Structure and Conformational Properties of the HIVThailand gp120 Immunodominant Epitope

NMR data and the previously developed theoretical method were used to determine the three-dimensional structure of the immunodominant epitope (IDE) of the HIV_Thailand protein gp120. The best energy IDE conformers consistent with the theoretical and experimental data were calculated, and their ensemble was shown to give rise to the main chain folds found earlier in examining the HIV_MN IDE structure. The gp120 IDE is supposed to behave as a metastable oligopeptide that, depending on the microenvironment, largely assumes one of the conformations from the ensemble. The results are discussed in the light of literature data on HIV-1 IDE structure.     

Study on conformational homology of the HIV-1 gp120 protein V3 loop. Structural analysis of the HIV-RF and HIV-thailand viral strains

Based on NMR spectroscopy data, conformation of the HIV-RF gp120 protein V3 loop giving rise to the virus principal neutralizing determinant and also determinants of cell tropism and syncytium formation was calculated by computer modeling approaches. Elements of the HIV-RF V3 loop secondary structure and conformational states of its irregular stretches were determined. The calculated structure was compared with the conformation of the homologous stretch of the HIV-Thailand protein gp120 V3 loop, and structural elements preserved in the two viral strains were identified. Conservative elements of the HIV-1 V3 loop structure are considered to be promising targets for deriving chemically modified forms of this loop with the enhanced immunogenicity and cross-reactivity of neutralizing antibodies and also for creation of effective antiviral drugs on this base.     

Structural analysis of the epitope of the anti-HIV antibody 2F5 sheds light into its mechanism of neutralization and HIV fusion

Inhibition of human immunodeficiency virus (HIV) fusion with the host cell has emerged as a viable therapeutic strategy, and rational design of inhibitors and vaccines, interfering with this process, is a prime target for antiviral research. To advance our knowledge of the structural biology of HIV fusion, we have studied the membrane-proximal region of the fusogenic envelope subunit gp41, which includes the epitope ELDKWA of the broadly neutralizing human antibody 2F5. The structural evidence available for this region is contradictory, with some studies suggesting an overall helical conformation, while the X-ray structure of the ELDKWAS peptide bound to the antibody shows it folded in a type I beta turn. We used a two-step strategy: Firstly, by a competition binding assay, we identified the proper boundaries of the domain recognized by 2F5, which we found considerably larger than the ELDKWAS hexapeptide. Secondly, we studied the structure of the resulting 13 amino acid residue peptide by collecting NMR data and analyzing them by our previously developed statistical method (NAMFIS). Our study revealed that the increase in binding affinity goes in parallel with stabilization of specific local and global conformational propensities, absent from the shorter epitope. When compounded with the available biological evidence, our structural analysis allows us to propose a specific role for the membrane-proximal region during HIV fusion, in terms of a conformational transition between the turn and the helical structure. At the same time, our hypothesis offers a structural explanation for the mechanism of neutralization of mAb 2F5.     

Determination of structurally conservative amino acids of the HIV-1 protein gp120 V3 loop as promising targets for drug design by protein engineering approaches

Based on the published NMR spectroscopy data, three-dimensional structures of the HIV-1 gp120 protein V3 loop were obtained by computer modeling in the viral strains HIV-Haiti and HIV-MN. In both cases, the secondary structure elements and conformations of irregular stretches were determined for the fragment representing the principal antigenic determinant of the virus, as well as determinants of the cellular tropism and syncytium formation. Notwithstanding the high variability of the amino acid sequence of gp120 protein, more than 50% of the V3 loop residues retained their conformations in the different HIV-1 virions. The combined analysis of the findings and the literature data on the biological activity of the individual residues of the HIV-1 V3 loop resulted in identification of its structurally conservative amino acids, which seem to be promising targets for antiviral drug design by protein engineering approaches.     

Surface expression of an immunodominant malaria protein B cell epitope by yellow fever virus.

The yellow fever 17D virus (YF17D) has several characteristics that are desirable for the development of new, live attenuated vaccines. We approached its development as a vector for heterologous antigens by studying the expression of a humoral epitope at the surface of the E protein based on the results of modelling its three-dimensional structure. This model indicated that the most promising insertion site is between beta-strands f and g, a site that is exposed at the external surface of the virus. The large deletion of six residues from the fg loop of the E protein from yellow fever virus, compared to tick-born encephalitis virus, leaves space at the dimer interface for a large insertion without creating steric hindrance. We have tested this hypothesis by inserting a model humoral epitope from the circumsporozoite protein of Plasmodium falciparum consisting of triple NANP repeats. Recombinant virus (17D/8) expressing this insertion flanked by two glycine residues at each end, is specifically neutralized by a monoclonal antibody to the model epitope. Furthermore, mouse antibodies raised to the recombinant virus recognize the parasite protein in an ELISA assay. Serial passage analysis confirmed the genetic stability of the insertion made in the viral genome and the resulting 17D/8 virus is significantly more attenuated in mouse neurovirulence tests than the 17DD vaccine. The fg loop belongs to the dimerization domain of the E protein and lies at the interface between monomers. This domain undergoes a low pH transition, which is related to the fusion of the viral envelope to the endosome membrane. It is conceivable that a slower rate of fusion, resulting from the insertion close to the dimer interface, may delay the onset of virus production and thereby lead to a milder infection of the host. This would account for the more attenuated phenotype of the recombinant virus in the mouse model and lower extent of replication in cultured cells. The vectorial capacity of the yellow fever virus is being further explored for the expression and presentation of other epitopes, including those mediating T-cell responses.     

Structural requirements for and consequences of an antiviral porphyrin binding to the V3 loop of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp120

Several porphyrin derivatives were reported to have anti-HIV-1 activity. Among them, meso-teta(4-carboxyphenyl)porphine (MYCPP) and other carboxyphenyl derivatives were the most potent inhibitors (EC₅₀ < 0.7 μM). MTCPP bound to the HIV-1 enveloope glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to HIV-1 envelop glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to regions on gp120 which cannot be mimicked by peptides. Further characterization of the binding domain for MTCPP is important for understanding the antiviral activity of porphyrins and for the design of anit-HIV-1 drugs interfering with functions of the virus envelope. Results presented here show that: (i) deletion of the V3 loop from the gp120 sequence resulted in drastically diminished MTCPP binding, suggesting that the V3 loop is the dominant if not the only target site on gp120; (ii) this site was only partially mimicked by full-length V3 loop peptides; (iii) MTCPP binding to the gp120 V3 loop elicited allosteric effects resulting in decreased accessibility of the CD4 receptor binding site; (iv) the binding site for MTCPP lies within the central portion of the V3 loop (KSIHIGPGRAFY for the HIV-1 subtype B consensus sequence) and does not involve directly the GPG apex of the loop. These results may help in designing antiviral compounds with improved activity.     

Oxidative stress and protein oxidation in the brain of water drinking and alcohol drinking rats administered the HIV envelope protein, gp120

Possible roles of oxidative stress and protein oxidation on alcohol-induced augmentation of cerebral neuropathy in gp120 administered alcohol preferring rats drinking either pure water (W rats) or a free-choice ethanol and water (E rats) for 90 days. This study showed that peripherally administered gp120 accumulated into the brain, liver, and RBCs samples from water drinking – gp120 administered rats (Wg rats) and ethanol drinking – gp120 administered rats (Eg rats), although gp120 levels in samples from Eg rats were significantly greater than the levels in samples from Wg rats. The brain samples from ethanol drinking-saline administered (EC) and Wg rats exhibited comparable levels of free radicals that were significantly lower than the levels in Eg rats. Peroxiredoxin-I (PrxI) activity in the brain samples exhibited the following pattern: Wg ≫ ≫ WC ≫ EC > Eg. Total protein-carbonyl and carbonylated hippocampal cholinergic neurostimulating peptide precursor protein levels, but not N -acetylaspartate or N -acetyl aspartylglutamate or total protein-thiol levels, paralleled the free radical levels in the brain of all four groups. This suggests PrxI inhibition may be more sensitive indicator of oxidative stress than measuring free radicals or metabolites. As PrxI oxidation in WC, Wg, and EC rats was reversible, while PrxI oxidation in Eg rats was not, we suggest that alcohol drinking and gp120 together hyperoxidized and inactivated PrxI that suppressed free radical neutralization in the brain of Eg rats. In conclusion, chronic alcohol drinking, by carbonylating and hyperoxidizing free radical neutralization proteins, augmented the gp120-induced oxidative stress that may be associated with an increase in severity of the brain neuropathy.     

Amyloid-beta-anti-amyloid-beta complex structure reveals an extended conformation in the immunodominant B-cell epitope

Alzheimer's disease (AD) is the most common form of dementia. Amyloid-β (Aβ) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on Aβ, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-Aβ antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the Aβ peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the Aβ peptide. The structures reveal the molecular basis for WO2 recognition and binding of Aβ. The Aβ peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound Aβ peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of Aβ, such as WO2, hold promise for therapeutic development.     

NMR study and comparison of the antigenic properties of a peptide recognized by two HIV-1 neutralizing antibodies

Fab–peptide complexes formed between a 15 residue peptide derived from the HIV-1 gp120 V3 loop and two of its cognate monoclonal antibodies, 5023A and 5025A, were studied using isotope-edited solution nuclear magnetic resonance (NMR) techniques. Since these antibodies neutralize HIV-1 virus with different strain specificities, this study was conducted to better understand the nature of these differences. The amide proton and nitrogen NMR resonances of specific residues were used to monitor the backbone of this peptide in these complexes. Three central residues of this peptide (‘RAF’) were found to be strongly affected by binding to both antibodies. Several other peptide residues were affected by binding to antibody 5023A but not 5025A. The antibody epitopes mapped by NMR are similar to those obtained previously via PEPSCAN at higher pH. One main difference between the PEPSCAN and NMR determined epitopes for 5023A involved two glycine residues of the peptide. By NMR, one of these glycines was more dramatically affected by antibody binding than predicted by PEPSCAN, while the other was much less so. © 1998 John Wiley & Sons, Ltd.     

Activation of gp120 of human immunodeficiency virus by their V3 loop-derived peptides

V3 loop peptides from three different human immunodeficiency virus type 1 (HIV-1) strains were synthesized. BH10, ADA, and 89.6 strains whose infections are dependent on CXCR4, CCR5, and both, respectively, were selected. Co-transfection of luciferase reporter gene and corresponding envelope genes (HXB2, ADA, and 89.6) generate pseudotype viruses (HXB2/Luc, ADA/Luc, and 89.6/Luc). The effects of each peptide on the infection of U87 cells expressing CD4 and one of the coreceptors with all pseudotype viruses were evaluated. V3 loop peptide from BH10 (V3-BH10) alone increased the HXB2/Luc infection by 93% at 10 microM. Both V3-ADA and V3-89.6 enhanced ADA/Luc infection by 38% and by 55% at 10 microM, respectively. For 89.6/Luc infection, only V3-89.6 enhanced the infections on both target cells. V3-BH10 modulated the epitopes of coreceptor binding site and V2 loop of gp120 on HIV-1 IIIB infected H9 cells, indicating that V3 loop peptide activates viral gp120 and enhances infectivity.     

人类免疫缺陷病毒1型gp41结构与功能的研究进展

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

Epistasis among Deleterious Mutations in the HIV-1 Protease

A central goal in molecular evolution is to understand how genetic interactions between protein mutations shape protein function and fitness. While intergenic epistasis has been extensively explored in eukaryotes, bacteria, and viruses, intragenic epistatic interactions have been insufficiently studied. Here, we employ a model system in which lambda phage fitness correlates with the enzymatic activity of human immunodeficiency virus type 1 (HIV-1) protease to systematically determine the epistatic interactions between intragenic pairs of deleterious protein substitutions. We generated 114 genotypes of the HIV-1 protease, each carrying pairs of nucleotide substitution mutations whose separated and combined deleterious effects on fitness were then determined. A high proportion (39%) of pairs displayed lethality. Several pairs exhibited significant interactions for fitness, including positive and negative epistasis. Significant negative epistatic interactions predominated (15%) over positive interactions (2%). However, the average ± SD epistatic effect, ē = 0.0025 ± 0.1334, was not significantly different from zero (p = 0.8368). Notably, epistatic interactions, regardless of epistatic direction, tend to be more frequent in the context of less deleterious mutations. In the present study, the high frequencies of lethality and negative epistasis indicate that the HIV-1 protease is highly sensitive to the effects of deleterious mutations. Therefore, proteins may not be as robust to mutational change as is usually expected.     

Core oligosaccharide structure from the highly phytopathogenic Agrobacterium tumefaciens TT111 and conformational analysis of the putative rhamnan epitope

The structure of the complex mixture of the core oligosaccharide components of the lipooligosaccharide (LOS) fraction of Agrobacterium tumefaciens strain TT111 was determined directly on the deacetylated products by means of spectroscopical methods. The rhamnan oligosaccharide elongating the inner Kdo residue shares structural features with other polysaccharides from well-known plant pathogenic bacteria. Its conformation was determined through extensive molecular dynamic (MD) analysis and presents an epitope similar to that recognized from the plant defense system.     

Localization of the HIV-1 gp120 Conformational Epitope Recognized by Virus-Neutralizing Monoclonal Antibodies 2G12

A phage peptide library was used to select peptides interacting with virus-neutralizing monoclonal antibodies (mAb) 2G12 which recognize a discontinuous surface epitope of HIV-1 gp120. With the published X-ray data, gp120 regions involved in the antigenic determinant were predicted. Binding with mAb 2G12 was ascribed to Thr297, Phe383, Tyr384, Arg419, Ile240, Thr415, Leu416, Pro417, Lys421, and Trp112. Though distant in the gp120 sequence, these residues are close in space and form the 2G12 epitope on the gp120 surface.     

Immunoreactivity and conformation of the immunodominant domain of HTLV-I envelope surface glycoprotein

Summary The envelope of the human retrovirus HTLV-I (human T-cell leukemia virus type I), like those of other retroviruses, plays an important role in viral infection. One of the major immunodominant domains of HTLV-I surface glycoprotein (gp46), inducing antibody reactions in over 90% of infected individuals, is bounded by amino acids 175 and 199. As compared to HTLV-I prototype strain MT-2, few amino acid substitutions have been described in this region; the most frequently observed is the replacement of a proline by a serine at position 192. In order to investigate the antigenic impact of this variation, we analysed the reactivity of synthetic peptides, harbouring either a proline or a serine residue, towards antibody containing HTLV-I positive sera in enzyme linked immunosorbent assays. The possible influence of this amino acid substitution on the conformational behaviour has been examined by studying the solution structure of two model peptides (corresponding to the 175–199 region) using two-dimensional¹H NMR spectroscopy. The results of this work should allow us to find out whether this amino acid substitution has to be taken into account for the design of a future peptide-based vaccine against HTLV-I infection.     

Immunoreactivity and conformation of the immunodominant domain of HTLV-I envelope surface glycoprotein

The envelope of the human retrovirus HTLV-I (humanT-cell leukemia virus type I), like those of otherretroviruses, plays an important role in viralinfection. One of the major immunodominant domains ofHTLV-I surface glycoprotein (gp46), inducing antibodyreactions in over 90% of infected individuals, isbounded by amino acids 175 and 199. As compared toHTLV-I prototype strain MT-2, few amino acidsubstitutions have been described in this region; themost frequently observed is the replacement of aproline by a serine at position 192. In order toinvestigate the antigenic impact of this variation, weanalysed the reactivity of synthetic peptides,harbouring either a proline or a serine residue,towards antibody containing HTLV-I positive sera inenzyme linked immunosorbent assays. The possibleinfluence of this amino acid substitution on theconformational behaviour has been examined by studyingthe solution structure of two model peptides(corresponding to the 175–199 region) usingtwo-dimensional ¹H NMR spectroscopy. The resultsof this work should allow us to find out whether thisamino acid substitution has to be taken into accountfor the design of a future peptide-based vaccineagainst HTLV-I infection.