-associating amphipathic helixes. The peptides solubilized turbid suspensions of DMPC vesicles, and electron microscopy of peptide-DMPC mixtures revealed the formation of discoidal complexes, suggesting that the peptides bind to and perturb lipid bilayers. The peptides were found to lyse lipid vesicles and caused carboxyfluorescein leakage from dye-entrapped egg phosphatidylcholine liposomes. The peptides also lysed human erythrocytes and were found to be toxic to cell cultures. At subtoxic concentrations, the peptides effectively inhibited the fusion of CD4+ cells infected with recombinant vaccinia virus expressing human immunodeficiency virus (HIV)-1 envelope proteins. Based on these results, and reported studies on the mutational analysis of HIV envelope proteins, we suggest that the amphipathic helical segments near the carboxyl terminus of HIV envelope proteins may play a role in lysis of HIV-infected cells and also may modulate the extent of cell fusion observed during HIV infection of CD4+ cells.     

Structural and functional roles of HIV-1 gp41 pretransmembrane sequence segmentation

The membrane-proximal segment connecting the helical core with the transmembrane anchor of human immunodeficiency virus type 1 gp41 is accessible to broadly neutralizing antibodies and plays a crucial role in fusion activity. New predictive approaches including computation of interfacial affinity and the corresponding hydrophobic moments suggest that this region is functionally segmented into two consecutive subdomains: one amphipathic at the N-terminal side and one fully interfacial at the C-terminus. The N-terminal subdomain would extend alpha-helices from the preceding carboxy-terminal heptad repeat and provide, at the same time, a hydrophobic-at-interface surface. Experiments were performed to compare a wild-type representing pretransmembrane peptide with a nonamphipathic defective sequence, which otherwise conserved interfacial hydrophobicity at the carboxy-subdomain. Results confirmed that both penetrated equally well into lipid monolayers and both were able to partition into membrane interfaces. However only the functional sequence: 1), adopted helical structures in solution and in membranes; 2), formed homo-oligomers in solution and membranes; and 3), inhibited gp41-induced cell-cell fusion. These data support two roles for gp41 aromatic-rich pretransmembrane sequence: 1), oligomerization of gp41; and 2), immersion into the viral membrane interface. Accessibility to membrane interfaces and subsequent adoption of the low-energy structure may augment helical bundle formation and perhaps be related to a concomitant loss of immunoreactivity. These results may have implications in the development of HIV-1 fusion inhibitors and vaccines.     

The hydrophobic moment and its use in the classification of amphiphilic structures (review)

Amphiphilic alpha-helices play a major role in membrane dependent processes and are manifested in the primary structure of a protein by the periodic appearance of hydrophobic residues. Based on these periodic sequences, the hydrophobic moment was introduced, , which essentially treats the hydrophobicity of amino acid residues as a two-dimensional vector sum and provides a measure of amphiphilicity within regular repeat structures. To identify putative amphiphilic alpha-helix forming sequences, hydrophobic moment analysis assumes an amino acid residue periodicity of 100 and scans protein primary structures to find the 11-residue window with maximal . Taken with the window's mean hydrophobicity, , hydrophobic moment plot analysis uses the coordinate pair, [, ] to classify alpha-helices as either surface active, globular or transmembrane. More recently, this latter analysis has been extended to recognize candidate oblique orientated alpha-helices. Here, the hydrophobic moment is reviewed and data to query the logic of using a fixed window length and a fixed residue angular periodicity in hydrophobic moment analysis are provided. In addition, problems associated with the use of such analysis to predict alpha-helix structure/function relationships are considered.     

Influence of alpha-helices on the emulsifying properties of proteins

A peptide derived from apomyoglobin by cyanogen bromide cleavage was found to be an active emulsifier. This molecule, peptide 1-55, has two potential amphipathic alpha-helices and a hydrophilic C-terminal domain. The importance of each of these domains to the emulsifying properties of this molecule was investigated by testing the products of gene constructs based on the sequence of peptide 1-55, but lacking one of the three domains. The emulsifying activity of the peptides lacking either of the alpha-helices was correlated with the hydrophobic moments of their respective helices. The hydrophobic moment is a measure of the amphipathicity of alpha-helices; a hydrophobic moment analysis of other emulsifying peptides supports the hypothesis that a high hydrophobic moment contributes to good emulsifying properties in a molecule which contains alpha-helices.     

Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus

The envelope protein of human immunodeficiency virus (HIV) is synthesized as a polyprotein (gp160) and cleaved intracellularly to a gp120-gp41 heterodimer. In this study, the tryptic-like endoproteolytic cleavage site was removed by site-directed mutagenesis and replaced with a chymotryptic-like site. The resultant mutant, RIP7/mut10, was found to be indistinguishable from wild-type HIV when analyzed at the level of proviral replication, RNA processing, protein expression, and viral assembly. However, the gp160 polyprotein was not cleaved and the mutated virions were biologically inactive, until and unless they were exposed to limiting concentrations of chymotrypsin. As is the case for other enveloped mammalian viruses, endoproteolytic cleavage of the HIV envelope protein and release of a unique hydrophobic domain appear to be necessary for the full expression of viral infectivity.     

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV.

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.     

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

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