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Sample records for glycoprotein gp41 contributes

  1. Residues in the gp41 Ectodomain Regulate HIV-1 Envelope Glycoprotein Conformational Transitions Induced by gp120-Directed Inhibitors.

    PubMed

    Pacheco, Beatriz; Alsahafi, Nirmin; Debbeche, Olfa; Prévost, Jérémie; Ding, Shilei; Chapleau, Jean-Philippe; Herschhorn, Alon; Madani, Navid; Princiotto, Amy; Melillo, Bruno; Gu, Christopher; Zeng, Xin; Mao, Youdong; Smith, Amos B; Sodroski, Joseph; Finzi, Andrés

    2017-03-01

    Interactions between the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer maintain the metastable unliganded form of the viral spike. Binding of gp120 to the receptor, CD4, changes the Env conformation to promote gp120 interaction with the second receptor, CCR5 or CXCR4. CD4 binding also induces the transformation of Env into the prehairpin intermediate, in which the gp41 heptad repeat 1 (HR1) coiled coil is assembled at the trimer axis. In nature, HIV-1 Envs must balance the requirements to maintain the noncovalent association of gp120 with gp41 and to evade the host antibody response with the need to respond to CD4 binding. Here we show that the gp41 HR1 region contributes to gp120 association with the unliganded Env trimer. Changes in particular amino acid residues in the gp41 HR1 region decreased the efficiency with which Env moved from the unliganded state. Thus, these gp41 changes decreased the sensitivity of HIV-1 to cold inactivation and ligands that require Env conformational changes to bind efficiently. Conversely, these gp41 changes increased HIV-1 sensitivity to small-molecule entry inhibitors that block Env conformational changes induced by CD4. Changes in particular gp41 HR1 amino acid residues can apparently affect the relative stability of the unliganded state and CD4-induced conformations. Thus, the gp41 HR1 region contributes to the association with gp120 and regulates Env transitions from the unliganded state to downstream conformations.IMPORTANCE The development of an efficient vaccine able to prevent HIV infection is a worldwide priority. Knowledge of the envelope glycoprotein structure and the conformational changes that occur after receptor engagement will help researchers to develop an immunogen able to elicit antibodies that block HIV-1 transmission. Here we identify residues in the HIV-1 transmembrane envelope glycoprotein that stabilize the unliganded state by modulating the

  2. The cholesterol-binding motif of the HIV-1 glycoprotein gp41 regulates lateral sorting and oligomerization.

    PubMed

    Schwarzer, Roland; Levental, Ilya; Gramatica, Andrea; Scolari, Silvia; Buschmann, Volker; Veit, Michael; Herrmann, Andreas

    2014-10-01

    Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO-K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO-K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell-to-cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes.

  3. Feline immunodeficiency virus envelope glycoprotein mediates apoptosis in activated PBMC by a mechanism dependent on gp41 function

    SciTech Connect

    Garg, Himanshu; Joshi, Anjali; Tompkins, Wayne A. . E-mail: Wayne_Tompkins@ncsu.edu

    2004-12-20

    Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function.

  4. Role for the Terminal Clasp of HIV-1 gp41 Glycoprotein in the Initiation of Membrane Fusion*

    PubMed Central

    Lay, Chan-Sien; Ludlow, Louise E.; Stapleton, David; Bellamy-McIntyre, Anna K.; Ramsland, Paul A.; Drummer, Heidi E.; Poumbourios, Pantelis

    2011-01-01

    The binding by HIV-1 gp120 to CD4 and a chemokine receptor activates the membrane fusion glycoprotein, gp41. The fusion function of gp41 involves the refolding of its core into a 6-helix bundle, which apposes the lipophilic termini (the fusion peptide and transmembrane domain) and the associated cell and viral membranes, leading to their fusion. In this study, we examined the functional role of the polar segment and membrane proximal external region (MPER), which link the fusion peptide and transmembrane domain, respectively, to the core domain and interact to form a terminal clasp adjacent to the core. Limited proteolysis indicated that the terminal clasp is destabilized by simultaneous I535A/V539G mutations within the polar segment and mutations within the MPER. The destabilizing effects of I535A/V539G correlated with defective cell-cell fusion, viral entry, and viral replication. By using lipophilic and cytoplasmic fluorescent dye transfer assays, we found that terminal clasp destabilization is linked to a block in the lipid mixing/hemifusion phase of the membrane fusion cascade. Because the biosynthesis of the prefusion gp120-gp41 complex did not appear to be affected by I535A/V539G, we infer that the hemifusion block is due to a specific effect on the trimer of hairpins conformation of gp41. By contrast, the decreased fusion function of the MPER mutants correlated with a decrease in the interfacial hydropathy of the MPER sequence, suggesting that the prefusion Env complex had been adversely affected in these cases. These findings reveal a novel conserved functional target for the discovery of fusion inhibitors. PMID:21976663

  5. N-terminal substitutions in HIV-1 gp41 reduce the expression of non-trimeric envelope glycoproteins on the virus

    SciTech Connect

    Dey, Antu K.; David, Kathryn B.; Ray, Neelanjana; Ketas, Thomas J.; Klasse, Per J.; Doms, Robert W.; Moore, John P.

    2008-03-01

    The native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120-gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified five amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Env-transfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B.

  6. Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein.

    PubMed

    Vassell, Russell; He, Yong; Vennakalanti, Prasad; Dey, Antu K; Zhuang, Min; Wang, Wei; Sun, Yide; Biron-Sorek, Zohar; Srivastava, Indresh K; LaBranche, Celia C; Montefiori, David C; Barnett, Susan W; Weiss, Carol D

    2015-01-01

    The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.

  7. An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

    SciTech Connect

    Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2005-07-20

    The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate.

  8. Resistance to neutralization by broadly reactive antibodies to the human immunodeficiency virus type 1 gp120 glycoprotein conferred by a gp41 amino acid change.

    PubMed Central

    Thali, M; Charles, M; Furman, C; Cavacini, L; Posner, M; Robinson, J; Sodroski, J

    1994-01-01

    A neutralization-resistant variant of human immunodeficiency virus type 1 (HIV-1) that emerged during in vitro propagation of the virus in the presence of neutralizing serum from an infected individual has been described. A threonine-for-alanine substitution at position 582 in the gp41 transmembrane envelope glycoprotein of the variant virus was responsible for the neutralization-resistant phenotype (M.S. Reitz, Jr., C. Wilson, C. Naugle, R. C. Gallo, and M. Robert-Guroff, Cell 54:57-63, 1988). The mutant virus also exhibited reduced sensitivity to neutralization by 30% of HIV-1-positive sera that neutralized the parental virus, suggesting that a significant fraction of the neutralizing activity within these sera can be affected by the amino acid change in gp41 (C. Wilson, M. S. Reitz, Jr., K. Aldrich, P. J. Klasse, J. Blomberg, R. C. Gallo, and M. Robert-Guroff, J. Virol. 64:3240-3248, 1990). It is shown here that the change of alanine 582 to threonine specifically confers resistance to neutralizing by antibodies directed against both groups of discontinuous, conserved epitopes related to the CD4 binding site on the gp120 exterior envelope glycoprotein. Only minor differences in binding of these antibodies to wild-type and mutant envelope glycoproteins were observed. Thus, the antigenic structure of gp120 can be subtly affected by an amino acid change in gp41, with important consequences for sensitivity to neutralization. Images PMID:7507184

  9. Tryptophan dendrimers that inhibit HIV replication, prevent virus entry and bind to the HIV envelope glycoproteins gp120 and gp41.

    PubMed

    Rivero-Buceta, Eva; Doyagüez, Elisa G; Colomer, Ignacio; Quesada, Ernesto; Mathys, Leen; Noppen, Sam; Liekens, Sandra; Camarasa, María-José; Pérez-Pérez, María-Jesús; Balzarini, Jan; San-Félix, Ana

    2015-12-01

    Dendrimers containing from 9 to 18 tryptophan residues at the peryphery have been efficiently synthesized and tested against HIV replication. These compounds inhibit an early step of the replicative cycle of HIV, presumably virus entry into its target cell. Our data suggest that HIV inhibition can be achieved by the preferred interaction of the compounds herein described with glycoproteins gp120 and gp41 of the HIV envelope preventing interaction between HIV and the (co)receptors present on the host cells. The results obtained so far indicate that 9 tryptophan residues on the periphery are sufficient for efficient gp120/gp41 binding and anti-HIV activity. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  10. Effects of the I559P gp41 change on the conformation and function of the human immunodeficiency virus (HIV-1) membrane envelope glycoprotein trimer.

    PubMed

    Alsahafi, Nirmin; Debbeche, Olfa; Sodroski, Joseph; Finzi, Andrés

    2015-01-01

    The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P) in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env spikes and I559P

  11. Development of HIV-1 fusion inhibitors targeting gp41.

    PubMed

    Lu, K; Asyifah, M R; Shao, F; Zhang, D

    2014-06-01

    The HIV-1 envelope protein glycoprotein 41 (gp41) is crucial in the HIV-1 infection process, therefore gp41 has emerged as an attractive target for drug design against AIDS. During the past few decades, tremendous efforts have been made on developing inhibitors that can prevent the HIV-1 entry process via suppressing functional gp41. In this review, the development of HIV-1 fusion inhibitors targeting gp41 including peptide inhibitors, small molecule inhibitors, vaccines and neutralized antibodies will be discussed.

  12. Structural and functional properties of the membranotropic HIV-1 glycoprotein gp41 loop region are modulated by its intrinsic hydrophobic core.

    PubMed

    Qiu, Jiayin; Ashkenazi, Avraham; Liu, Shuwen; Shai, Yechiel

    2013-10-04

    The gp41 disulfide loop region switches from a soluble state to a membrane-bound state during the human immunodeficiency virus type 1 (HIV-1) envelope-mediated membrane fusion process. The loop possesses a hydrophobic core at the center of the region with an unusual basic residue (Lys-601). Furthermore, two loop core mutations, K601A and L602A, are found to inhibit HIV-1 infectivity while keeping wild type-like levels of the envelope, implying that they exert an inhibitory effect on gp41 during the membrane fusion event. Here, we investigated the mode of action of these mutations on the loop region. We show that the K601A mutation, but not the L602A mutation, abolished the binding of a loop-specific monoclonal antibody to a loop domain peptide. Additionally, the K601A, but not the L602A, impaired disulfide bond formation in the peptides. This was correlated with changes in the circular dichroism spectrum imposed by the K601A mutation. In the membrane, however, the L602A, but not the K601A, reduced the lipid mixing ability of the loop peptides, which was correlated with decreased α-helical content of the L602A mutant. The results suggest that the Lys-601 residue provides a moderate hydrophobicity level within the gp41 loop core that contributes to the proper structure and function of the loop inside and outside the membrane. Because basic residues are found between the loop Cys residues of several lentiviral fusion proteins, the findings may contribute to understanding the fusion mechanism of other viruses as well.

  13. Structural and Functional Properties of the Membranotropic HIV-1 Glycoprotein gp41 Loop Region Are Modulated by Its Intrinsic Hydrophobic Core*

    PubMed Central

    Qiu, Jiayin; Ashkenazi, Avraham; Liu, Shuwen; Shai, Yechiel

    2013-01-01

    The gp41 disulfide loop region switches from a soluble state to a membrane-bound state during the human immunodeficiency virus type 1 (HIV-1) envelope-mediated membrane fusion process. The loop possesses a hydrophobic core at the center of the region with an unusual basic residue (Lys-601). Furthermore, two loop core mutations, K601A and L602A, are found to inhibit HIV-1 infectivity while keeping wild type-like levels of the envelope, implying that they exert an inhibitory effect on gp41 during the membrane fusion event. Here, we investigated the mode of action of these mutations on the loop region. We show that the K601A mutation, but not the L602A mutation, abolished the binding of a loop-specific monoclonal antibody to a loop domain peptide. Additionally, the K601A, but not the L602A, impaired disulfide bond formation in the peptides. This was correlated with changes in the circular dichroism spectrum imposed by the K601A mutation. In the membrane, however, the L602A, but not the K601A, reduced the lipid mixing ability of the loop peptides, which was correlated with decreased α-helical content of the L602A mutant. The results suggest that the Lys-601 residue provides a moderate hydrophobicity level within the gp41 loop core that contributes to the proper structure and function of the loop inside and outside the membrane. Because basic residues are found between the loop Cys residues of several lentiviral fusion proteins, the findings may contribute to understanding the fusion mechanism of other viruses as well. PMID:23960077

  14. The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

    SciTech Connect

    Hollier, Mark J.; Dimmock, Nigel J. . E-mail: n.j.dimmock@warwick.ac.uk

    2005-07-05

    In addition to the major ectodomain, the gp41 transmembrane glycoprotein of HIV-1 is now known to have a minor ectodomain that is part of the long C-terminal tail. Both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. However, data have so far been biologically based, and derived solely from T cell line-adapted (TCLA), B clade viruses. Here we have carried out sequence and theoretically based structural analyses of 357 gp41 C-terminal sequences of mainly primary isolates of HIV-1 clades A, B, C, and D. Data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, {beta}-sheet, membrane-spanning domains (MSDs). This means that the first (N-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. However, we suggest that only a minority of cell-associated gp41 molecules - those destined for incorporation into virions - has 3 MSDs and the minor ectodomain. Most intracellular gp41 has the conventional single MSD, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. The gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize HIV-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell.

  15. Design and evaluation of antiretroviral peptides corresponding to the C-terminal heptad repeat region (C-HR) of human immunodeficiency virus type 1 envelope glycoprotein gp41

    SciTech Connect

    Soonthornsata, Bongkot; Tian, Yu-Shi; Utachee, Piraporn; Sapsutthipas, Sompong; Isarangkura-na-ayuthaya, Panasda; Auwanit, Wattana; Takagi, Tatsuya; Ikuta, Kazuyoshi; Sawanpanyalert, Pathom; Kawashita, Norihito; Kameoka, Masanori

    2010-09-15

    Two {alpha}-helical heptad repeats, N-HR and C-HR, located in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41, play an important role in membrane fusion by forming a 6-helix bundle. C34, a peptide mimicking C-HR, inhibits the formation of the 6-helix bundle; thus, it has potential as a novel antiretroviral compound. In order to improve the inhibitory effect of C34 on HIV-1 replication, we designed new C34-derived peptides based on computational analysis of the stable conformation of the 6-helix bundle. Newly designed peptides showed a stronger inhibitory effect on the replication of recombinant viruses containing CRF01{sub A}E, subtype B or subtype C Env than C34 or a fusion inhibitor, T-20. In addition, these peptides inhibited the replication of a T-20-resistant virus. We propose that these peptides could be applied to develop novel antiretroviral compounds to inhibit the replication of various subtypes of HIV-1 as well as of T-20-resistant variants.

  16. The Role of HIV-1 gp41 Glycoprotein in Infectious Tropism Inferred from Physico-Chemical Properties of its Amino Acid Sequence

    NASA Astrophysics Data System (ADS)

    Figueroa, E.; Villarreal, C.; Huerta, L.; Cocho, G.

    2006-09-01

    We performed a statistical analysis of the amino acid sequence of the gp41 ectodomain of the Human Immunodeficiency Virus type 1. We found strong correlations between physicochemical properties of highly variable residues and the viral infectious tropism.

  17. Characterization of Humoral Immune Responses against Capsid Protein p24 and Transmembrane Glycoprotein gp41 of Human Immunodeficiency Virus Type 1 in China.

    PubMed

    Li, Xiufen; Wu, Yue; Ren, Xuqi; Deng, Shuyun; Hu, Guifang; Yu, Shouyi; Tang, Shixing

    2016-01-01

    The objective of this study was to extend our previous research and to further characterize the humoral immune responses against HIV-1 p24, gp41 and the specific peptides carrying the immunodominant epitopes (IDEs) that react with human serum samples from HIV-1-infected individuals in China. We found that the majority (90.45%, 180/199) of the samples did not react with any of the three HIV-1 p24 peptides carrying IDEs, but did react with the recombinant full-length p24, suggesting that these samples tested in China were primarily directed against the conformational epitopes of HIV-1 p24. In contrast, 84.54% (164/194) of the samples reacted with at least one HIV-1 linear gp41 peptide, in particular the gp41-p1 peptide (amino acids 560-616). Both recently and long-term HIV-1-infected individuals displayed similar humoral immune responses against the recombinant gp41. However, samples from long-term HIV-1-infected subjects but not from recently infected subjects, showed a very strong reaction against the gp41-p1 peptide. The different response patterns observed for the two groups against the gp41 and the peptide gp41-p1 were statistically significant (P<0.01, Chi-square test). These results have direct relevance and importance for design of improved HIV-1 p24 detection assays and the gp41- based immunoassay that can be used to reliably distinguish recent and long-term HIV-1 infection.

  18. Designing a Soluble Near Full-Length HIV-1 GP41 Trimer

    DTIC Science & Technology

    2012-11-26

    envelope; gp41 trimer; bacteriophage T4 display; prehairpin fusion intermediate. Background: The envelope glycoprotein gp41 is a key component of...protein into trimers and defined oligomers. These gp41 trimers were displayed on bacteriophage T4 capsid nanoparticles by attaching to the small...gp41 Trimers on the Bacteriophage T4 Nanoparticle—Eight hundred and seventy copies of a small outer capsid protein, Soc (9 kDa), decorate the surface

  19. Expression in yeast of a cDNA clone encoding a transmembrane glycoprotein gp41 fragment (a.a. 591-642) bearing the major immunodominant domain of human immunodeficiency virus.

    PubMed

    Gairin, J E; Madaule, P; Traincard, F; Barrès, E; Rossier, J

    1991-04-01

    A cDNA clone corresponding to the gp41 gene fragment nucl. 7573-7730 of the human immunodeficiency virus type 1 (HIV-1) was selected from a random HIV-1 genomic library expressed in yeast. This clone encodes a 52-residue long peptide (amino acid (a.a.)) 591-642) bearing the major immunodominant domain (a.a. 598-609) of the HIV-1 transmembrane glycoprotein gp41. Expression of the recombinant peptide pSE-env591-642 was driven by the alpha-mating factor leader sequence contained in a plasmid pSE-x allowing the synthesis and secretion of foreign gene product in Saccharomyces cerevisiae. Time-course analysis of the secretion into culture medium revealed an optimal production of the glycoprotein fragment at 28-30 h with no observable cytotoxicity. The secreted peptide is highly glycosylated with NH2-terminal heterogeneity probably due to different post-translational modifications. The secreted peptide shows an extreme antigenicity since in ELISA assays, as few as 5 microliters/well of crude supernatant are sufficient to obtain a strong detection by monoclonal antibodies or by 100% of sera from HIV-infected individuals. The purified glycopeptide pSE-env591-642 binds to a monoclonal antibody directed against the immunodominant epitope (a.a. 603-609) with an affinity similar to that of the complete glycoprotein gp160 (Kd values within the 10(-10) M range) and with a 100-fold higher affinity than that of a linear peptide fragment SP-env584-609. These results indicate that overexpression in yeast can efficiently provide an abundant source of highly antigenic gp41 protein fragment pSE-env591-642 which retains the antigenic properties of the native gp160 protein. Such a recombinant peptide should therefore be considered as a good candidate for antigen in HIV detection tests.

  20. The convertases furin and PC1 can both cleave the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp160 into gp120 (HIV-1 SU) and gp41 (HIV-I TM).

    PubMed

    Decroly, E; Vandenbranden, M; Ruysschaert, J M; Cogniaux, J; Jacob, G S; Howard, S C; Marshall, G; Kompelli, A; Basak, A; Jean, F

    1994-04-22

    Intracellular proteolytic processing of human immunodeficiency virus envelope glycoprotein precursor (gp160) is an essential step for virus infectivity. Northern blot analysis provided evidence that furin and PC1, but not PC2, are expressed in the CD4+ human lymphoblastoid H9 cell line, suggesting the possible participation of these convertases in human immunodeficiency virus (HIV) gp160 proteolytic processing. Purified PC1 and furin cleaved specifically in vitro gp160 into gp120 (HIV-I SU) and gp41 (HIV-I TM). NH2-terminal sequence analysis of the produced gp41 (HIV-I TM) demonstrated that the cleavage occurred within the sequence Arg-Glu-Lys-Arg decreases Ala-Val-Gly-Ile, which is identical to the bond cleaved in vivo. Transition state analog peptides were designed and tested in vitro for their ability to inhibit the PC1- or furin-mediated gp160 cleavage. The best inhibitor was decanoyl-Arg-Lys-Arg-Arg-psi [CH2NH]-Phe-Leu-Gly-Phe-NH2.

  1. Sequences in Glycoprotein gp41, the CD4 Binding Site, and the V2 Domain Regulate Sensitivity and Resistance of HIV-1 to Broadly Neutralizing Antibodies

    PubMed Central

    O'Rourke, Sara M.; Schweighardt, Becky; Phung, Pham; Mesa, Kathryn A.; Vollrath, Aaron L.; Tatsuno, Gwen P.; To, Briana; Sinangil, Faruk; Limoli, Kay; Wrin, Terri

    2012-01-01

    The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals. PMID:22933284

  2. Designing a soluble near full-length HIV-1 gp41 trimer.

    PubMed

    Gao, Guofen; Wieczorek, Lindsay; Peachman, Kristina K; Polonis, Victoria R; Alving, Carl R; Rao, Mangala; Rao, Venigalla B

    2013-01-04

    The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors, whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been extensively studied, the detailed mechanism is still poorly understood. Design of gp41 recombinants that mimic key intermediates is essential to elucidate the mechanism as well as to develop potent therapeutics and vaccines. Here, using molecular genetics and biochemical approaches, a series of hypotheses was tested to overcome the extreme hydrophobicity of HIV-1 gp41 and design a soluble near full-length gp41 trimer. The two long heptad repeat helices HR1 and HR2 of gp41 ectodomain were mutated to disrupt intramolecular HR1-HR2 interactions but not intermolecular HR1-HR1 interactions. This resulted in reduced aggregation and improved solubility. Attachment of a 27-amino acid foldon at the C terminus and slow refolding channeled gp41 into trimers. The trimers appear to be stabilized in a prehairpin-like structure, as evident from binding of a HR2 peptide to exposed HR1 grooves, lack of binding to hexa-helical bundle-specific NC-1 mAb, and inhibition of virus neutralization by broadly neutralizing antibodies 2F5 and 4E10. Fusion to T4 small outer capsid protein, Soc, allowed display of gp41 trimers on the phage nanoparticle. These approaches for the first time led to the design of a soluble gp41 trimer containing both the fusion peptide and the cytoplasmic domain, providing insights into the mechanism of entry and development of gp41-based HIV-1 vaccines.

  3. Inhibition of HIV-1 gp41 expression with hammerhead ribozymes.

    PubMed

    Fedoruk-Wyszomirska, Agnieszka; Szymański, Maciej; Głodowicz, Paweł; Gabryelska, Marta; Wyszko, Eliza; Estrin, William J; Barciszewski, Jan

    2015-10-01

    Despite great progress in the treatment of AIDS, HIV-1 remains one of the major concerns as a human pathogen. One of the therapeutic strategies against viral infections is the application of catalytic ribonucleic acids (ribozymes) that can significantly reduce expression of a target gene by site-specific hydrolysis of its mRNA. In the present paper, we report a study on the activity of several variants of hammerhead ribozymes targeting a conserved region within mRNA encoding HIV-1 envelope glycoprotein gp41. On the basis of the data from in vitro assays and gene silencing in the cultured cells, we propose a new hammerhead ribozyme targeting the gp41-encoding sequence that can be potentially used as a therapeutic agent in AIDS treatment. Moreover, we demonstrate that the hydrolytic activity of the ribozyme in the intracellular environment cannot be inferred solely from the results of in vitro experiments.

  4. A fusion intermediate gp41 immunogen elicits neutralizing antibodies to HIV-1.

    PubMed

    Lai, Rachel P J; Hock, Miriam; Radzimanowski, Jens; Tonks, Paul; Hulsik, David Lutje; Effantin, Gregory; Seilly, David J; Dreja, Hanna; Kliche, Alexander; Wagner, Ralf; Barnett, Susan W; Tumba, Nancy; Morris, Lynn; LaBranche, Celia C; Montefiori, David C; Seaman, Michael S; Heeney, Jonathan L; Weissenhorn, Winfried

    2014-10-24

    The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41(int)-Cys) and show that it folds into an elongated ∼ 12-nm-long extended structure based on small angle x-ray scattering data. Gp41(int)-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41(int)-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140(CA018) in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140(CA018) was higher than that induced by gp41(int)-Cys, the majority of animals immunized with gp41(int)-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols.

  5. Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope

    PubMed Central

    Penn-Nicholson, Adam; Han, Dong P.; Kim, Soon J.; Park, Hanna; Ansari, Rais; Montefiori, David C.; Cho, Michael W.

    2008-01-01

    Human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein gp41 is targeted by broadly-reactive neutralizing antibodies 2F5 and 4E10, making it an attractive target for vaccine development. To better assess immunogenic properties of gp41, we generated five soluble glutathione S-transferase fusion proteins encompassing C-terminal 30, 64, 100, 142, or 172 (full-length) amino acids of gp41 ectodomain from M group consensus envelope sequence. Antibody responses in HIV-1-infected patients were evaluated using these proteins and overlapping peptides. We found (i) antibody responses against different regions of gp41 varied tremendously among individual patients, (ii) patients with stronger antibody responses against membrane-proximal external region exhibit broader and more potent neutralizing activity, and (iii) several patients mounted antibodies against epitopes that are near, or overlap with, those targeted by 2F5 or 4E10. These soluble gp41 fusion proteins could be an important source of antigens for future vaccine development efforts. PMID:18068750

  6. Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope

    SciTech Connect

    Penn-Nicholson, Adam; Han, Dong P.; Kim, Soon J.; Park, Hanna; Ansari, Rais; Montefiori, David C.; Cho, Michael W.

    2008-03-15

    Human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein gp41 is targeted by broadly-reactive neutralizing antibodies 2F5 and 4E10, making it an attractive target for vaccine development. To better assess immunogenic properties of gp41, we generated five soluble glutathione S-transferase fusion proteins encompassing C-terminal 30, 64, 100, 142, or 172 (full-length) amino acids of gp41 ectodomain from M group consensus envelope sequence. Antibody responses in HIV-1-infected patients were evaluated using these proteins and overlapping peptides. We found (i) antibody responses against different regions of gp41 varied tremendously among individual patients, (ii) patients with stronger antibody responses against membrane-proximal external region exhibit broader and more potent neutralizing activity, and (iii) several patients mounted antibodies against epitopes that are near, or overlap with, those targeted by 2F5 or 4E10. These soluble gp41 fusion proteins could be an important source of antigens for future vaccine development efforts.

  7. Receptor Activation of HIV-1 Env Leads to Asymmetric Exposure of the gp41 Trimer

    PubMed Central

    2016-01-01

    Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in a nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI. PMID:27992602

  8. Receptor Activation of HIV-1 Env Leads to Asymmetric Exposure of the gp41 Trimer.

    PubMed

    Khasnis, Mukta D; Halkidis, Konstantine; Bhardwaj, Anshul; Root, Michael J

    2016-12-01

    Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in a nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI.

  9. Role for the disulfide-bonded region of human immunodeficiency virus type 1 gp41 in receptor-triggered activation of membrane fusion function

    SciTech Connect

    Bellamy-McIntyre, Anna K.; Baer, Severine; Ludlow, Louise; Drummer, Heidi E.; Poumbourios, Pantelis

    2010-04-16

    The conserved disulfide-bonded region (DSR) of the human immunodeficiency virus type 1 (HIV-1) fusion glycoprotein, gp41, mediates association with the receptor-binding glycoprotein, gp120. Interactions between gp120, CD4 and chemokine receptors activate the fusion activity of gp41. The introduction of W596L and W610F mutations to the DSR of HIV-1{sub QH1549.13} blocked viral entry and hemifusion without affecting gp120-gp41 association. The fusion defect correlated with inhibition of CD4-triggered gp41 pre-hairpin formation, consistent with the DSR mutations having decoupled receptor-induced conformational changes in gp120 from gp41 activation. Our data implicate the DSR in sensing conformational changes in the gp120-gp41 complex that lead to fusion activation.

  10. Molecular basis for epitope recognition by non-neutralizing anti-gp41 antibody F240

    PubMed Central

    Gohain, Neelakshi; Tolbert, William D.; Orlandi, Chiara; Richard, Jonathan; Ding, Shilei; Chen, Xishan; Bonsor, Daniel A.; Sundberg, Eric J.; Lu, Wuyuan; Ray, Krishanu; Finzi, Andrés; Lewis, George K.; Pazgier, Marzena

    2016-01-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) by non-neutralizing antibodies (nnAbs) specific to the HIV envelope (Env) glycoproteins present at the surface of virus sensitized or infected cells plays a role in the effective adaptive immune response to HIV. Here, we explore the molecular basis for the epitope at the disulfide loop region (DLR) of the principal immunodominant domain of gp41, recognized by the well-known nnAb F240. Our structural studies reveal details of the F240-gp41 interface and describe a structure of DLR that is distinct from known conformations of this region studied in the context of either CD4-unliganded Env trimer or the gp41 peptide in the unbound state. These data coupled with binding and functional analyses indicate that F240 recognizes non-trimeric Env forms which are significantly overexpressed on intact virions but poorly represented at surfaces of cells infected with infectious molecular clones and endogenously-infected CD4 T cells from HIV-1-infected individuals. Furthermore, although we detect ADCC activities of F240 against cells spinoculated with intact virions, our data suggest that these activities result from F240 recognition of gp41 stumps or misfolded Env variants present on virions rather than its ability to recognize functional gp41 transition structures emerging on trimeric Env post CD4 receptor engagement. PMID:27827447

  11. Molecular basis for epitope recognition by non-neutralizing anti-gp41 antibody F240.

    PubMed

    Gohain, Neelakshi; Tolbert, William D; Orlandi, Chiara; Richard, Jonathan; Ding, Shilei; Chen, Xishan; Bonsor, Daniel A; Sundberg, Eric J; Lu, Wuyuan; Ray, Krishanu; Finzi, Andrés; Lewis, George K; Pazgier, Marzena

    2016-11-09

    Antibody-dependent cell-mediated cytotoxicity (ADCC) by non-neutralizing antibodies (nnAbs) specific to the HIV envelope (Env) glycoproteins present at the surface of virus sensitized or infected cells plays a role in the effective adaptive immune response to HIV. Here, we explore the molecular basis for the epitope at the disulfide loop region (DLR) of the principal immunodominant domain of gp41, recognized by the well-known nnAb F240. Our structural studies reveal details of the F240-gp41 interface and describe a structure of DLR that is distinct from known conformations of this region studied in the context of either CD4-unliganded Env trimer or the gp41 peptide in the unbound state. These data coupled with binding and functional analyses indicate that F240 recognizes non-trimeric Env forms which are significantly overexpressed on intact virions but poorly represented at surfaces of cells infected with infectious molecular clones and endogenously-infected CD4 T cells from HIV-1-infected individuals. Furthermore, although we detect ADCC activities of F240 against cells spinoculated with intact virions, our data suggest that these activities result from F240 recognition of gp41 stumps or misfolded Env variants present on virions rather than its ability to recognize functional gp41 transition structures emerging on trimeric Env post CD4 receptor engagement.

  12. HIV-1 gp41 fusion intermediate: a target for HIV therapeutics.

    PubMed

    Pan, Chungen; Liu, Shuwen; Jiang, Shibo

    2010-02-01

    Human immunodeficiency virus (HIV)-1 infection is initiated by the binding of gp120 envelope glyco-protein to its cell receptor (CD4) and a coreceptor (CXCR4 or CCR5), followed by a series of conformational changes in the gp41 transmembrane subunit. These changes include insertion of fusion peptide into the target cell membrane and association of C-heptad repeat (CHR) peptide with the N-heptad repeat (NHR) trimer, a pre-hairpin fusion intermediate. A stable six-helix bundle core is then formed, bringing the viral envelope and target cell membrane into close proximity for fusion. Peptides derived from the CHR region, such as T20 and C34, inhibit HIV-1 fusion by interacting with the gp41 fusion intermediate. A number of anti-HIV-1 peptides and small molecule compounds targeting the gp41 NHR-trimer have been identified. By combining HIV fusion/entry inhibitors targeting different sites in the gp41 fusion intermediate, a potent synergistic effect takes place, resulting in a potential new therapeutic strategy for the HIV infection/AIDS. Here, we present an overview of the current development of anti-HIV drugs, particularly those targeting the gp41 fusion intermediate.

  13. Role of a Putative gp41 Dimerization Domain in Human Immunodeficiency Virus Type 1 Membrane Fusion

    SciTech Connect

    Liu, J.; Deng, Y; Li, Q; Dey, A; Moore, J; Lu, M

    2010-01-01

    The entry of human immunodeficiency virus type 1 (HIV-1) into a target cell entails a series of conformational changes in the gp41 transmembrane glycoprotein that mediates the fusion of the viral and target cell membranes. A trimer-of-hairpins structure formed by the association of two heptad repeat (HR) regions of the gp41 ectodomain has been implicated in a late step of the fusion pathway. Earlier native and intermediate states of the protein are postulated to mediate the antiviral activity of the fusion inhibitor enfuvirtide and of broadly neutralizing monoclonal antibodies (NAbs), but the details of these structures remain unknown. Here, we report the identification and crystal structure of a dimerization domain in the C-terminal ectodomain of gp41 (residues 630 to 683, or C54). Two C54 monomers associate to form an asymmetric, antiparallel coiled coil with two distinct C-terminal {alpha}-helical overhangs. This dimer structure is conferred largely by interactions within a central core that corresponds to the sequence of enfuvirtide. The mutagenic alteration of the dimer interface severely impairs the infectivity of Env-pseudotyped viruses. Moreover, the C54 structure binds tightly to both the 2F5 and 4E10 NAbs and likely represents a potential intermediate conformation of gp41. These results should enhance our understanding of the molecular basis of the gp41 fusogenic structural transitions and thereby guide rational, structure-based efforts to design new fusion inhibitors and vaccine candidates intended to induce broadly neutralizing antibodies.

  14. Reorientation of the helix of the tryptophan-rich gp41W peptide from HIV-1 at interfaces

    NASA Astrophysics Data System (ADS)

    Matar, Gladys; Benichou, Emmanuel; Nasir, Mehmet Nail; Harfouch, Yara El; Brevet, Pierre-François; Besson, Françoise

    2013-12-01

    The glycoprotein gp41 from the Human Immunodeficiency Virus type 1 (HIV-1) has an amino acid sequence enriched in tryptophan residues, the so-called gp41W peptide (i.e., KWASLWNWFNITNWLWYIK) and plays a crucial role in HIV-1 host cell infection. Using the coupling of Second Harmonic Generation targeting the tryptophan residues with lateral surface tension measurements, we investigate the interaction of gp41W with a neat air/water and a lipid/water interfaces. At the air/water interface, gp41W presents a well-defined orientation and this orientation is strongly modified at the lipid/water interface, depending on the surface pressure. These results show that this strategy is well suited to monitor tryptophan containing α-helices orientation at lipid/water interfaces.

  15. Kinetically coupled folding of a single HIV-1 glycoprotein 41 complex in viral membrane fusion and inhibition.

    PubMed

    Jiao, Junyi; Rebane, Aleksander A; Ma, Lu; Gao, Ying; Zhang, Yongli

    2015-06-02

    HIV-1 glycoprotein 41 (gp41) mediates viral entry into host cells by coupling its folding energy to membrane fusion. Gp41 folding is blocked by fusion inhibitors, including the commercial drug T20, to treat HIV/AIDS. However, gp41 folding intermediates, energy, and kinetics are poorly understood. Here, we identified the folding intermediates of a single gp41 trimer-of-hairpins and measured their associated energy and kinetics using high-resolution optical tweezers. We found that folding of gp41 hairpins was energetically independent but kinetically coupled: Each hairpin contributed a folding energy of ∼-23 kBT, but folding of one hairpin successively accelerated the folding rate of the next one by ∼20-fold. Membrane-mimicking micelles slowed down gp41 folding and reduced the stability of the six-helix bundle. However, the stability was restored by cooperative folding of the membrane-proximal external region. Surprisingly, T20 strongly inhibited gp41 folding by actively displacing the C-terminal hairpin strand in a force-dependent manner. The inhibition was abolished by a T20-resistant gp41 mutation. The energetics and kinetics of gp41 folding established by us provides a basis to understand viral membrane fusion, infection, and therapeutic intervention.

  16. Statistical correlation of nonconservative substitutions of HIV gp41 variable amino acid residues with the R5X4 HIV-1 phenotype.

    PubMed

    Pacheco-Martínez, Elena; Figueroa-Medina, Evangelina; Villarreal, Carlos; Cocho, Germinal; Medina-Franco, José L; Méndez-Lucio, Oscar; Huerta, Leonor

    2016-02-16

    The interaction of the envelope glycoprotein of HIV-1 (gp120/gp41) with coreceptor molecules has important implications for specific cellular targeting and pathogenesis. Experimental and theoretical evidences have shown a role for gp41 in coreceptor tropism, although there is no consensus about the positions involved. Here we analyze the association of physicochemical properties of gp41 amino acid residues with viral tropism (X4, R5, and R5X4) using a large set of HIV-1 sequences. Under the assumption that conserved regions define the complex structural features essential for protein function, we focused our search only on amino acids in the gp41 variable regions. Gp41 amino acid sequences of 2823 HIV-1 strains from all clades with known coreceptor tropism were retrieved from Los Alamos HIV Database. Consensus sequences were constructed for homologous sequences (those obtained from the same patient and having the same tropism) in order to avoid bias due to sequence overrepresentation, and the variability (entropy) per site was determined. Comparisons of hydropathy index (HI) and charge (Q) of amino acid residues at highly variable positions between coreceptor groups were performed using two non-parametrical tests and Benjamini-Hochberg correction. Pearson's correlation analysis was performed to determine covariance of HI and Q values. Calculation of variability per site rendered 58 highly variable amino acid positions. Of these, statistical analysis rendered significantly different HI or Q only for the R5 vs. R5X4 comparison at twelve positions: 535, 602, 619, 636, 640, 641, 658, 662, 667, 723, 756 and 841. The largest differences in particular amino acid frequencies between coreceptor groups were found at 619, 636, 640, 641, 662, 723 and 756. A hydrophobic tendency of residues 619, 640, 641, 723 and 756, along with a hydrophilic/charged tendency at residues 636 and 662 was observed in R5X4 with respect to R5 sequences. HI of position 640 covariated with that of

  17. Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41.

    PubMed

    Lu, Lu; Yu, Fei; Cai, Lifeng; Debnath, Asim K; Jiang, Shibo

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein surface subunit gp120 and transmembrane subunit gp41 play important roles in HIV-1 entry, thus serving as key targets for the development of HIV-1 entry inhibitors. T20 peptide (enfuvirtide) is the first U.S. FDA-approved HIV entry inhibitor; however, its clinical application is limited by the lack of oral availability. Here, we have described the structure and function of the HIV-1 gp120 and gp41 subunits and reviewed advancements in the development of small-molecule HIV entry inhibitors specifically targeting these two Env glycoproteins. We then compared the advantages and disadvantages of different categories of HIV entry inhibitor candidates and further predicted the future trend of HIV entry inhibitor development.

  18. Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41

    PubMed Central

    Lu, Lu; Yu, Fei; Cai, Lifeng; Debnath, Asim K.; Jiang, Shibo

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein surface subunit gp120 and transmembrane subunit gp41 play important roles in HIV-1 entry, thus serving as key targets for the development of HIV-1 entry inhibitors. T20 peptide (enfuvirtide) is the first U.S. FDA-approved HIV entry inhibitor; however, its clinical application is limited by the lack of oral availability. Here, we have described the structure and function of the HIV-1 gp120 and gp41 subunits and reviewed advancements in the development of small-molecule HIV entry inhibitors specifically targeting these two Env glycoproteins. We then compared the advantages and disadvantages of different categories of HIV entry inhibitor candidates and further predicted the future trend of HIV entry inhibitor development. PMID:26324044

  19. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike.

    PubMed

    Lee, Jeong Hyun; Leaman, Daniel P; Kim, Arthur S; Torrents de la Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R; Nussenzweig, Michel C; Poignard, Pascal; Moore, John P; Klasse, Per Johan; Sanders, Rogier W; Zwick, Michael B; Wilson, Ian A; Ward, Andrew B

    2015-09-25

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120-gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120-gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes.

  20. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

    PubMed Central

    Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de la Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

    2015-01-01

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120–gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120–gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes. PMID:26404402

  1. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

    NASA Astrophysics Data System (ADS)

    Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de La Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

    2015-09-01

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120-gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120-gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes.

  2. Immunologic NO synthase: elevation in severe AIDS dementia and induction by HIV-1 gp41.

    PubMed

    Adamson, D C; Wildemann, B; Sasaki, M; Glass, J D; McArthur, J C; Christov, V I; Dawson, T M; Dawson, V L

    1996-12-13

    Indirect mechanisms are implicated in the pathogenesis of the dementia associated with human immunodeficiency virus-type 1 (HIV-1) infection. Proinflammatory molecules such as tumor necrosis factor alpha and eicosanoids are elevated in the central nervous system of patients with HIV-1-related dementia. Nitric oxide (NO) is a potential mediator of neuronal injury, because cytokines may activate the immunologic (type II) isoform of NO synthase (iNOS). The levels of iNOS in severe HIV-1-associated dementia coincided with increased expression of the HIV-1 coat protein gp41. Furthermore, gp41 induced iNOS in primary cultures of mixed rat neuronal and glial cells and killed neurons through a NO-dependent mechanism. Thus, gp41-induced NO formation may contribute to the severe cognitive dysfunction associated with HIV-1 infection.

  3. HIV-1 envelope protein gp41: an NMR study of dodecyl phosphocholine embedded gp41 reveals a dynamic prefusion intermediate conformation.

    PubMed

    Lakomek, Nils-Alexander; Kaufman, Joshua D; Stahl, Stephen J; Wingfield, Paul T

    2014-09-02

    Human immunodeficiency viral (HIV-1) fusion is mediated by the viral envelope gp120/gp41 complex (ENVelope glycoprotein). After gp120 shedding, gp41 is exposed and elicits membrane fusion via a cascade of conformational changes. In contrast to prefusion and postfusion conformation, little is known about any intermediate conformation. We report on a solution NMR investigation of homotrimeric HIV-1 gp41(27-194), comprising the transmembrane region and reconstituted in dodecyl phosphocholine (DPC) micelles. The protein is mainly α-helical, but experiences internal dynamics on the nanosecond and micro to millisecond time scale and transient α-helical behavior for certain residues in the N-terminal heptad repeat (NHR). Strong lipid interactions are observed, in particular for C-terminal residues of the NHR and imunodominant loop region connecting NHR and C-terminal heptad repeat (CHR). Our data indicate an extended conformation with features anticipated for a prefusion intermediate, presumably in exchange with a lowly populated postfusion six-helical bundle conformation.

  4. Role of a Putative gp41 Dimerization Domain in Human Immunodeficiency Virus Type 1 Membrane Fusion▿

    PubMed Central

    Liu, Jie; Deng, Yiqun; Li, Qunnu; Dey, Antu K.; Moore, John P.; Lu, Min

    2010-01-01

    The entry of human immunodeficiency virus type 1 (HIV-1) into a target cell entails a series of conformational changes in the gp41 transmembrane glycoprotein that mediates the fusion of the viral and target cell membranes. A trimer-of-hairpins structure formed by the association of two heptad repeat (HR) regions of the gp41 ectodomain has been implicated in a late step of the fusion pathway. Earlier native and intermediate states of the protein are postulated to mediate the antiviral activity of the fusion inhibitor enfuvirtide and of broadly neutralizing monoclonal antibodies (NAbs), but the details of these structures remain unknown. Here, we report the identification and crystal structure of a dimerization domain in the C-terminal ectodomain of gp41 (residues 630 to 683, or C54). Two C54 monomers associate to form an asymmetric, antiparallel coiled coil with two distinct C-terminal α-helical overhangs. This dimer structure is conferred largely by interactions within a central core that corresponds to the sequence of enfuvirtide. The mutagenic alteration of the dimer interface severely impairs the infectivity of Env-pseudotyped viruses. Moreover, the C54 structure binds tightly to both the 2F5 and 4E10 NAbs and likely represents a potential intermediate conformation of gp41. These results should enhance our understanding of the molecular basis of the gp41 fusogenic structural transitions and thereby guide rational, structure-based efforts to design new fusion inhibitors and vaccine candidates intended to induce broadly neutralizing antibodies. PMID:19846514

  5. Structural Characterization of HIV gp41 with the Membrane-proximal External Region

    SciTech Connect

    Shi, W.; Bohon, J; Han, D; Habte, H; Qin, Y; Cho, M; Chance, M

    2010-01-01

    Human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein (gp120/gp41) plays a critical role in virus infection and pathogenesis. Three of the six monoclonal antibodies considered to have broadly neutralizing activities (2F5, 4E10, and Z13e1) bind to the membrane-proximal external region (MPER) of gp41. This makes the MPER a desirable template for developing immunogens that can elicit antibodies with properties similar to these monoclonal antibodies, with a long term goal of developing antigens that could serve as novel HIV vaccines. In order to provide a structural basis for rational antigen design, an MPER construct, HR1-54Q, was generated for x-ray crystallographic and x-ray footprinting studies to provide both high resolution atomic coordinates and verification of the solution state of the antigen, respectively. The crystal structure of HR1-54Q reveals a trimeric, coiled-coil six-helical bundle, which probably represents a postfusion form of gp41. The MPER portion extends from HR2 in continuation of a slightly bent long helix and is relatively flexible. The structures observed for the 2F5 and 4E10 epitopes agree well with existing structural data, and enzyme-linked immunosorbent assays indicate that the antigen binds well to antibodies that recognize the above epitopes. Hydroxyl radical-mediated protein footprinting of the antigen in solution reveals specifically protected and accessible regions consistent with the predictions based on the trimeric structure from the crystallographic data. Overall, the HR1-54Q antigen, as characterized by crystallography and footprinting, represents a postfusion, trimeric form of HIV gp41, and its structure provides a rational basis for gp41 antigen design suitable for HIV vaccine development.

  6. A novel enzyme-linked immunosorbent assay for screening HIV-1 fusion inhibitors targeting HIV-1 Gp41 core structure.

    PubMed

    Pang, Wei; Wang, Rui-Rui; Gao, Yue-Dong; Yang, Liu-Meng; Sun, Yi; Huang, Jing-Fei; Tien, Po; Zheng, Yong-Tang

    2011-02-01

    The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein mediates the fusion of viral and host cell membranes. As the HIV-1 enters the host cells, the 2 helical regions, HR1 and HR2, in the ectodomain of gp41 can form a 6-helix bundle, which brings the viral and target cell membranes to close proximity and serves as an attractive target for developing HIV-1 fusion inhibitors. Now, there are several cell- and molecule-based assays to identify potential HIV-1 fusion inhibitors targeting gp41. However, these assays cannot be used universally because they are time-consuming, inconvenient, and expensive. In the present study, the authors expressed and purified GST-HR121 and C43-30a proteins that were derived from the HIV-1 gp41 ectodomain region. GST-HR121 has a function similar to the HR1 peptide of gp41, whereas C43-30a is an HR2-derived peptide that added 50 amino acid residues (aa) in the N-terminal of C43. Further research found they could interact with each other, and a potential HIV-1 fusion inhibitor could inhibit this interaction. On the basis of this fact, a novel, rapid, and economic enzyme-linked immunosorbent assay was established, which can be developed for high-throughput screening of HIV-1 fusion inhibitors.

  7. Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41.

    PubMed

    Gong, Zhen; Kessans, Sarah A; Song, Lusheng; Dörner, Katerina; Lee, Ho-Hsien; Meador, Lydia R; LaBaer, Joshua; Hogue, Brenda G; Mor, Tsafrir S; Fromme, Petra

    2014-11-01

    The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the post-fusion forms. Structural information on the TM domain of gp41 is scant and at low resolution. Here we describe the design, expression and purification of a protein construct that includes MPR and the transmembrane domain of gp41 (MPR-TMTEV-6His), which reacts with the broadly neutralizing antibodies 2F5 and 4E10 and thereby may represent an immunologically relevant conformation mimicking a prehairpin intermediate of gp41. The expression level of MPR-TMTEV-6His was improved by fusion to the C-terminus of Mistic protein, yielding ∼ 1 mg of pure protein per liter. The isolated MPR-TMTEV-6His protein was biophysically characterized and is a monodisperse candidate for crystallization. This work will enable further investigation into the structure of MPR-TMTEV-6His, which will be important for the structure-based design of a mucosal vaccine against HIV-1.

  8. Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion.

    PubMed Central

    Schaal, H; Klein, M; Gehrmann, P; Adams, O; Scheid, A

    1995-01-01

    An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion

  9. Targeting HIV-1 gp41 in close proximity to the membrane using antibody and other molecules.

    PubMed

    Gach, Johannes S; Leaman, Daniel P; Zwick, Michael B

    2011-12-01

    HIV-1 envelope glycoprotein (Env) spikes are supported at the viral membrane interface by a highly conserved and hydrophobic region of gp41, designated the membrane-proximal external region (MPER). The MPER is mandatory for infection of host cells by HIV-1, and is the target of some of the most broadly neutralizing antibodies described to date. As such, the MPER is also of considerable interest for HIV vaccine design. However, structural models indicate that the MPER assumes distinct conformations prior to and leading up to Env-mediated fusion. Thus, the more of these distinct conformations that antibodies and inhibitors can recognize will likely be the better for antiviral potency. In addition to its flexibility, the MPER is lipophilic and its accessibility to bulky macromolecules is limited by steric and kinetic blocks that present particular challenges for eliciting HIV-1 neutralizing antibodies. Moreover, the ability of the MPER and viral membrane to combine as a complex has critical mechanistic implications for molecules that target lipid-bound and/or unbound states. Interestingly, membrane affinity frequently appears to enhance the potency of both fusion inhibitors and antibodies to different sites on gp41. We therefore highlight mechanisms to be harnessed in targeting membraneproximal sites on HIV gp41 for both vaccine and fusion inhibitor design. Such design efforts will likely need to draw upon knowledge of MPER structure and function, and may in turn inform analogous approaches to MPERs of other enveloped viruses and systems.

  10. Mechanism of HIV-1 neutralization by antibodies targeting a membrane-proximal region of gp41.

    PubMed

    Chen, Jia; Frey, Gary; Peng, Hanqin; Rits-Volloch, Sophia; Garrity, Jetta; Seaman, Michael S; Chen, Bing

    2014-01-01

    Induction of broadly neutralizing antibodies (bNAbs) is an important goal for HIV-1 vaccine development. Two autoreactive bNAbs, 2F5 and 4E10, recognize a conserved region on the HIV-1 envelope glycoprotein gp41 adjacent to the viral membrane known as the membrane-proximal external region (MPER). They block viral infection by targeting a fusion-intermediate conformation of gp41, assisted by an additional interaction with the viral membrane. Another MPER-specific antibody, 10E8, has recently been reported to neutralize HIV-1 with potency and breadth much greater than those of 2F5 or 4E10, but it appeared not to bind phospholipids and might target the untriggered envelope spikes, raising the hope that the MPER could be harnessed for vaccine design without major immunological concerns. Here, we show by three independent approaches that 10E8 indeed binds lipid bilayers through two hydrophobic residues in its CDR H3 (third heavy-chain complementarity-determining region). Its weak affinity for membranes in general and preference for cholesterol-rich membranes may account for its great neutralization potency, as it is less likely than other MPER-specific antibodies to bind cellular membranes nonspecifically. 10E8 binds with high affinity to a construct mimicking the fusion intermediate of gp41 but fails to recognize the envelope trimers representing the untriggered conformation. Moreover, we can improve the potency of 4E10 without affecting its binding to gp41 by a modification of its lipid-interacting CDR H3. These results reveal a general mechanism of HIV-1 neutralization by MPER-specific antibodies that involves interactions with viral lipids.

  11. Identification of a human protein-derived HIV-1 fusion inhibitor targeting the gp41 fusion core structure.

    PubMed

    Chao, Lijun; Lu, Lu; Yang, Hengwen; Zhu, Yun; Li, Yuan; Wang, Qian; Yu, Xiaowen; Jiang, Shibo; Chen, Ying-Hua

    2013-01-01

    The HIV-1 envelope glycoprotein (Env) gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR) of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462-521) of the human POB1 (the partner of RalBP1), designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR) of gp41, and to the six-helix bundle (6-HB) formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.

  12. Fertilization competence of the egg-coating envelope is regulated by direct interaction of dicalcin and gp41, the Xenopus laevis ZP3.

    PubMed

    Miwa, Naofumi; Ogawa, Motoyuki; Hanaue, Mayu; Takamatsu, Ken

    2015-08-05

    Fertilization begins with species-restricted interaction of sperm and the egg-coating envelope, which includes a three-dimensional meshwork of filaments composed of glycoproteins (called ZP proteins). Growing evidence has unveiled the molecular nature of ZP proteins; however, the structural property conferring fertilization competence to the egg-coating envelope remains unknown. Here, we show the molecular mechanism that mediates direct interaction between dicalcin, a novel fertilization-suppressive ZP protein-associated protein, and gp41, a Xenopus laevis ortholog of mammalian ZP3, and subsequently demonstrate the structural basis of the envelope for fertilization competence. The interactive regions between dicalcin and gp41 comprised five and nine amino acid residues within dicalcin and twenty-three within gp41 [corrected]. Synthetic peptides corresponding to these regions dramatically affected fertilization: treatment with dicalcin- or gp41-derived peptides decreased or increased fertilization rates, respectively. Prior application of these peptides caused distinct alterations in the in vivo lectin-staining pattern of the envelope as well. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide induced the formation of a well-organized meshwork, whereas the gp41-derived peptide caused the formation of a significantly disorganized meshwork. These findings indicated that the fertilization competence of the egg-coating envelope is crucially regulated by the direct interaction between dicalcin and gp41.

  13. Fertilization competence of the egg-coating envelope is regulated by direct interaction of dicalcin and gp41, the Xenopus laevis ZP3

    PubMed Central

    Miwa, Naofumi; Ogawa, Motoyuki; Hanaue, Mayu; Takamatsu, Ken

    2015-01-01

    Fertilization begins with species-restricted interaction of sperm and the egg-coating envelope, which includes a three-dimensional meshwork of filaments composed of glycoproteins (called ZP proteins). Growing evidence has unveiled the molecular nature of ZP proteins; however, the structural property conferring fertilization competence to the egg-coating envelope remains unknown. Here, we show the molecular mechanism that mediates direct interaction between dicalcin, a novel fertilization-suppressive ZP protein-associated protein, and gp41, a Xenopus laevis ortholog of mammalian ZP3, and subsequently demonstrate the structural basis of the envelope for fertilization competence. The interactive regions between dicalcin and gp41 comprised six and nine amino acid residues within dicalcin and twenty-three within gp41. Synthetic peptides corresponding to these regions dramatically affected fertilization: treatment with dicalcin- or gp41-derived peptides decreased or increased fertilization rates, respectively. Prior application of these peptides caused distinct alterations in the in vivo lectin-staining pattern of the envelope as well. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide induced the formation of a well-organized meshwork, whereas the gp41-derived peptide caused the formation of a significantly disorganized meshwork. These findings indicated that the fertilization competence of the egg-coating envelope is crucially regulated by the direct interaction between dicalcin and gp41. PMID:26243547

  14. Conditional trimerization and lytic activity of HIV-1 gp41 variants containing the membrane-associated segments.

    PubMed

    Dai, Zhou; Tao, Yisong; Liu, Nina; Brenowitz, Michael D; Girvin, Mark E; Lai, Jonathan R

    2015-03-03

    Fusion of host and viral membranes is a critical step during infection by membrane-bound viruses. The HIV-1 glycoproteins gp120 (surface subunit) and gp41 (fusion subunit) represent the prototypic system for studying this process; in the prevailing model, the gp41 ectodomain forms a trimeric six-helix bundle that constitutes a critical intermediate and provides the energetic driving force for overcoming barriers associated with membrane fusion. However, most structural studies of gp41 variants have been performed either on ectodomain constructs lacking one or more of the membrane-associated segments (the fusion peptide, FP, the membrane-proximal external region, MPER, and the transmembrane domain, TM) or on variants consisting of these isolated segments alone without the ectodomain. Several recent reports have suggested that the HIV-1 ectodomain, as well as larger construct containing the membrane-bound segments, dissociates from a trimer to a monomer in detergent micelles. Here we compare the properties of a series of gp41 variants to delineate the roles of the ectodomain, FP, and MPER and TM, all in membrane-mimicking environments. We find that these proteins are prone to formation of a monomer in detergent micelles. In one case, we observed exclusive monomer formation at pH 4 but conditional trimerization at pH 7 even at low micromolar (∼5 μM) protein concentrations. Liposome release assays demonstrate that these gp41-related proteins have the capacity to induce content leakage but that this activity is also strongly modulated by pH with much higher activity at pH 4. Circular dichroism, nuclear magnetic resonance, and binding assays with antibodies specific to the MPER provide insight into the structural and functional roles of the FP, MPER, and TM and their effect on structure within the larger context of the fusion subunit.

  15. Antibody to gp41 MPER alters functional properties of HIV-1 Env without complete neutralization.

    PubMed

    Kim, Arthur S; Leaman, Daniel P; Zwick, Michael B

    2014-07-01

    Human antibody 10E8 targets the conserved membrane proximal external region (MPER) of envelope glycoprotein (Env) subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design.

  16. Antibody to gp41 MPER Alters Functional Properties of HIV-1 Env without Complete Neutralization

    PubMed Central

    Kim, Arthur S.; Leaman, Daniel P.; Zwick, Michael B.

    2014-01-01

    Human antibody 10E8 targets the conserved membrane proximal external region (MPER) of envelope glycoprotein (Env) subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design. PMID:25058619

  17. Modulation of cytokine release and gene expression by the immunosuppressive domain of gp41 of HIV-1.

    PubMed

    Denner, Joachim; Eschricht, Magdalena; Lauck, Michael; Semaan, Marwan; Schlaermann, Philipp; Ryu, Hyunmi; Akyüz, Levent

    2013-01-01

    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.

  18. Insights into vaccine development for acquired immune deficiency syndrome from crystal structures of human immunodeficiency virus-1 gp41 and equine infectious anemia virus gp45.

    PubMed

    Duan, Liangwei; Du, Jiansen; Liu, Xinqi

    2015-10-01

    An effective vaccine against acquired immune deficiency syndrome is still unavailable after dozens of years of striving. The glycoprotein gp41 of human immunodeficiency virus is a good candidate as potential immunogen because of its conservation and relatively low glycosylation. As a reference of human immunodeficiency virus gp41, gp45 from equine infectious anemia virus (EIAV) could be used for comparison because both wild-type and vaccine strain of EIAV have been extensively studied. From structural studies of these proteins, the conformational changes during viral invasion could be unveiled, and a more effective acquired immune deficiency syndrome vaccine immunogen might be designed based on this information.

  19. Nonneutralizing Antibodies Induced by the HIV-1 gp41 NHR Domain Gain Neutralizing Activity in the Presence of the HIV Fusion Inhibitor Enfuvirtide: a Potential Therapeutic Vaccine Strategy.

    PubMed

    Wang, Qian; Bi, Wenwen; Zhu, Xiaojie; Li, Haoyang; Qi, Qianqian; Yu, Fei; Lu, Lu; Jiang, Shibo

    2015-07-01

    A key barrier against developing preventive and therapeutic human immunodeficiency virus (HIV) vaccines is the inability of viral envelope glycoproteins to elicit broad and potent neutralizing antibodies. However, in the presence of fusion inhibitor enfuvirtide, we show that the nonneutralizing antibodies induced by the HIV type 1 (HIV-1) gp41 N-terminal heptad repeat (NHR) domain (N63) exhibit potent and broad neutralizing activity against laboratory-adapted HIV-1 strains, including the drug-resistant variants, and primary HIV-1 isolates with different subtypes, suggesting the potential of developing gp41-targeted HIV therapeutic vaccines.

  20. The Transmembrane Domain of HIV-1 gp41 Inhibits T-Cell Activation by Targeting Multiple T-Cell Receptor Complex Components through Its GxxxG Motif.

    PubMed

    Rotem, Etai; Reuven, Eliran Moshe; Klug, Yoel A; Shai, Yechiel

    2016-02-23

    To successfully infect and persist within its host, HIV-1 utilizes several immunosuppressive motifs within its gp41 envelope glycoprotein to manipulate and evade the immune system. The transmembrane domain (TMD) of gp41 downregulates T-cell receptor (TCR) signaling through a hitherto unknown mechanism. Interactions between TMDs within the membrane milieu have been shown to be typically mediated by particular amino acids, such as interactions between basic and acidic residues and dimerization motifs as GxxxG. The HIV-1 TMD exhibits both a polar arginine (Arg(696)) residue and a GxxxG motif, making them ideal candidates for mediators of TMD-TCR interaction. Using a primary T-cell activation assay and biochemical and biophysical methods, we demonstrate that the gp41 TMD directly interacts with TMDs of the TCR and the CD3 coreceptors (δ, γ, and ε) within the membrane, presumably leading to impairment of complex assembly. Additionally, we reveal that although Arg(696) does not affect TMD immunosuppression, the GxxxG motif is crucial in mediating gp41's TMD interaction with the CD3 coreceptors of the TCR. These findings suggest that compared with other gp41 immunosuppressive motifs, the gp41 TMD has multiple targets within the TCR complex, suggesting less susceptibility to evolutionary pressure and consequently being advantageous for the virus over the host immune response. Furthermore, as the GxxxG motif mediates interactions of the gp41 TMD with multiple receptors, it emerges as an attractive drug target. This multitarget inhibitory mechanism might be a strategy utilized by HIV to interfere with the function of additional host receptors.

  1. Anti-HIV-1 Activity Prediction of Novel Gp41 Inhibitors Using Structure-Based Virtual Screening and Molecular Dynamics Simulation.

    PubMed

    Sepehri, Saghi; Saghaie, Lotfollah; Fassihi, Afshin

    2017-03-01

    The fusion of viral and host cell membranes is mediated using gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. As the HIV-1 enters the host cells, the two helical regions (HR1 and HR2) in the ectodomain of gp41 form a six-helix bundle, which carries the target and viral cell membranes to close proximity. Steps of this process serve as attractive targets for developing HIV-1 fusion inhibitors. Identification of some novel HIV fusion inhibitors with the goal of blocking the formation of the six-helix bundle was accomplished by computer-aided drug design techniques. A virtual screening strategy was employed to recognize small molecules presumably able to bind the gp41 at the internal interface of the NHR helices at the core native viral six-helix. This study was carried out in two stages. In the first stage, a library of more than seven thousand compounds was collected from ZINC, PubChem and BindingDB databases and protein data bank. Key contacts of known inhibitors with gp41 binding site residues were considered as the collecting criteria. In the second stage series of filtering processes were performed on this library in subsequent steps to find the potential gp41 inhibitors. The filtering criteria included pharmacokinetic and ADMET properties as well as in silico anti-HIV-1 prediction. Molecular docking simulation was carried out to identify interactions of the filtered molecules with the key residues in the gp41 binding site. Finally, molecular dynamics simulation indicates the superior inhibitory ability of three selected compounds over the known gp41inhibitor, NB-64.

  2. Membrane-Anchored Inhibitory Peptides Capture Human Immunodeficiency Virus Type 1 gp41 Conformations That Engage the Target Membrane prior to Fusion

    PubMed Central

    Melikyan, Gregory B.; Egelhofer, Marc; von Laer, Dorothee

    2006-01-01

    Soluble peptides derived from the C-terminal heptad repeat domain of human immunodeficiency virus type 1 (HIV-1) gp41 are potent inhibitors of HIV-1 entry and gp41-induced fusion. Target membrane-anchored variants of these peptides have been shown to retain inhibitory activity. Both soluble and membrane-anchored C peptides (MACs) are thought to block fusion by binding to the N-terminal coiled coil domain of gp41 and preventing formation of the final six-helix bundle structure. However, interactions of target MACs with gp41 must be restricted to a subset of trimers that have their hydrophobic fusion peptides inserted into the target membrane. This unique feature of MACs was used to identify the intermediate step of fusion at which gp41 engaged the target membrane. Fusion between HIV envelope-expressing effector cells and target cells was measured by fluorescence microscopy. Expression of MACs in target cells led to less than twofold reduction in the extent of fusion. However, when reaction was first arrested by adding lysolipids that disfavored membrane merger, and the lipids were subsequently removed by washing, control cells supported fusion, whereas those that expressed MACs did not. The drastically improved potency of MACs implies that, at lipid-arrested stage, gp41 bridges the viral and target cell membranes and therefore more optimally binds the membrane-anchored peptides. Experimental demonstration of this intermediate shows that, similar to fusion induced by many other viral glycoproteins, engaging the target membrane by HIV-1 gp41 permits coupling between six-helix bundle formation and membrane merger. PMID:16537592

  3. A novel bispecific peptide HIV-1 fusion inhibitor targeting the N-terminal heptad repeat and fusion peptide domains in gp41.

    PubMed

    Jiang, Xifeng; Jia, Qiyan; Lu, Lu; Yu, Fei; Zheng, Jishen; Shi, Weiguo; Cai, Lifeng; Jiang, Shibo; Liu, Keliang

    2016-12-01

    HIV-1 fusion with the target cell is initiated by the insertion of the gp41 fusion peptide (FP) into the target cell membrane and the interaction between the gp41 N- and C-terminal heptad repeats (NHR and CHR), followed by the formation of the six-helix bundle (6-HB) fusion core. Therefore, both FP and NHR are important targets for HIV-1 fusion inhibitors. Here, we designed and synthesized a dual-target peptidic HIV-1 fusion inhibitor, 4HR-LBD-VIRIP, in which 4HR-LBD is able to bind to the gp41 NHR domain, while VIRIP is able to interact with gp41 FP. We found that 4HR-LBD-VIRIP is about tenfold more potent than 4HR-LBD and VIRIP in inhibiting HIV-1IIIB infection and HIV-1 envelope glycoprotein (Env)-mediated cell-cell fusion, suggesting that this dual-target HIV-1 fusion inhibitor possesses a strong synergistic antiviral effect. A biophysical analysis indicates that 4HR-LBD-VIRIP can interact with N70 peptide that contains the gp41 NHR and FP domains and binds with lipid membrane. This study provides a new approach for designing novel viral fusion inhibitors against HIV and other enveloped viruses with class I membrane fusion proteins.

  4. Increased infectivity in human cells and resistance to antibody-mediated neutralization by truncation of the SIV gp41 cytoplasmic tail.

    PubMed

    Kuwata, Takeo; Kaori, Takaki; Enomoto, Ikumi; Yoshimura, Kazuhisa; Matsushita, Shuzo

    2013-01-01

    The role of antibodies in protecting the host from human immunodeficiency virus type 1 (HIV-1) infection is of considerable interest, particularly because the RV144 trial results suggest that antibodies contribute to protection. Although infection of non-human primates with simian immunodeficiency virus (SIV) is commonly used as an animal model of HIV-1 infection, the viral epitopes that elicit potent and broad neutralizing antibodies to SIV have not been identified. We isolated a monoclonal antibody (MAb) B404 that potently and broadly neutralizes various SIV strains. B404 targets a conformational epitope comprising the V3 and V4 loops of Env that intensely exposed when Env binds CD4. B404-resistant variants were obtained by passaging viruses in the presence of increasing concentration of B404 in PM1/CCR5 cells. Genetic analysis revealed that the Q733stop mutation, which truncates the cytoplasmic tail of gp41, was the first major substitution in Env during passage. The maximal inhibition by B404 and other MAbs were significantly decreased against a recombinant virus with a gp41 truncation compared with the parental SIVmac316. This indicates that the gp41 truncation was associated with resistance to antibody-mediated neutralization. The infectivities of the recombinant virus with the gp41 truncation were 7,900-, 1,000-, and 140-fold higher than those of SIVmac316 in PM1, PM1/CCR5, and TZM-bl cells, respectively. Immunoblotting analysis revealed that the gp41 truncation enhanced the incorporation of Env into virions. The effect of the gp41 truncation on infectivity was not obvious in the HSC-F macaque cell line, although the resistance of viruses harboring the gp41 truncation to neutralization was maintained. These results suggest that viruses with a truncated gp41 cytoplasmic tail were selected by increased infectivity in human cells and by acquiring resistance to neutralizing antibody.

  5. Characterization of a trimeric MPER containing HIV-1 gp41 antigen

    SciTech Connect

    Hinz, Andreas; Schoehn, Guy; Quendler, Heribert; Hulsik, David Lutje; Stiegler, Gabi; Katinger, Hermann; Seaman, Michael S.; Montefiori, David; Weissenhorn, Winfried

    2009-08-01

    The membrane-proximal external region (MPER) of gp41 is considered as a prime target for the induction of neutralizing antibodies, since it contains the epitopes for three broadly neutralizing antibodies (2F5, 4E10 and Z13). Here we present a novel gp41 construct (HA-gp41) comprising gp41 HR2 and MPER fused to two triple-stranded coiled-coil domains at both ends. HA-gp41 is trimeric, has a high helical content in solution and forms rod-like structures as revealed by negative staining electron microscopy. Immunization of rabbits with HA-gp41 induced antibodies directed against MPER, which failed to exert significant neutralization capacity against envelopes from primary isolates. Thus trimerisation of MPER regions does not suffice to induce a potent neutralizing antibody response specific for conserved regions within gp41.

  6. All-atom models of the membrane-spanning domain of HIV-1 gp41 from metadynamics.

    PubMed

    Gangupomu, Vamshi K; Abrams, Cameron F

    2010-11-17

    The 27-residue membrane-spanning domain (MSD) of the HIV-1 glycoprotein gp41 bears conserved sequence elements crucial to the biological function of the virus, in particular a conserved GXXXG motif and a midspan arginine. However, structure-based explanations for the roles of these and other MSD features remain unclear. Using molecular dynamics and metadynamics calculations of an all-atom, explicit solvent, and membrane-anchored model, we study the conformational variability of the HIV-1 gp41 MSD. We find that the MSD peptide assumes a stable tilted α-helical conformation in the membrane. However, when the side chain of the midspan Arg (694) "snorkels" to the outer leaflet of the viral membrane, the MSD assumes a metastable conformation where the highly-conserved N-terminal core (between Lys(681) and Arg(694) and containing the GXXXG motif) unfolds. In contrast, when the Arg(694) side chain snorkels to the inner leaflet, the MSD peptide assumes a metastable conformation consistent with experimental observations where the peptide kinks at Phe(697) to facilitate Arg(694) snorkeling. Both of these models suggest specific ways that gp41 may destabilize viral membrane, priming the virus for fusion with a target cell.

  7. Structure-based design of a protein immunogen that displays an HIV-1 gp41 neutralizing epitope.

    PubMed

    Stanfield, Robyn L; Julien, Jean-Philippe; Pejchal, Robert; Gach, Johannes S; Zwick, Michael B; Wilson, Ian A

    2011-12-02

    Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K(d) of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.

  8. Membrane-Proximal External HIV-1 gp41 Motif Adapted for Destabilizing the Highly Rigid Viral Envelope

    PubMed Central

    Apellániz, Beatriz; Ivankin, Andrey; Nir, Shlomo; Gidalevitz, David; Nieva, José L.

    2011-01-01

    Electron microscopy structural determinations suggest that the membrane-proximal external region (MPER) of glycoprotein 41 (gp41) may associate with the HIV-1 membrane interface. It is further proposed that MPER-induced disruption and/or deformation of the lipid bilayer ensue during viral fusion. However, it is predicted that the cholesterol content of this membrane (∼45 mol %) will act against MPER binding and restructuring activity, in agreement with alternative structural models proposing that the MPER constitutes a gp41 ectodomain component that does not insert into the viral membrane. Here, using MPER-based peptides, we test the hypothesis that cholesterol impedes the membrane association and destabilizing activities of this gp41 domain. To that end, partitioning and leakage assays carried out in lipid vesicles were combined with x-ray reflectivity and grazing-incidence diffraction studies of monolayers. CpreTM, a peptide combining the carboxyterminal MPER sequence with aminoterminal residues of the transmembrane domain, bound and destabilized effectively cholesterol-enriched membranes. Accordingly, virion incubation with this peptide inhibited cell infection potently but nonspecifically. Thus, CpreTM seems to mimic the envelope-perturbing function of the MPER domain and displays antiviral activity. As such, we infer that CpreTM bound to cholesterol-enriched membranes would represent a relevant target for anti-HIV-1 immunogen and inhibitor development. PMID:22098741

  9. Structure-Based Design of a Protein Immunogen that Displays an HIV-1 gp41 Neutralizing Epitope

    SciTech Connect

    Stanfield, Robyn L.; Julien, Jean-Philippe; Pejchal, Robert; Gach, Johannes S.; Zwick, Michael B.; Wilson, Ian A.

    2012-06-27

    Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K{sub d} of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.

  10. Stabilizing the Native Trimer of HIV-1 Env by Destabilizing the Heterodimeric Interface of the gp41 Postfusion Six-Helix Bundle

    PubMed Central

    Kesavardhana, Sannula

    2014-01-01

    ABSTRACT The HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers and is essential for viral entry. The gp41 subunit in native, prefusion trimeric Env exists in a metastable conformation and attains a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives the fusion of viral and cellular membranes. We attempted to stabilize native Env trimers by incorporation of mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The mutations V570D and I573D stabilize native Env of the HIV-1 JRFL strain and occlude nonneutralizing epitopes to a greater extent than the previously identified I559P mutation that is at the interface of the NHR trimers in the 6-HB. The mutations prevent soluble-CD4 (sCD4)-induced gp120 shedding and 6-HB formation. In the context of cell surface-expressed JRFL Env, introduction of a previously reported additional disulfide between residues A501 and T605 perturbs the native conformation, though this effect is partially alleviated by furin coexpression. The data suggest that positions 570 and 573 are surface proximal in native Env and that the NHR homotrimeric coiled coil in native Env terminates before or close to residue 573. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the postfusion 6-HB conformation. These mutations can be used to stabilize Env in a DNA vaccine format. IMPORTANCE The major protein on the surface of HIV-1 is the envelope (Env) glycoprotein. Env is a trimer of gp120-gp41 heterodimers. gp120 is involved in receptor/coreceptor binding and gp41 in the fusion of viral and cellular membranes. Like many other viral fusion proteins, the gp41 subunit in native trimeric Env exists in a metastable conformation. gp41 readily forms a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers

  11. Molecular and Physicochemical Factors Governing Solubility of the HIV gp41 Ectodomain.

    PubMed

    Manssour-Triedo, Fadia; Crespillo, Sara; Morel, Bertrand; Casares, Salvador; Mateo, Pedro L; Notka, Frank; Roger, Marie G; Mouz, Nicolas; El-Habib, Raphaelle; Conejero-Lara, Francisco

    2016-08-23

    The HIV gp41 ectodomain (e-gp41) is an attractive target for the development of vaccines and drugs against HIV because of its crucial role in viral fusion to the host cell. However, because of the high insolubility of e-gp41, most biophysical and structural analyses have relied on the production of truncated versions removing the loop region of gp41 or the utilization of nonphysiological solubilizing conditions. The loop region of gp41 is also known as principal immunodominant domain (PID) because of its high immunogenicity, and it is essential for gp41-mediated HIV fusion. In this study we identify the aggregation-prone regions of the amino acid sequence of the PID and engineer a highly soluble mutant that preserves the trimeric structure of the wild-type e-gp41 under physiological pH. Furthermore, using a reverse mutagenesis approach, we analyze the role of mutated amino acids upon the physicochemical factors that govern solubility of e-gp41. On this basis, we propose a molecular model for e-gp41 self-association, which can guide the production of soluble e-gp41 mutants for future biophysical analyses and biotechnological applications.

  12. N-butyldeoxynojirimycin-mediated inhibition of human immunodeficiency virus entry correlates with impaired gp120 shedding and gp41 exposure.

    PubMed Central

    Fischer, P B; Karlsson, G B; Dwek, R A; Platt, F M

    1996-01-01

    The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although an NB-DNJ-mediated change in viral envelope N-glycan composition inhibits HIV entry at the level of post-CD4 binding, the exact mechanism of inhibition remains to be established. In this study we have examined the effects of NB-DNJ on virion envelope composition and CD4-induced gp120 shedding and gp41 exposure. Virion composition analysis revealed an NB-DNJ-mediated reduction of 15% in overall virion envelope glycoprotein content and a reduction of 26% in the proteolytic maturation of virion gp160. Taken together, these two effects resulted in a reduction of approximately 40% in virion gp120 content. CD4-induced shedding of gp120 from the surfaces of envelope-transfected Cos cells was undetectable when gp120 was expressed in the presence of NB-DNJ. Similarly, the shedding of virion-associated gp120 was reduced 7.4-fold. CD4-induced exposure of cryptic gp41 epitopes on the surfaces of HIV-expressing ACH-2 cells was also greatly impaired, and the exposure of virion-associated gp41 epitopes was reduced 4.0-fold. Finally, CD4-induced increases in the binding of antibodies to the V3 loop of ACH-2-cell-expressed envelope glycoproteins were reduced 25-fold when the glycoproteins were expressed in the presence of NB-DNJ. These results suggest that the NB-DNJ-mediated retention of glycosylated N-glycans inhibits HIV entry by a combined effect of a reduction in virion gp120 content and a qualitative defect within the remaining gp120, preventing it from undergoing conformational changes after CD4 binding. PMID:8794362

  13. Viremic HIV Infected Individuals with High CD4 T Cells and Functional Envelope Proteins Show Anti-gp41 Antibodies with Unique Specificity and Function

    PubMed Central

    Curriu, Marta; Fausther-Bovendo, Hughes; Pernas, María; Massanella, Marta; Carrillo, Jorge; Cabrera, Cecilia; López-Galíndez, Cecilio; Clotet, Bonaventura; Debré, Patrice; Vieillard, Vincent; Blanco, Julià

    2012-01-01

    Background CD4 T-cell decay is variable among HIV-infected individuals. In exceptional cases, CD4 T-cell counts remain stable despite high plasma viremia. HIV envelope glycoprotein (Env) properties, namely tropism, fusion or the ability to induce the NK ligand NKp44L, or host factors that modulate Env cytopathic mechanisms may be modified in such situation. Methods We identified untreated HIV-infected individuals showing non-cytopathic replication (VL>10,000 copies/mL and CD4 T-cell decay<50 cells/µL/year, Viremic Non Progressors, VNP) or rapid progression (CD4 T-cells<350 cells/µL within three years post-infection, RP). We isolated full-length Env clones and analyzed their functions (tropism, fusion activity and capacity to induce NKp44L expression on CD4 cells). Anti-Env humoral responses were also analyzed. Results Env clones isolated from VNP or RP individuals showed no major phenotypic differences. The percentage of functional clones was similar in both groups. All clones tested were CCR5-tropic and showed comparable expression and fusogenic activity. Moreover, no differences were observed in their capacity to induce NKp44L expression on CD4 T cells from healthy donors through the 3S epitope of gp41. In contrast, anti- Env antibodies showed clear functional differences: plasma from VNPs had significantly higher capacity than RPs to block NKp44L induction by autologous viruses. Consistently, CD4 T-cells isolated from VNPs showed undetectable NKp44L expression and specific antibodies against a variable region flanking the highly conserved 3S epitope were identified in plasma samples from these patients. Conversely, despite continuous antigen stimulation, VNPs were unable to mount a broad neutralizing response against HIV. Conclusions Env functions (fusion and induction of NKp44L) were similar in viremic patients with slow or rapid progression to AIDS. However, differences in humoral responses against gp41 epitopes nearby 3S sequence may contribute to the lack

  14. ADS-J1 inhibits human immunodeficiency virus type 1 entry by interacting with the gp41 pocket region and blocking fusion-active gp41 core formation.

    PubMed

    Wang, Hongtao; Qi, Zhi; Guo, Angi; Mao, Qinchao; Lu, Hong; An, Xiuli; Xia, Chenglai; Li, Xiaojuan; Debnath, Asim K; Wu, Shuguang; Liu, Shuwen; Jiang, Shibo

    2009-12-01

    We previously identified a small-molecule anti-human immunodeficiency virus type 1 (anti-HIV-1) compound, ADS-J1, using a computer-aided molecular docking technique for primary screening and a sandwich enzyme-linked immunosorbent assay (ELISA) as a secondary screening method. In the present study, we demonstrated that ADS-J1 is an HIV-1 entry inhibitor, as determined by a time-of-addition assay and an HIV-1-mediated cell fusion assay. Further mechanism studies confirmed that ADS-J1 does not block gp120-CD4 binding and exhibits a marginal interaction with the HIV-1 coreceptor CXCR4. However, ADS-J1 inhibited the fusion-active gp41 core formation mimicked by peptides derived from the viral gp41 N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), as determined by ELISA, native polyacrylamide gel electrophoresis, and circular dichroism analysis. Moreover, using a surface plasmon resonance assay, we found that ADS-J1 could bind directly to IQN17, a trimeric peptide containing the gp41 pocket region, resulting in the conformational change of IQN17 and the blockage of its interaction with a short D peptide, PIE7. The positively charged residue (K574) located in the gp41 pocket region is critical for the binding of ADS-J1 to NHR. These results suggest that ADS-J1 may bind to the viral gp41 NHR region through its hydrophobic and ionic interactions with the hydrophobic and positively charged resides located in the pocket region, subsequently blocking the association between the gp41 NHR and CHR regions to form the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry.

  15. Contribution of Intrinsic Reactivity of the HIV-1 Envelope Glycoproteins to CD4-Independent Infection and Global Inhibitor Sensitivity

    PubMed Central

    Haim, Hillel; Strack, Bettina; Kassa, Aemro; Madani, Navid; Wang, Liping; Courter, Joel R.; Princiotto, Amy; McGee, Kathleen; Pacheco, Beatriz; Seaman, Michael S.; Smith, Amos B.; Sodroski, Joseph

    2011-01-01

    Human immunodeficiency virus (HIV-1) enters cells following sequential activation of the high-potential-energy viral envelope glycoprotein trimer by target cell CD4 and coreceptor. HIV-1 variants differ in their requirements for CD4; viruses that can infect coreceptor-expressing cells that lack CD4 have been generated in the laboratory. These CD4-independent HIV-1 variants are sensitive to neutralization by multiple antibodies that recognize different envelope glycoprotein epitopes. The mechanisms underlying CD4 independence, global sensitivity to neutralization and the association between them are still unclear. By studying HIV-1 variants that differ in requirements for CD4, we investigated the contribution of CD4 binding to virus entry. CD4 engagement exposes the coreceptor-binding site and increases the “intrinsic reactivity” of the envelope glycoproteins; intrinsic reactivity describes the propensity of the envelope glycoproteins to negotiate transitions to lower-energy states upon stimulation. Coreceptor-binding site exposure and increased intrinsic reactivity promote formation/exposure of the HR1 coiled coil on the gp41 transmembrane glycoprotein and allow virus entry upon coreceptor binding. Intrinsic reactivity also dictates the global sensitivity of HIV-1 to perturbations such as exposure to cold and the binding of antibodies and small molecules. Accordingly, CD4 independence of HIV-1 was accompanied by increased susceptibility to inactivation by these factors. We investigated the role of intrinsic reactivity in determining the sensitivity of primary HIV-1 isolates to inhibition. Relative to the more common neutralization-resistant (“Tier 2-like”) viruses, globally sensitive (“Tier 1”) viruses exhibited increased intrinsic reactivity, i.e., were inactivated more efficiently by cold exposure or by a given level of antibody binding to the envelope glycoprotein trimer. Virus sensitivity to neutralization was dictated both by the efficiency of

  16. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1

    PubMed Central

    Mühle, Michael; Lehmann, Melissa; Hoffmann, Kerstin; Stern, Daniel; Kroniger, Tobias; Luttmann, Werner

    2017-01-01

    The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus—1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1

  17. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1.

    PubMed

    Mühle, Michael; Lehmann, Melissa; Hoffmann, Kerstin; Stern, Daniel; Kroniger, Tobias; Luttmann, Werner; Denner, Joachim

    2017-01-01

    The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus-1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1

  18. HIV DNA-Adenovirus Multiclade Envelope Vaccine Induces Gp41 Antibody Immunodominance in Rhesus Macaques.

    PubMed

    Han, Qifeng; Williams, Wilton B; Saunders, Kevin O; Seaton, Kelly E; Wiehe, Kevin J; Vandergrift, Nathan; Von Holle, Tarra; Trama, Ashley M; Parks, Robert J; Luo, Kan; Gurley, Thaddeus C; Kepler, Thomas B; Marshall, Dawn J; Montefiori, David C; Sutherland, Laura L; Alam, Munir S; Whitesides, John F; Bowman, Cindy; Permar, Sallie R; Graham, Barney S; Mascola, John R; Seed, Patrick C; Van Rompay, Koen K A; Tomaras, Georgia D; Moody, Michael A; Haynes, Barton F

    2017-08-09

    Dominant antibody responses in vaccinees who received the multiclade (A, B and C) envelope (Env) DNA/rAd5 vaccine studied in the HIV-1 vaccine trials network (HVTN) efficacy trial 505 (HVTN 505), targeted Env gp41 and cross-reacted with microbial antigens. In this study, we asked if the DNA/rAd5 vaccine induced a similar antibody response in rhesus macaques (RMs) that are commonly used as an animal model for human HIV-1 infections and for testing candidate HIV-1 vaccines. We also asked if gp41 immunodominance could be avoided by immunization of neonatal RMs during the early stages of microbial colonization. We found that the DNA/rAd5 vaccine elicited a higher frequency of gp41-reactive memory B cells compared to gp120-memory B cells in adult and neonatal RMs. Analysis of the vaccine-induced Env-reactive B cell repertoire revealed that the majority of HIV-1 Env-reactive antibodies in both adult and neonatal RMs were targeted to gp41. Interestingly, a subset of gp41-reactive antibodies isolated from RMs cross-reacted with host antigens, including autologous intestinal microbiota. Thus, gp41-containing DNA/rAd5 vaccine induced dominant gp41-microbiota cross-reactive antibodies derived from blood memory B cells in RMs as observed in the HVTN 505 efficacy trial. These data demonstrated that RMs can be used to investigate the gp41 immunodominance in candidate HIV-1 vaccines. Moreover, colonization of neonatal RMs occurred within the first week of life, and immunization of neonatal RMs during this time also induced a dominant gp41-reactive antibody response.IMPORTANCE Our results are critical to current work in the HIV-1 vaccine field evaluating the phenomenon of gp41 immunodominance induced by HIV-1 Env gp140 in RMs and humans. Our data demonstrate that RMs are an appropriate animal model to study this phenomenon and to determine the immunogenicity in new HIV-1 Env trimer vaccine designs. The demonstration of gp41 immunodominance in memory B cells of both adult and

  19. HIV-1 Fusion Is Blocked through Binding of GB Virus C E2D Peptides to the HIV-1 gp41 Disulfide Loop

    PubMed Central

    Eissmann, Kristin; Mueller, Sebastian; Sticht, Heinrich; Jung, Susan; Zou, Peng; Jiang, Shibo; Gross, Andrea; Eichler, Jutta; Fleckenstein, Bernhard; Reil, Heide

    2013-01-01

    A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors. PMID:23349893

  20. HIV-1 fusion is blocked through binding of GB Virus C E2-derived peptides to the HIV-1 gp41 disulfide loop [corrected].

    PubMed

    Eissmann, Kristin; Mueller, Sebastian; Sticht, Heinrich; Jung, Susan; Zou, Peng; Jiang, Shibo; Gross, Andrea; Eichler, Jutta; Fleckenstein, Bernhard; Reil, Heide

    2013-01-01

    A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors.

  1. A functional interaction between gp41 and gp120 is observed for monomeric but not oligomeric, uncleaved HIV-1 Env gp140.

    PubMed

    Guttman, Miklos; Lee, Kelly K

    2013-11-01

    The envelope glycoprotein (Env) is the sole antigenic feature on the surface of HIV and the target for the humoral immune system. Soluble, uncleaved gp140 Env constructs truncated at the transmembrane domain are being investigated intensively as potential vaccine immunogens by many groups, and understanding their structural properties is essential. We used hydrogen/deuterium-exchange mass spectrometry and small-angle X-ray scattering to probe structural order in a panel of commonly used gp140 constructs and matched gp120 monomers. We observed that oligomeric forms of uncleaved gp140, generally presumed to be trimeric, contain a protease-resistant form of gp41 akin to the postfusion, helical bundle conformation and appear to lack specific interactions between gp120 and gp41. In contrast, the monomeric form of gp140 shows significant stabilization of the gp120 inner domain imparted by the gp41 region, demonstrating excellent agreement with past mutagenesis studies. Moreover, the gp140 monomers respond to CD4 binding in manner that is consistent with the initial stages of Env activation: CD4 binding induces structural ordering throughout gp120 while loosening its association with gp41. The results indicate that uncleaved gp140 oligomers do not represent an authentic prefusion form of Env, whereas gp140 monomers isolated from the same glycoprotein preparations in many ways exhibit function and internal structural order that are consistent with expectations for certain aspects of native Env. gp140 monomers may thus be a useful reagent for advancing structural and functional studies.

  2. The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region.

    PubMed

    Apellániz, Beatriz; Rujas, Edurne; Serrano, Soraya; Morante, Koldo; Tsumoto, Kouhei; Caaveiro, Jose M M; Jiménez, M Ángeles; Nieva, José L

    2015-05-22

    The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted α-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct subdomains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments.

  3. Dissociation of the trimeric gp41 ectodomain at the lipid-water interface suggests an active role in HIV-1 Env-mediated membrane fusion.

    PubMed

    Roche, Julien; Louis, John M; Grishaev, Alexander; Ying, Jinfa; Bax, Adriaan

    2014-03-04

    The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. The actual fusion process involves a switch from a homotrimeric prehairpin intermediate conformation, consisting of parallel coiled-coil helices, to a postfusion state where the ectodomains are arranged as a trimer of helical hairpins, adopting a six-helix bundle (6HB) state. Here, we show by solution NMR spectroscopy that a water-soluble 6HB gp41 ectodomain binds to zwitterionic detergents that contain phosphocholine or phosphatidylcholine head groups and phospholipid vesicles that mimic T-cell membrane composition. Binding results in the dissociation of the 6HB and the formation of a monomeric state, where its two α-helices, N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), become embedded in the lipid-water interface of the virus and host cell. The atomic structure of the gp41 ectodomain monomer, based on NOE distance restraints and residual dipolar couplings, shows that the NHR and CHR helices remain mostly intact, but they completely lose interhelical contacts. The high affinity of the ectodomain helices for phospholipid surfaces suggests that unzippering of the prehairpin intermediate leads to a state where the NHR and CHR helices become embedded in the host cell and viral membranes, respectively, thereby providing a physical force for bringing these membranes into close juxtaposition before actual fusion.

  4. Biochemistry and Biophysics of HIV-1 gp41 – membrane interactions

    PubMed Central

    Cai, Lifeng; Gochin, Miriam; Liu, Keliang

    2011-01-01

    Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein – mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), N-terminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors. PMID:22044229

  5. Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41.

    PubMed Central

    Caffrey, M; Cai, M; Kaufman, J; Stahl, S J; Wingfield, P T; Covell, D G; Gronenborn, A M; Clore, G M

    1998-01-01

    The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e-gp41), consisting of residues 27-149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e-gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N-terminal (residues 30-80) and C-terminal (residues 107-147) helices connected by a 26 residue loop (residues 81-106). The N-terminal helices of each subunit form a parallel coiled-coil structure in the interior of the complex which is surrounded by the C-terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non-sequential intramolecular contacts. The helical core of SIV e-gp41 is similar to recent X-ray structures of truncated constructs of the helical core of HIV-1 e-gp41. The present structure establishes unambiguously the connectivity of the N- and C-terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e-gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e-gp41 represents one of the largest protein structures determined by NMR to date. PMID:9707417

  6. Correlation of Antibody Responses to a Peptide Antigen gp120-C5(501-512)/gp41(732-744) with HIV Disease Progression.

    PubMed

    Sørensen, Birger; Sommerfelt, Maja A; Stjernholm, Grete; Smith, Peter Lawrence; Ökvist, Mats; Hovden, Arnt-Ove; Hoddevik, Gunnar; Redfield, Robert; Ustina, Valentina; Jelmert, Øyvind; Zeldis, Jerome; Dalgleish, Angus

    2017-01-31

    Antibodies to the carboxy-terminal constant (C5) region 5 of the HIV-1 envelope glycoprotein gp120 have previously been associated with slow disease progression. This is one of the regions on gp120 that interact with the transmembrane glycoprotein, gp41, anchoring it to the viral and infected cell membrane. This study analyzed humoral responses to a novel heterodimeric peptide construct comprising the C5(501-512) region and a compatible region on gp41(732-744). Antibody levels to C5(501-512)/gp41(732-744) were associated with slow disease progression in a treatment naive historical longitudinal cohort from Norway (n = 32; p = .00001). Elevated anti-C5(501-512)/gp41(732-744) antibody levels correlated with moderate viral load (VL) (50-10,000 copies/ml) in a cohort, including natural viral suppressors (NVS) in the Unites States (n = 58; p = .002). Analysis of HIV-positive sera from treatment naive patients in Estonia (n = 300) showed an inverse correlation between anti-C5(501-512)/gp41(732-744) antibodies and VL when comparing VL 2,000-10,000 copies/ml with VL >10,000 (p = .050). Further mapping using peptide inhibition of antibody binding revealed that responses to the C5(501-506) subdomain correlated with preserved CD4 counts (n = 55; p = .0012) irrespective of VL in this cohort. The C5 region encompassing C5(501-506) shows sequence similarity to the shared epitope (SE) of certain HLA-DR associated with immune dysfunction. Partial antigenic cross-reactivity between SE and C5 is indicated by partial inhibition of NVS antibody binding using SE 15-mer peptide (median 65% inhibition), the C5(501-506) 6-mer peptide (79% inhibition), and binding of rheumatoid arthritis patient sera to both SE and C5 peptide sequences. The potential influence of these observations on HIV-1 pathogenesis remains to be determined.

  7. Diversion of HIV-1 Vaccine-induced Immunity by gp41-Microbiota Cross-reactive Antibodies

    PubMed Central

    Williams, Wilton B; Liao, Hua-Xin; Moody, M. Anthony; Kepler, Thomas B.; Alam, S Munir; Gao, Feng; Wiehe, Kevin; Trama, Ashley M.; Jones, Kathryn; Zhang, Ruijun; Song, Hongshuo; Marshall, Dawn J; Whitesides, John F; Sawatzki, Kaitlin; Hua, Axin; Liu, Pinghuang; Tay, Matthew Z; Seaton, Kelly; Shen, Xiaoying; Foulger, Andrew; Lloyd, Krissey E.; Parks, Robert; Pollara, Justin; Ferrari, Guido; Yu, Jae-Sung; Vandergrift, Nathan; Montefiori, David C.; Sobieszczyk, Magdalena E; Hammer, Scott; Karuna, Shelly; Gilbert, Peter; Grove, Doug; Grunenberg, Nicole; McElrath, Julie; Mascola, John R.; Koup, Richard A; Corey, Lawrence; Nabel, Gary J.; Morgan, Cecilia; Churchyard, Gavin; Maenza, Janine; Keefer, Michael; Graham, Barney S.; Baden, Lindsey R.; Tomaras, Georgia D.; Haynes, Barton F.

    2015-01-01

    A HIV-1 DNA prime-recombinant Adenovirus Type 5 (rAd5) boost vaccine failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells was to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies (mAbs) were non-neutralizing, and frequently polyreactive with host and environmental antigens including intestinal microbiota (IM). Next generation sequencing of an IGHV repertoire prior to vaccination revealed an Env-IM cross-reactive Ab that was clonally-related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy. PMID:26229114

  8. Initiation of immune tolerance–controlled HIV gp41 neutralizing B cell lineages

    PubMed Central

    Zhang, Ruijun; Verkoczy, Laurent; Wiehe, Kevin; Alam, S. Munir; Nicely, Nathan I.; Santra, Sampa; Bradley, Todd; Pemble, Charles W.; Zhang, Jinsong; Gao, Feng; Montefiori, David C.; Bouton-Verville, Hilary; Kelsoe, Garnett; Larimore, Kevin; Greenberg, Phillip D.; Parks, Robert; Foulger, Andrew; Peel, Jessica N.; Luo, Kan; Lu, Xiaozhi; Trama, Ashley M.; Vandergrift, Nathan; Tomaras, Georgia D.; Kepler, Thomas B.; Moody, M. Anthony; Liao, Hua-Xin; Haynes, Barton F.

    2016-01-01

    Development of an HIV vaccine is a global priority. A major roadblock to a vaccine is an inability to induce protective broadly neutralizing antibodies (bnAbs). HIV gp41 bnAbs have characteristics that predispose them to be controlled by tolerance. We used gp41 2F5 bnAb germline knock-in mice and macaques vaccinated with immunogens reactive with germline precursors to activate neutralizing antibodies. In germline knock-in mice, bnAb precursors were deleted, with remaining anergic B cells capable of being activated by germline-binding immunogens to make gp41-reactive immunoglobulin M (IgM). Immunized macaques made B cell clonal lineages targeted to the 2F5 bnAb epitope, but 2F5-like antibodies were either deleted or did not attain sufficient affinity for gp41-lipid complexes to achieve the neutralization potency of 2F5. Structural analysis of members of a vaccine-induced antibody lineage revealed that heavy chain complementarity-determining region 3 (HCDR3) hydrophobicity was important for neutralization. Thus, gp41 bnAbs are controlled by immune tolerance, requiring vaccination strategies to transiently circumvent tolerance controls. PMID:27122615

  9. Viroporin potential of the lentivirus lytic peptide (LLP) domains of the HIV-1 gp41 protein

    PubMed Central

    Costin, Joshua M; Rausch, Joshua M; Garry, Robert F; Wimley, William C

    2007-01-01

    Background Mechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development. Virally induced changes in membrane ionic permeability induced by lytic viruses of many families contribute to cytopathogenesis. HIV-1 induces disturbances in plasma membrane ion transport. The carboxyl terminus of TM (gp41) contains potential amphipathic α-helical motifs identified through their structural similarities to naturally occurring cytolytic peptides. These sequences have been dubbed lentiviral lytic peptides (LLP) -1, -2, and -3. Results Peptides corresponding to the LLP domains (from a clade B virus) partition into lipid membranes, fold into α-helices and disrupt model membrane permeability. A peptide corresponding to the LLP-1 domain of a clade D HIV-1 virus, LLP-1D displayed similar activity to the LLP-1 domain of the clade B virus in all assays, despite a lack of amino acid sequence identity. Conclusion These results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin. Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs. PMID:18028545

  10. Computational study of bindings of olive leaf extract (OLE) to HIV-1 fusion protein gp41.

    PubMed

    Bao, J; Zhang, D W; Zhang, J Z H; Huang, P Lee; Huang, P Lin; Lee-Huang, S

    2007-06-12

    Recent experimental study found that OLE (olive leaf extract) has anti-HIV activity by blocking the HIV virus entry to host cells [Lee-Huang, S., Zhang, L., Huang, P.L., Chang, Y. and Huang, P.L. (2003) Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment. Biochem. Biophys. Res. Commun. 307, 1029; Lee-Huang, S., Huang, P.L., Zhang, D., Lee, J.W., Bao, J., Sun, Y., Chang, Y.-Tae, Zhang, J.Z.H. and Huang, P.L. (2007) Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol. Biochem. Biophys. Res. Commun. 354, 872-878, 879-884]. As part of a joint experimental and theoretical effort, we report here computational study to help identify and characterize the binding complexes of several main compounds of OLE (olive leaf extract) to HIV-1 envelop protein gp41. A number of possible binding modes are found by docking oleuropein and its metabolites, aglycone, elenolic acid and hydroxytyrosol, onto the hydrophobic pocket on gp41. Detailed OLE-gp41 binding interactions and free energies of binding are obtained through molecular dynamics simulation and MM-PBSA calculation. Specific molecular interactions in our predicted OLE/gp41 complexes are identified and hydroxytyrosol is identified to be the main moiety for binding to gp41. This computational study complements the corresponding experimental investigation and helps establish a good starting point for further refinement of OLE-based gp41 inhibitors.

  11. Approaches for identification of HIV-1 entry inhibitors targeting gp41 pocket.

    PubMed

    Yu, Fei; Lu, Lu; Du, Lanying; Zhu, Xiaojie; Debnath, Asim K; Jiang, Shibo

    2013-01-11

    The hydrophobic pocket in the HIV-1 gp41 N-terminal heptad repeat (NHR) domain plays an important role in viral fusion and entry into the host cell, and serves as an attractive target for development of HIV-1 fusion/entry inhibitors. The peptide anti-HIV drug targeting gp41 NHR, T-20 (generic name: enfuvirtide; brand name: Fuzeon), was approved by the U.S. FDA in 2003 as the first HIV fusion/entry inhibitor for treatment of HIV/AIDS patients who fail to respond to the current antiretroviral drugs. However, because T20 lacks the pocket-binding domain (PBD), it exhibits low anti-HIV-1 activity and short half-life. Therefore, several next-generation HIV fusion inhibitory peptides with PBD have been developed. They possess longer half-life and more potent antiviral activity against a broad spectrum of HIV-1 strains, including the T-20-resistant variants. Nonetheless, the clinical application of these peptides is still limited by the lack of oral availability and the high cost of production. Thus, development of small molecule compounds targeting the gp41 pocket with oral availability has been promoted. This review describes the main approaches for identification of HIV fusion/entry inhibitors targeting the gp41 pocket and summarizes the latest progress in developing these inhibitors as a new class of anti-HIV drugs.

  12. Striking HIV-1 Entry by Targeting HIV-1 gp41. But, Where Should We Target?

    PubMed Central

    Teixeira, Cátia; Barbault, Florent; Couesnon, Thierry; Gomes, José R. B.; Gomes, Paula; Maurel, François

    2016-01-01

    HIV-1 gp41 facilitates the viral fusion through a conformational switch involving the association of three C-terminal helices along the conserved hydrophobic grooves of three N-terminal helices coiled-coil. The control of these structural rearrangements is thought to be central to HIV-1 entry and, therefore, different strategies of intervention are being developed. Herewith, we describe a procedure to simulate the folding of an HIV-1 gp41 simplified model. This procedure is based on the construction of plausible conformational pathways, which describe protein transition between non-fusogenic and fusogenic conformations. The calculation of the paths started with 100 molecular dynamics simulations of the non-fusogenic conformation, which were found to converge to different intermediate states. Those presenting defined criteria were selected for separate targeted molecular dynamics simulations, subjected to a force constant imposing a movement towards the gp41 fusogenic conformation. Despite significant diversity, a preferred sequence of events emerged when the simulations were analyzed in terms of the formation, breakage and evolution of the contacts. We pointed out 29 residues as the most relevant for the movement of gp41; also, 2696 possible interactions were reduced to only 48 major interactions, which reveals the efficiency of the method. The analysis of the evolution of the main interactions lead to the detection of four main behaviors for those contacts: stable, increasing, decreasing and repulsive interactions. Altogether, these results suggest a specific small cavity of the HIV-1 gp41 hydrophobic groove as the preferred target to small molecules. PMID:26785380

  13. Striking HIV-1 Entry by Targeting HIV-1 gp41. But, Where Should We Target?

    PubMed

    Teixeira, Cátia; Barbault, Florent; Couesnon, Thierry; Gomes, José R B; Gomes, Paula; Maurel, François

    2016-01-01

    HIV-1 gp41 facilitates the viral fusion through a conformational switch involving the association of three C-terminal helices along the conserved hydrophobic grooves of three N-terminal helices coiled-coil. The control of these structural rearrangements is thought to be central to HIV-1 entry and, therefore, different strategies of intervention are being developed. Herewith, we describe a procedure to simulate the folding of an HIV-1 gp41 simplified model. This procedure is based on the construction of plausible conformational pathways, which describe protein transition between non-fusogenic and fusogenic conformations. The calculation of the paths started with 100 molecular dynamics simulations of the non-fusogenic conformation, which were found to converge to different intermediate states. Those presenting defined criteria were selected for separate targeted molecular dynamics simulations, subjected to a force constant imposing a movement towards the gp41 fusogenic conformation. Despite significant diversity, a preferred sequence of events emerged when the simulations were analyzed in terms of the formation, breakage and evolution of the contacts. We pointed out 29 residues as the most relevant for the movement of gp41; also, 2696 possible interactions were reduced to only 48 major interactions, which reveals the efficiency of the method. The analysis of the evolution of the main interactions lead to the detection of four main behaviors for those contacts: stable, increasing, decreasing and repulsive interactions. Altogether, these results suggest a specific small cavity of the HIV-1 gp41 hydrophobic groove as the preferred target to small molecules.

  14. Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated

    PubMed Central

    Chen, Xi; Munshaw, Supriya; Zhang, Ruijun; Marshall, Dawn J.; Vandergrift, Nathan; Whitesides, John F.; Lu, Xiaozhi; Yu, Jae-Sung; Hwang, Kwan-Ki; Gao, Feng; Markowitz, Martin; Heath, Sonya L.; Bar, Katharine J.; Goepfert, Paul A.; Montefiori, David C.; Shaw, George C.; Alam, S. Munir; Margolis, David M.; Denny, Thomas N.; Boyd, Scott D.; Marshal, Eleanor; Egholm, Michael; Simen, Birgitte B.; Hanczaruk, Bozena; Fire, Andrew Z.; Voss, Gerald; Kelsoe, Garnett; Tomaras, Georgia D.; Moody, M. Anthony; Kepler, Thomas B.

    2011-01-01

    The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non–HIV-1 antigens. PMID:21987658

  15. A highly conserved sequence associated with the HIV gp41 loop region is an immunomodulator of antigen-specific T cells in mice.

    PubMed

    Ashkenazi, Avraham; Faingold, Omri; Kaushansky, Nathali; Ben-Nun, Avraham; Shai, Yechiel

    2013-03-21

    Modulation of T-cell responses by HIV occurs via distinct mechanisms, 1 of which involves inactivation of T cells already at the stage of virus-cell fusion. Hydrophobic portions of the gp41 protein of the viral envelope that contributes to membrane fusion may modulate T-cell responsiveness. Here we found a highly conserved sequence (termed "ISLAD") that is associated with the membranotropic gp41 loop region. We showed that ISLAD has the ability to bind the T-cell membrane and to interact with the T-cell receptor (TCR) complex. Furthermore, ISLAD inhibited T-cell proliferation and interferon-γ secretion that resulted from TCR engagement through antigen-presenting cells. Moreover, administering ISLAD (10 µg per mouse) to an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis reduced the severity of the disease. This was related to the inhibition of pathogenic T-cell proliferation and to reduced pro-inflammatory cytokine secretion in the lymph nodes of ISLAD-treated EAE mice. The data suggest that T-cell inactivation by HIV during membrane fusion may lie in part in this conserved sequence associated with the gp41 loop region.

  16. Identification of fragments targeting an alternative pocket on HIV-1 gp41 by NMR screening and similarity searching.

    PubMed

    Chu, Shidong; Gochin, Miriam

    2013-09-15

    The HIV-1 envelope glycoprotein gp41 fusion intermediate is a promising drug target for inhibiting viral entry. However, drug development has been impeded by challenges inherent in mediating the underlying protein-protein interaction. Here we report on the identification of fragments that bind to a C-terminal sub-pocket adjacent to the well-known hydrophobic pocket on the NHR coiled coil. Using a specifically designed assay and ligand-based NMR screening of a fragment library, we identified a thioenylaminopyrazole compound with a dissociation constant of ~500 μM. Interaction with the C-terminal sub-pocket was confirmed by paramagnetic relaxation enhancement NMR experiments, which also yielded the binding mode. Shape-based similarity searching detected additional phenylpyrazole and phenyltriazole fragments within the library, enriching the hit rate over random screening, and revealing molecular features required for activity. Discovery of the novel scaffolds and binding mechanism suggests avenues for extending the interaction surface and improving the potency of a hydrophobic pocket binding inhibitor.

  17. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface

    SciTech Connect

    Huang, Jinghe; Kang, Byong H.; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Imamichi, Hiromi; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald; Klasse, Per-Johan; Migueles, Stephen A.; Bailer, Robert T.; Alam, Munir; Pugach, Pavel; Haynes, Barton F.; Wyatt, Richard T.; Sanders, Rogier W.; Binley, James M.; Ward, Andrew B.; Mascola, John R.; Kwong, Peter D.; Connors, Mark

    2015-10-15

    The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC50) <50 μg ml-1. The median IC50 of neutralized viruses was 0.033 μg ml-1, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.

  18. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-120 interface

    PubMed Central

    Huang, Jinghe; Kang, Byong H.; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald; Klasse, Per-Johan; Migueles, Stephen A.; Bailer, Robert T.; Alam, Munir; Pugach, Pavel; Haynes, Barton F.; Wyatt, Richard T.; Sanders, Rogier W.; Binley, James M.; Ward, Andrew B.; Mascola, John R.; Kwong, Peter D.; Connors, Mark

    2014-01-01

    The isolation of human monoclonal antibodies (mAbs) is providing important insights regarding the specificities that underlie broad neutralization of HIV-1 (reviewed in1). Here we report a broad and extremely potent HIV-specific mAb, termed 35O22, which binds novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with an IC50<50 μg/ml. The median IC50 of neutralized viruses was 0.033 μg/ml, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed it to bind a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current mAb-based approaches to immunotherapies, prophylaxis, and vaccine design. PMID:25186731

  19. Evaluation of "credit card" libraries for inhibition of HIV-1 gp41 fusogenic core formation.

    PubMed

    Xu, Yang; Lu, Hong; Kennedy, Jack P; Yan, Xuxia; McAllister, Laura A; Yamamoto, Noboru; Moss, Jason A; Boldt, Grant E; Jiang, Shibo; Janda, Kim D

    2006-01-01

    Protein-protein interactions are of critical importance in biological systems, and small molecule modulators of such protein recognition and intervention processes are of particular interest. To investigate this area of research, we have synthesized small-molecule libraries that can disrupt a number of biologically relevant protein-protein interactions. These library members are designed upon planar motif, appended with a variety of chemical functions, which we have termed "credit-card" structures. From two of our "credit-card" libraries, a series of molecules were uncovered which act as inhibitors against the HIV-1 gp41 fusogenic 6-helix bundle core formation, viral antigen p24 formation, and cell-cell fusion at low micromolar concentrations. From the high-throughput screening assays we utilized, a selective index (SI) value of 4.2 was uncovered for compound 2261, which bodes well for future structure activity investigations and the design of more potent gp41 inhibitors.

  20. Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape.

    PubMed

    Wibmer, Constantinos Kurt; Gorman, Jason; Ozorowski, Gabriel; Bhiman, Jinal N; Sheward, Daniel J; Elliott, Debra H; Rouelle, Julie; Smira, Ashley; Joyce, M Gordon; Ndabambi, Nonkululeko; Druz, Aliaksandr; Asokan, Mangai; Burton, Dennis R; Connors, Mark; Abdool Karim, Salim S; Mascola, John R; Robinson, James E; Ward, Andrew B; Williamson, Carolyn; Kwong, Peter D; Morris, Lynn; Moore, Penny L

    2017-01-01

    A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing

  1. Monoclonal Antibodies to V2, V3, the CD4-binding site and gp41 HIV-1 Mediate Phagocytosis in a Dose-dependent Manner.

    PubMed

    Musich, Thomas; Li, Liuzhe; Liu, Lily; Zolla-Pazner, Susan; Robert-Guroff, Marjorie; Gorny, Miroslaw K

    2017-01-25

    In light of weak or absent neutralizing activity mediated by anti-V2 monoclonal Abs (mAbs), we tested whether they can mediate Ab-dependent cellular phagocytosis (ADCP) which is an important element of anti-HIV-1 immunity. We tested six anti-V2 mAbs and compared them with 21 mAbs specific for V3, the CD4-binding site (CD4bs), and gp41 derived from HIV-1 chronically infected individuals and produced by hybridoma cells. ADCP activity was measured by flow cytometry using uptake by THP-1 monocytic cells of fluorescent beads coated with gp120, gp41, BG505 SOSIP.664 or BG505 DS-SOSIP.664 complexed with mAbs. The ADCP activity measured by the area under the curve showed significantly higher activity of anti-gp41 mAbs compared to three other groups of mAbs tested using beads coated with monomeric gp41 or gp120; anti-V2 mAbs were dominant over anti-V3 and anti-CD4bs against clade C gp120ZM109. ADCP mediated by V2 and V3 mAbs was positive against stabilized DS-SOSIP.664 trimer but negligible against SOSIP.664 targets, suggesting a closed envelope conformation better exposes the variable loops. Two IgG3 mAbs against V2 and V3 regions displayed dominant ADCP activity over a panel of IgG1 mAbs. This superior ADCP activity was confirmed when two of three recombinant IgG3 anti-V2 mAbs were compared to IgG1 counterparts. The study demonstrated dominant ADCP activity of anti-gp41 against monomers but not trimers with some higher activity of anti-V2 mAbs over anti-V3 and anti-CD4bs mAbs. The ability to mediate ADCP suggests a mechanism by which anti-HIV-1 envelope Abs can contribute to protective efficacy.

  2. Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape

    PubMed Central

    Elliott, Debra H.; Rouelle, Julie; Smira, Ashley; Ndabambi, Nonkululeko; Druz, Aliaksandr; Williamson, Carolyn

    2017-01-01

    A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing

  3. Characterization of the water defect at the HIV-1 gp41 membrane spanning domain in bilayers with and without cholesterol using molecular simulations

    PubMed Central

    Baker, Michelle K.; Gangupomu, Vamshi K.; Abrams, Cameron F.

    2014-01-01

    The membrane spanning domain (MSD) of human immunodeficiency virus 1 (HIV-1) envelope glycoprotein gp41 is important for fusion and infection. We used molecular dynamics (MD) simulations (3.4 μs total) to relate membrane and peptide properties that lead to water solvation of the α-helical gp41 MSD’s midspan arginine in pure dipalmitoylphosphatidylcholine (DPPC) and in 50/50 DPPC/cholesterol membranes. We find that the midspan arginine is solvated by water that penetrates the inner leaflet, leading to a so-called water defect. The water defect is surprisingly robust across initial conditions and membrane compositions, but the presence of cholesterol modulates its behavior in several key ways. In the cholesterol-containing membranes, fluctuations in membrane thickness and water penetration depth are localized near the midspan arginine, and the MSD helices display a tightly regulated tilt angle. In the cholesterol-free membranes, thickness fluctuations are not as strongly correlated to the peptide position and tilt angles vary significantly depending on protein position relative to boundaries between domains of differing thickness. Cholesterol in an HIV-1 viral membrane is required for infection. Therefore, this work suggests that the colocalized water defect and membrane thickness fluctuations in cholesterol-containing viral membranes play an important role in fusion by bringing the membrane closer to a stability limit that must be crossed for fusion to occur. PMID:24440660

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

    NASA Technical Reports Server (NTRS)

    Lim, Kap; Ho, Joseph X.; Keeling, Kim; Gilliland, Gary L.; Ji, Xinhua; Rueker, Florian; Carter, Daniel C.

    1994-01-01

    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) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class 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(sub 3)2(sub 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.

  5. Characterization of the water defect at the HIV-1 gp41 membrane spanning domain in bilayers with and without cholesterol using molecular simulations.

    PubMed

    Baker, Michelle K; Gangupomu, Vamshi K; Abrams, Cameron F

    2014-05-01

    The membrane spanning domain (MSD) of human immunodeficiency virus 1 (HIV-1) envelope glycoprotein gp41 is important for fusion and infection. We used molecular dynamics (MD) simulations (3.4 μs total) to relate membrane and peptide properties that lead to water solvation of the α-helical gp41 MSD's midspan arginine in pure dipalmitoylphosphatidylcholine (DPPC) and in 50/50 DPPC/cholesterol membranes. We find that the midspan arginine is solvated by water that penetrates the inner leaflet, leading to a so-called water defect. The water defect is surprisingly robust across initial conditions and membrane compositions, but the presence of cholesterol modulates its behavior in several key ways. In the cholesterol-containing membranes, fluctuations in membrane thickness and water penetration depth are localized near the midspan arginine, and the MSD helices display a tightly regulated tilt angle. In the cholesterol-free membranes, thickness fluctuations are not as strongly correlated to the peptide position and tilt angles vary significantly depending on protein position relative to boundaries between domains of differing thickness. Cholesterol in an HIV-1 viral membrane is required for infection. Therefore, this work suggests that the colocalized water defect and membrane thickness fluctuations in cholesterol-containing viral membranes play an important role in fusion by bringing the membrane closer to a stability limit that must be crossed for fusion to occur.

  6. Conformation of trimeric envelope glycoproteins: the CD4-dependent membrane fusion mechanism of HIV-1.

    PubMed

    Yingliang, Wu; Hong, Yi; Zhijian, Cao; Wenxin, Li

    2007-08-01

    The HIV-1 envelope glycoproteins are assembled by the trimeric gp120s and gp41s proteins. The gp120 binds sequentially to CD4 and coreceptor for initiating virus entry. Because of noncovalent interaction and heavy glycosylation for envelope glycoproteins, it is highly difficult to determine entire envelope glycoproteins structure now. Such question extremely limits our good understanding of HIV-1 membrane fusion mechanism. Here, a novel and reasonable assembly model of trimeric gp120s and gp41s was proposed based on the conformational dynamics of trimeric gp120-gp41 complex and gp41, respectively. As for gp41, the heptad repeat sequences in the gp41 C-terminal is of enormous flexibility. On the contrary, the heptad repeat sequences in the gp41 N-terminal likely present stable three-helical bundle due to strong nonpolar interaction, and they were predicted to associate three alpha1 helixes from the non-neutralizing face of the gp120 inner domain, which is quite similar to gp41 fusion core structure. Such interaction likely leads to the formation of noncovalent gp120-gp41 complex. In the proposed assembly of trimeric gp120-gp41 complex, three gp120s present not only perfectly complementary and symmetrical distribution around the gp41, but also different flexibility degree in the different structural domains. Thus, the new model can well explain numerous experimental phenomena, present plenty of structural information, elucidate effectively HIV-1 membrane fusion mechanism, and direct to further develop vaccine and novel fusion inhibitors.

  7. Structure-guided Alterations of the gp41-directed HIV-1 Broadly Neutralizing Antibody 2F5 Reveal New Properties Regarding its Neutralizing Function

    PubMed Central

    Guenaga, Javier; Wyatt, Richard T.

    2012-01-01

    The broadly neutralizing HIV-1 antibody 2F5 recognizes an epitope in the gp41 membrane proximal external region (MPER). The MPER adopts a helical conformation as free peptide, as post-fusogenic forms of gp41, and when bound to the 4E10 monoclonal antibody (Mab). However, when bound to 2F5, the epitope is an extended-loop. The antibody-peptide structure reveals binding between the heavy and light chains with most the long, hydrophobic CDRH3 not contacting peptide. However, mutagenesis identifies this loop as critical for binding, neutralization and for putative hydrophobic membrane interactions. Here, we examined length requirements of the 2F5 CDRH3 and plasticity regarding binding and neutralization. We generated 2F5 variants possessing either longer or shorter CDRH3s and assessed function. The CDRH3 tolerated elongations and reductions up to four residues, displaying a range of binding affinities and retaining some neutralizing capacity. 2F5 antibody variants selective recognition of conformationally distinctive MPER probes suggests a new role for the CDRH3 loop in destabilizing the helical MPER. Binding and neutralization were enhanced by targeted tryptophan substitutions recapitulating fully the activities of the wild-type 2F5 antibody in a shorter CDRH3 variant. MPER alanine scanning revealed binding contacts of this variant downstream of the 2F5 core epitope, into the 4E10 epitope region. This variant displayed increased reactivity to cardiolipin-beta-2-glycoprotein. Tyrosine replacements maintained neutralization while eliminating cardiolipin-beta-2-glycoprotein interaction. The data suggest a new mechanism of action, important for vaccine design, in which the 2F5 CDRH3 contacts and destabilizes the MPER helix downstream of its core epitope to allow induction of the extended-loop conformation. PMID:22829767

  8. Structure-guided alterations of the gp41-directed HIV-1 broadly neutralizing antibody 2F5 reveal new properties regarding its neutralizing function.

    PubMed

    Guenaga, Javier; Wyatt, Richard T

    2012-01-01

    The broadly neutralizing HIV-1 antibody 2F5 recognizes an epitope in the gp41 membrane proximal external region (MPER). The MPER adopts a helical conformation as free peptide, as post-fusogenic forms of gp41, and when bound to the 4E10 monoclonal antibody (Mab). However, when bound to 2F5, the epitope is an extended-loop. The antibody-peptide structure reveals binding between the heavy and light chains with most the long, hydrophobic CDRH3 not contacting peptide. However, mutagenesis identifies this loop as critical for binding, neutralization and for putative hydrophobic membrane interactions. Here, we examined length requirements of the 2F5 CDRH3 and plasticity regarding binding and neutralization. We generated 2F5 variants possessing either longer or shorter CDRH3s and assessed function. The CDRH3 tolerated elongations and reductions up to four residues, displaying a range of binding affinities and retaining some neutralizing capacity. 2F5 antibody variants selective recognition of conformationally distinctive MPER probes suggests a new role for the CDRH3 loop in destabilizing the helical MPER. Binding and neutralization were enhanced by targeted tryptophan substitutions recapitulating fully the activities of the wild-type 2F5 antibody in a shorter CDRH3 variant. MPER alanine scanning revealed binding contacts of this variant downstream of the 2F5 core epitope, into the 4E10 epitope region. This variant displayed increased reactivity to cardiolipin-beta-2-glycoprotein. Tyrosine replacements maintained neutralization while eliminating cardiolipin-beta-2-glycoprotein interaction. The data suggest a new mechanism of action, important for vaccine design, in which the 2F5 CDRH3 contacts and destabilizes the MPER helix downstream of its core epitope to allow induction of the extended-loop conformation.

  9. Genetic analysis and natural polymorphisms in HIV-1 gp41 isolates from Maputo City, Mozambique.

    PubMed

    Ismael, Nália; Bila, Dulce; Mariani, Diana; Vubil, Adolfo; Mabunda, Nedio; Abreu, Celina; Jani, Ilesh; Tanuri, Amilcar

    2014-06-01

    Enfuvirtide was the first fusion inhibitor approved by the Food and Drug Administration (FDA) in 2003 for HIV-1 infection in treatment-experienced patient. It is the first approved antiviral agent to attack the HIV life cycle in its early stages. For HIV fusion to occur, the HR1 and HR2 domains in the gp41 region need to interact. Enfuvirtide is a synthetic peptide that corresponds to 36 amino acids of the HR2, which competitively binds to HR1 inhibiting the interaction with the HR2 domain thus preventing fusogenic conformation and inhibiting viral entry into host cells. Resistance to enfuvirtide is conferred by mutations occurring in the HR1 region involving residues 36-45. Mozambique, a sub-Saharan country, with an HIV prevalence of 11.5%, provides first line and second line antiretroviral therapy (ART)-based treatment. In poor resource settings such as Mozambique the lack of adequate infrastructures, the high costs of viral load tests, and the availability of salvage treatment have hindered the intended objective of monitoring HIV treatment, suggesting an important concern regarding the development of drug resistance. The general aim of this study was to evaluate naturally occurring polymorphisms and resistance-associated mutations in the gp41 region of HIV-1 isolates from Mozambique. The study included 78 patients naive to ARV treatment and 28 patients failing first line regimen recruited from Centro de Saúde Alto-Maé situated in Maputo. The gp41 gene from 103 patients was sequenced and resistance-associated mutations for enfuvirtide were screened. Subtype analysis revealed that 96% of the sequences were classified as subtype C, 2% as subtype G, 1% as subtype A1, and the other 1% as a mosaic form composed of A1/C. No enfuvirtide resistance-associated mutations in HR1 of gp41 were detected. The major polymorphisms in the HR1 were N42S, L54M, A67T, and V72I. This study suggests that this new class of antiviral drug may be effective as a salvage therapy in

  10. Discovery of HIV fusion inhibitors targeting gp41 using a comprehensive α-helix mimetic library

    PubMed Central

    Whitby, Landon R.; Boyle, Kristopher E.; Cai, Lifeng; Yu, Xiaoqian; Gochin, Miriam; Boger, Dale L.

    2012-01-01

    The evaluation of a comprehensive α-helix mimetic library for binding the gp41 NHR hydrophobic pocket recognizing an intramolecular CHR α-helix provided a detailed depiction of structural features required for binding and led to the discovery of small molecule inhibitors (Ki 0.6–1.3 µM) that not only match or exceed the potency of those disclosed over the past decade, but that also exhibit effective activity in a cell–cell fusion assay (IC50 5–8 µM). PMID:22424973

  11. Genetic Analysis and Natural Polymorphisms in HIV-1 gp41 Isolates from Maputo City, Mozambique

    PubMed Central

    Ismael, Nália; Bila, Dulce; Mariani, Diana; Vubil, Adolfo; Mabunda, Nedio; Abreu, Celina; Jani, Ilesh

    2014-01-01

    Abstract Enfuvirtide was the first fusion inhibitor approved by the Food and Drug Administration (FDA) in 2003 for HIV-1 infection in treatment-experienced patient. It is the first approved antiviral agent to attack the HIV life cycle in its early stages. For HIV fusion to occur, the HR1 and HR2 domains in the gp41 region need to interact. Enfuvirtide is a synthetic peptide that corresponds to 36 amino acids of the HR2, which competitively binds to HR1 inhibiting the interaction with the HR2 domain thus preventing fusogenic conformation and inhibiting viral entry into host cells. Resistance to enfuvirtide is conferred by mutations occurring in the HR1 region involving residues 36–45. Mozambique, a sub-Saharan country, with an HIV prevalence of 11.5%, provides first line and second line antiretroviral therapy (ART)-based treatment. In poor resource settings such as Mozambique the lack of adequate infrastructures, the high costs of viral load tests, and the availability of salvage treatment have hindered the intended objective of monitoring HIV treatment, suggesting an important concern regarding the development of drug resistance. The general aim of this study was to evaluate naturally occurring polymorphisms and resistance-associated mutations in the gp41 region of HIV-1 isolates from Mozambique. The study included 78 patients naive to ARV treatment and 28 patients failing first line regimen recruited from Centro de Saúde Alto-Maé situated in Maputo. The gp41 gene from 103 patients was sequenced and resistance-associated mutations for enfuvirtide were screened. Subtype analysis revealed that 96% of the sequences were classified as subtype C, 2% as subtype G, 1% as subtype A1, and the other 1% as a mosaic form composed of A1/C. No enfuvirtide resistance-associated mutations in HR1 of gp41 were detected. The major polymorphisms in the HR1 were N42S, L54M, A67T, and V72I. This study suggests that this new class of antiviral drug may be effective as a salvage

  12. Antibodies to gp41 and nef in otherwise HIV-negative homosexual man with Kaposi's sarcoma.

    PubMed

    Bowden, F J; McPhee, D A; Deacon, N J; Cumming, S A; Doherty, R R; Sonza, S; Lucas, C R; Crowe, S M

    1991-06-01

    A homosexual man with histologically confirmed Kaposi's sarcoma remained seronegative for HIV-1, HIV-2, and HTLV-1 on conventional tests over a 4-year period. HIV cultures were also negative on thirteen separate occasions. However, serum antibodies to synthetic peptide analogues of the gp41 and nef regions of HIV-1 were consistently detected on an enzyme immunoassay. Tests with the polymerase chain reaction with primers directed to the gag and env regions were negative. The antigens to which the antibodies were produced might have come from a defective HIV mutant, another retrovirus, or a hitherto unknown "agent of Kaposi's sarcoma" with similar antigenic epitopes.

  13. Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion.

    PubMed

    Lai, Alex L; Moorthy, Anna Eswara; Li, Yinling; Tamm, Lukas K

    2012-04-20

    The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy.  The fusion domain formed an α-helix in membranes containing less than 30 mol% cholesterol and  formed β-sheet secondary structure in membranes containing ≥30 mol% cholesterol. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of α-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical form and its β-sheet form. High cholesterol, which induced β-sheets, promoted fusion; however, acidic lipids, which promoted relatively deep membrane insertion as an α-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided that their membrane insertion is deep, α-helical and β-sheet conformations contribute to membrane fusion. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Broadly neutralizing anti-HIV-1 antibodies disrupt a hinge-related function of gp41 at the membrane interface

    PubMed Central

    Song, Likai; Sun, Zhen-Yu J.; Coleman, Kate E.; Zwick, Michael B.; Gach, Johannes S.; Wang, Jia-huai; Reinherz, Ellis L.; Wagner, Gerhard; Kim, Mikyung

    2009-01-01

    A vaccine capable of stimulating protective antiviral antibody responses is needed to curtail the global AIDS epidemic caused by HIV-1. Although rarely elicited during the course of natural infection or upon conventional vaccination, the membrane-proximal ectodomain region (MPER) of the HIV-1 glycoprotein of Mr 41,000 (gp41) envelope protein subunit is the target of 3 such human broadly neutralizing antibodies (BNAbs): 4E10, 2F5, and Z13e1. How these BNAbs bind to their lipid-embedded epitopes and mediate antiviral activity is unclear, but such information might offer important insight into a worldwide health imperative. Here, EPR and NMR techniques were used to define the manner in which these BNAbs differentially recognize viral membrane-encrypted residues configured within the L-shaped helix–hinge–helix MPER segment. Two distinct modes of antibody-mediated interference of viral infection were identified. 2F5, like 4E10, induces large conformational changes in the MPER relative to the membrane. However, although 4E10 straddles the hinge and extracts residues W672 and F673, 2F5 lifts up residues N-terminal to the hinge region, exposing L669 and W670. In contrast, Z13e1 effects little change in membrane orientation or conformation, but rather immobilizes the MPER hinge through extensive rigidifying surface contacts. Thus, BNAbs disrupt HIV-1 MPER fusogenic functions critical for virus entry into human CD4 T cells and macrophages either by preventing hinge motion or by perturbing MPER orientation. HIV-1 MPER features important for targeted vaccine design have been revealed, the implications of which extend to BNAb targets on other viral fusion proteins. PMID:19458040

  15. DNA unwinding by ring-shaped T4 helicase gp41 is hindered by tension on the occluded strand.

    PubMed

    Ribeck, Noah; Saleh, Omar A

    2013-01-01

    The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, 'occluded' strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.

  16. Design, synthesis and activity evaluation of novel peptide fusion inhibitors targeting HIV-1 gp41.

    PubMed

    Tan, Jianjun; Su, Min; Zeng, Yi; Wang, Cunxin

    2016-01-15

    Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes about 2 million people to death every year. Fusion inhibitors targeted the envelope protein (gp41) represent a novel and alternative approach for anti-AIDS therapy, which terminates the HIV-1 life cycle at an early stage. Using CP621-652 as a template, a series of peptides were designed, synthesized and evaluated in vitro assays. An interesting phenomenon was found that the substitution of hydrophobic residues at solvent accessible sites could increase the anti-HIV activity when the C-terminal sequence was extended with an enough numbers of amino acids. After the active peptides was synthesized and evaluated, peptide 8 showed the best anti-HIV-1 IIIB whole cell activity (MAGI IC50=53.02 nM). Further study indicated that peptide 8 bound with the gp41 NHR helix, and then blocked the conformation of 6-helix, thus inhibited virus-cell membrane fusion. The results would be helpful for the design of peptide fusion inhibitors against HIV-1 infection.

  17. Investigating hexameric helicases: Single-molecule studies of DnaB and T4 gp41

    NASA Astrophysics Data System (ADS)

    Saleh, Omar; Ribeck, Noah; Berezney, John

    2011-03-01

    Hexameric, ring-shaped motor proteins serve as replicative helicases in many systems. They function by encircling and translocating along ssDNA, denaturing dsDNA in advance of its motion by sterically occluding the complementary strand to the outside of the ring. We investigate the helicase activity of two such motors using single-molecule measurements with magnetic tweezers. First, we measure the activity of the E. coli helicase DnaB complexed with the tau subunit of the Pol III holoenzyme. Tau is known from bulk measurements to stimulate DnaB activity (Kim et al., Cell, 1996); we investigate the means of this stimulation. Second, we measure helicase activity of the T4 phage helicase gp41 in multiple tethered DNA geometries. Previous work on DnaB showed a dependence of helicase activity on DNA geometry (Ribeck et al., Biophys. J., 2010); here, we test gp41 for similar behavior to see whether it is a common characteristic of hexameric helicases.

  18. Amino Acid Changes in the HIV-1 gp41 Membrane Proximal Region Control Virus Neutralization Sensitivity.

    PubMed

    Bradley, Todd; Trama, Ashley; Tumba, Nancy; Gray, Elin; Lu, Xiaozhi; Madani, Navid; Jahanbakhsh, Fatemeh; Eaton, Amanda; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey E; Sutherland, Laura L; Scearce, Richard M; Bowman, Cindy M; Barnett, Susan; Abdool-Karim, Salim S; Boyd, Scott D; Melillo, Bruno; Smith, Amos B; Sodroski, Joseph; Kepler, Thomas B; Alam, S Munir; Gao, Feng; Bonsignori, Mattia; Liao, Hua-Xin; Moody, M Anthony; Montefiori, David; Santra, Sampa; Morris, Lynn; Haynes, Barton F

    2016-10-01

    Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier-1) viruses or rare cases of vaccine-matched neutralization-resistant (tier-2) viruses, but no vaccine has induced antibodies that can broadly neutralize heterologous tier-2 viruses. In this study, we isolated antibodies from an HIV-1-infected individual that targeted the gp41 membrane-proximal external region (MPER) that may have selected single-residue changes in viral variants in the MPER that resulted in neutralization sensitivity to antibodies targeting distal epitopes on the HIV-1 Env. Similarly, a single change in the MPER in a second virus from another infected-individual also conferred enhanced neutralization sensitivity. These gp41 single-residue changes thus transformed tier-2 viruses into tier-1 viruses that were sensitive to vaccine-elicited tier-1 neutralizing antibodies. These data demonstrate that Env amino acid changes within the MPER bnAb epitope of naturally-selected escape viruses can increase neutralization sensitivity to multiple types of neutralizing antibodies, and underscore the critical importance of the MPER for maintaining the integrity of the tier-2 HIV-1 trimer.

  19. Differences in serum IgA responses to HIV-1 gp41 in elite controllers compared to viral suppressors on highly active antiretroviral therapy.

    PubMed

    Nabi, Rafiq; Moldoveanu, Zina; Wei, Qing; Golub, Elizabeth T; Durkin, Helen G; Greenblatt, Ruth M; Herold, Betsy C; Nowicki, Marek J; Kassaye, Seble; Cho, Michael W; Pinter, Abraham; Landay, Alan L; Mestecky, Jiri; Kozlowski, Pamela A

    2017-01-01

    A responses and affinity maturation of anti-gp41 IgA antibodies occurs to a greater extent in EC than in subjects on HAART. Future studies will be required to determine if IgA antibodies in EC may contribute in control of viral replication.

  20. Differences in serum IgA responses to HIV-1 gp41 in elite controllers compared to viral suppressors on highly active antiretroviral therapy

    PubMed Central

    Nabi, Rafiq; Moldoveanu, Zina; Wei, Qing; Golub, Elizabeth T.; Durkin, Helen G.; Greenblatt, Ruth M.; Herold, Betsy C.; Nowicki, Marek J.; Kassaye, Seble; Cho, Michael W.; Pinter, Abraham; Landay, Alan L.; Mestecky, Jiri

    2017-01-01

    A responses and affinity maturation of anti-gp41 IgA antibodies occurs to a greater extent in EC than in subjects on HAART. Future studies will be required to determine if IgA antibodies in EC may contribute in control of viral replication. PMID:28671952

  1. HIV-1 VACCINES. Diversion of HIV-1 vaccine-induced immunity by gp41-microbiota cross-reactive antibodies.

    PubMed

    Williams, Wilton B; Liao, Hua-Xin; Moody, M Anthony; Kepler, Thomas B; Alam, S Munir; Gao, Feng; Wiehe, Kevin; Trama, Ashley M; Jones, Kathryn; Zhang, Ruijun; Song, Hongshuo; Marshall, Dawn J; Whitesides, John F; Sawatzki, Kaitlin; Hua, Axin; Liu, Pinghuang; Tay, Matthew Z; Seaton, Kelly E; Shen, Xiaoying; Foulger, Andrew; Lloyd, Krissey E; Parks, Robert; Pollara, Justin; Ferrari, Guido; Yu, Jae-Sung; Vandergrift, Nathan; Montefiori, David C; Sobieszczyk, Magdalena E; Hammer, Scott; Karuna, Shelly; Gilbert, Peter; Grove, Doug; Grunenberg, Nicole; McElrath, M Juliana; Mascola, John R; Koup, Richard A; Corey, Lawrence; Nabel, Gary J; Morgan, Cecilia; Churchyard, Gavin; Maenza, Janine; Keefer, Michael; Graham, Barney S; Baden, Lindsey R; Tomaras, Georgia D; Haynes, Barton F

    2015-08-14

    An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy. Copyright © 2015, American Association for the Advancement of Science.

  2. [Subtype analysis of gp41 gene of HIV-1 among men who have sex with men in Zhengzhou].

    PubMed

    Song, Dan; Sun, Guo-qing; Zhang, Yan-min; Cui, Wei-guo; An, Hui-juan; An, Yu-hui; Wang, Zhe; Bao, Yu-zhou

    2012-08-01

    This study aimed to investigate the subtype distribution of gp41 gene of human immunodeficiency virus-1 (HIV-1) among men who have sex with men (MSM) in Zhengzhou. Thirty blood samples were collected from men who have sex with men infected by HIV. The complete gp41 gene was amplified by RT-PCR and nested-PCR and sequenced. All sequences were edited by Bioedit and subtyped with HIV sequence library US Los Alamos National Laboratory and online genotyping software provided by American National Center of Biotechnology Information. Phylogenetic analysis of gp41 gene was performed using the MEGA 3.1 software, and the genic dispersion rates among subtype of gp41 gene were analyzed. A total of eighteen gene sequences of HIV-1 gp41 gene were obtained from thirty men who have sex with men infected by HIV, which belonged to subtype CRF15-01B (50% (9/18)), CRF01-AE (22% (4/18)), CRF07-B (22% (4/18)) and B (6% (1/18)), respectively. The intersubtype HIV-1 strains aggregate with according reference strains. The genetic distance inter-subtype of subtype CRF15-01B, CRF01-AE and CRF07-B were 0.050 ± 0.007, 0.052 ± 0.009 and 0.082 ± 0.012, respectively. The prevalent subtypes of HIV-1 among among MSM in Zhengzhou was complicated and recombinant HIV-1 strains were the most prevalent strains.

  3. The HIV-1 gp41 ectodomain is cleaved by matriptase to produce a chemotactic peptide that acts through FPR2.

    PubMed

    Wood, Matthew P; Cole, Amy L; Eade, Colleen R; Chen, Li-Mei; Chai, Karl X; Cole, Alexander M

    2014-07-01

    Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. Whereas this chemotactic activity has been reported, it is not known how these peptides could be produced under biological conditions. The heptad repeat 1 (HR1) region of gp41 is exposed to the extracellular environment and could therefore be susceptible to proteolytic processing into smaller peptides. Matriptase is a serine protease expressed at the surface of most epithelia, including the prostate and mucosal surfaces. Here, we present evidence that matriptase efficiently cleaves the HR1 portion of gp41 into a 22-residue chemotactic peptide MAT-1, the sequence of which is highly conserved across HIV-1 clades. We found that MAT-1 induced migration of primary neutrophils and monocytes, the latter of which act as a cellular reservoir of HIV during early stage infection. We then used formyl peptide receptor 1 (FPR1) and FPR2 inhibitors, along with HEK 293 cells, to demonstrate that MAT-1 can induce chemotaxis specifically using FPR2, a receptor found on the surface of monocytes, macrophages and neutrophils. These findings are the first to identify a proteolytic cleavage product of gp41 with chemotactic activity and highlight a potential role for matriptase in HIV-1 transmission and infection at epithelial surfaces and within tissue reservoirs of HIV-1. © 2014 John Wiley & Sons Ltd.

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

    NASA Technical Reports Server (NTRS)

    Lim, K.; Ho, J. X.; Keeling, K.; Gilliland, G. L.; Ji, X.; Ruker, F.; Carter, D. C.

    1994-01-01

    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.

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

    NASA Technical Reports Server (NTRS)

    Lim, K.; Ho, J. X.; Keeling, K.; Gilliland, G. L.; Ji, X.; Ruker, F.; Carter, D. C.

    1994-01-01

    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.

  6. Conformational mapping of the N-terminal peptide of HIV-1 gp41 in lipid detergent and aqueous environments using 13C-enhanced Fourier transform infrared spectroscopy

    PubMed Central

    Gordon, Larry M.; Mobley, Patrick W.; Lee, William; Eskandari, Sepehr; Kaznessis, Yiannis N.; Sherman, Mark A.; Waring, Alan J.

    2004-01-01

    The N-terminal domain of HIV-1 glycoprotein 41,000 (gp41) participates in viral fusion processes. Here, we use physical and computational methodologies to examine the secondary structure of a peptide based on the N terminus (FP; residues 1–23) in aqueous and detergent environments. 12C-Fourier transform infrared (FTIR) spectroscopy indicated greater α-helix for FP in lipid-detergent sodium dodecyl sulfate (SDS) and aqueous phosphate-buffered saline (PBS) than in only PBS. 12C-FTIR spectra also showed disordered FP conformations in these two environments, along with substantial β-structure for FP alone in PBS. In experiments that map conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using FP peptides labeled with 13C-carbonyl. 13C-FTIR results on FP in SDS at low peptide loading indicated α-helix (residues 5 to 16) and disordered conformations (residues 1–4). Because earlier 13C-FTIR analysis of FP in lipid bilayers demonstrated α-helix for residues 1–16 at low peptide loading, the FP structure in SDS micelles only approximates that found for FP with membranes. Molecular dynamics simulations of FP in an explicit SDS micelle indicate that the fraying of the first three to four residues may be due to the FP helix moving to one end of the micelle. In PBS alone, however, electron microscopy of FP showed large fibrils, while 13C-FTIR spectra demonstrated antiparallel β-sheet for FP (residues 1–12), analogous to that reported for amyloid peptides. Because FP and amyloid peptides each exhibit plaque formation, α-helix to β-sheet interconversion, and membrane fusion activity, amyloid and N-terminal gp41 peptides may belong to the same superfamily of proteins. PMID:15044732

  7. Effect of point mutations in the N terminus of the lentivirus lytic peptide-1 sequence of human immunodeficiency virus type 1 transmembrane protein gp41 on Env stability.

    PubMed

    Lee, Sheau-Fen; Ko, Chiung-Yuan; Wang, Chin-Tien; Chen, Steve S-L

    2002-05-03

    To understand the role of the lentivirus lytic peptide-1 region of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp) 41 in viral infection, we examined the effects on virus replication of single amino acid deletions spanning this region in an infectious provirus of the HXB2 strain. Among the mutants analyzed, only the deletion of one of the two adjacent valine residues located at positions 832 and 833 (termed the Delta 833 mutant for simplicity) greatly reduced the steady-state, cell-associated levels of the Env precursor and gp120, as opposed to the wild-type virus. The altered Env phenotype resulted in severely impaired virus infectivity and gp120 incorporation into this mutant virion. Analyses of additional mutants with deletions at Ile-830, Ala-836, and Ile-840 demonstrated that the Delta 830 mutant exhibited the most significant inhibitory effect on Env steady-state expression. These results indicate that the N terminus of the lentivirus lytic peptide-1 region is critical for Env steady-state expression. Among the mutant viruses encoding Env proteins in which residues Val-832 and Val-833 were individually substituted by nonconserved amino acids Ala, Ser, or Pro, which were expected to disrupt the alpha-helical structure in the increasingly severe manner of Pro > Ser > Ala, only the 833P mutant exhibited significantly reduced steady-state Env expression. Pulse labeling and pulse-chase studies demonstrated that the Delta 830, Delta 833, and 833P mutants of Env proteins degraded more rapidly in a time-dependent manner after biosynthesis than did the wild-type Env. The results indicate that residue 830 and 833 mutations are likely to induce a conformational change in Env that targets the mutant protein for cellular degradation. Our study has implications about the structural determinants located at the N terminus of the lentivirus lytic peptide-1 sequence of gp41 that affect the fate of Env in virus-infected cells.

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

    PubMed Central

    Lim, K.; Ho, J. X.; Keeling, K.; Gilliland, G. L.; Ji, X.; Rüker, F.; Carter, D. C.

    1994-01-01

    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. PMID:7538846

  9. Expression and immunological characterization of cardamom mosaic virus coat protein displaying HIV gp41 epitopes.

    PubMed

    Damodharan, Subha; Gujar, Ravindra; Pattabiraman, Sathyamurthy; Nesakumar, Manohar; Hanna, Luke Elizabeth; Vadakkuppattu, Ramanathan D; Usha, Ramakrishnan

    2013-05-01

    The coat protein of cardamom mosaic virus (CdMV), a member of the genus Macluravirus, assembles into virus-like particles when expressed in an Escherichia coli expression system. The N and C-termini of the coat protein were engineered with the Kennedy peptide and the 2F5 and 4E10 epitopes of gp41 of HIV. The chimeric proteins reacted with sera from HIV positive persons and also stimulated secretion of cytokines by peripheral blood mononuclear cells from these persons. Thus, a system based on the coat protein of CdMV can be used to display HIV-1 antigens. © 2013 The Societies and Wiley Publishing Asia Pty Ltd.

  10. Multimerized HIV-gp41-derived peptides as fusion inhibitors and vaccines.

    PubMed

    Nomura, Wataru; Mizuguchi, Takaaki; Tamamura, Hirokazu

    2016-11-04

    To date, several antigens based on the amino-terminal leucine/isoleucine heptad repeat (NHR) region of an HIV-1 envelope protein gp41 and fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of gp41 have been reported. We have developed a synthetic antigen targeting the membrane-fusion mechanism of HIV-1. This uses a template designed with C3-symmetric linkers and mimics the trimeric form of the NHR-derived peptide N36. The antiserum obtained by immunization of the N36 trimeric antigen binds preferentially to the N36 trimer and blocks HIV-1 infection effectively, compared with the antiserum obtained by immunization of the N36 monomer. Using another template designed with different C3-symmetric linkers, we have also developed a synthetic peptide mimicking the trimeric form of the CHR-derived peptide C34, with ∼100 times the inhibitory activity against the HIV-1 fusion mechanism than that of the monomer C34 peptide. A dimeric derivative of C34 has potent inhibitory activity at almost the same levels as this C34 trimer mimic, suggesting that presence of a dimeric form of C34 is structurally critical for fusion inhibitors. As examples of rising mid-size drugs, this review describes an effective strategy for the design of HIV vaccines and fusion inhibitors based on a relationship with the native structure of proteins involved in HIV fusion mechanisms. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 622-628, 2016.

  11. Design, Synthesis, and Biological Evaluation of N-Carboxyphenylpyrrole Derivatives as Potent HIV Fusion Inhibitors Targeting gp41

    PubMed Central

    Liu, Kun; Lu, Hong; Hou, Ling; Qi, Zhi; Teixeira, Cátia; Barbault, Florent; Fan, Bo-Tao; Liu, Shuwen; Jiang, Shibo; Xie, Lan

    2009-01-01

    Based on the structures of small-molecule hits targeting the HIV-1 gp41, N-(4-carboxy-3-hydroxy)phenyl-2,5-dimethylpyrrole (2, NB-2) and N-(3-carboxy-4-chloro)phenylpyrrole (A1, NB-64), 42 N-carboxyphenylpyrrole derivatives in two categories (A and B series) were designed and synthesized. We found that 11 compounds exhibited promising anti-HIV-1 activity at micromolar level and their antiviral activity was correlated with their inhibitory activity on gp41 six-helix bundle formation, suggesting that these compounds block HIV fusion and entry by disrupting gp41 core formation. The structure-activity relationship and molecular docking analysis revealed that the carboxyl group could interact with either Arg579 or Lys574 to form salt bridges and two methyl groups on the pyrrole ring were favorable for interaction with the residues in gp41 pocket. The most active compound, N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrrole (A12), partially occupied the deep hydrophobic pocket, suggesting that enlarging the molecular size of A12 could improve its binding affinity and anti-HIV-1 activity for further development as a small-molecule HIV fusion and entry inhibitor. PMID:19053778

  12. Interaction between phage BFK20 helicase gp41 and its host Brevibacterium flavum primase DnaG.

    PubMed

    Solteszova, Barbora; Halgasova, Nora; Bukovska, Gabriela

    2015-01-22

    Protein-protein interactions have been identified between the replication proteins of corynephage BFK20 and its host Brevibacterium flavum CCM 251. We tested the interactions between phage proteins gp40, gp41, gp42, gp43 and gp44 and between these phage proteins and host replication proteins DnaZX, DnaN, Dnaδ, DnaG, DnaA, RecF, TrxC, TrxB1 and SSB using a bacterial two-hybrid system. Phage proteins gp41, gp42 and gp43 self-associated, demonstrating that these proteins are oligomers in vivo. Interactions were also detected between phage protein gp41 and host proteins DnaZX, DnaN, Dnaδ, DnaG and SSB. β-galactosidase activity measurements showed that the strongest interaction was between gp41 and DnaG. The interaction was studied further using 2-dimensional blue native SDS-polyacrylamide gel electrophoresis and Western blot analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. The crystal structure of HIV CRF07 B'/C gp41 reveals a hyper-mutant site in the middle of HR2 heptad repeat.

    PubMed

    Du, Jiansen; Xue, Hailing; Ma, Jing; Liu, Fang; Zhou, Jianhua; Shao, Yiming; Qiao, Wentao; Liu, Xinqi

    2013-11-01

    HIV CRF07 B'/C is a strain circulating mainly in northwest region of China. The gp41 region of CRF07 is derived from a clade C virus. In order to compare the difference of CRF07 gp41 with that of typical clade B virus, we solved the crystal structure of the core region of CRF07 gp41. Compared with clade B gp41, CRF07 gp41 evolved more basic and hydrophilic residues on its helix bundle surface. Based on sequence alignment, a hyper-mutant cluster located in the middle of HR2 heptads repeat was identified. The mutational study of these residues revealed that this site is important in HIV mediated cell-cell fusion and plays critical roles in conformational changes during viral invasion.

  14. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

    NASA Technical Reports Server (NTRS)

    He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  15. Analysis of a subclass-restricted HIV-1 gp41 epitope by omission peptides.

    PubMed Central

    Mathiesen, T; Chiodi, F; Broliden, P A; Albert, J; Houghten, R A; Utter, G; Wahren, B; Norrby, E

    1989-01-01

    To define the amino acids involved in IgG subclass reactivity to two overlapping HIV-1 gp41 (E34/32; amino acid positions 582-613) peptides, sera from 18 HIV-infected individuals were studied. Peptides mimicking E34 but with single amino acid deletions or glycine substitutions were used to define the amino acid residues necessary for antibody binding. Two dominating immunogenic epitopes, containing highly hydrophilic amino acids, were found on the original peptide. Further analysis was undertaken with two corresponding omission sets of dodecapeptides representing halves of the complete E34 plus a terminal cystein peptide. The subclass reactivities usually differed between the patients with regard to the epitopes with which the different IgG subclasses reacted and also to the importance of different amino acids in antibody binding. The 600 glycine and the 601 lysine were involved in the binding of all IgG1, 2 and 4 and most IgG3. The development of E34/32-reactive IgM and IgG subclasses showed different patterns in four patients with primary HIV infections, contradicting the existence of a general pattern for the development of IgG subclasses to this peptide. The findings suggest that different progenitor clones are selected for synthesis of the different subclasses. PMID:2472353

  16. Structural Plasticity in the Topology of the Membrane-Interacting Domain of HIV-1 gp41

    PubMed Central

    Kyrychenko, Alexander; Freites, J. Alfredo; He, Jing; Tobias, Douglas J.; Wimley, William C.; Ladokhin, Alexey S.

    2014-01-01

    We use a number of computational and experimental approaches to investigate the membrane topology of the membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein. Several putative transmembrane regions are identified using hydrophobicity analysis based on the Wimley-White scales, including the membrane-proximal external region (MPER). The MPER region is an important target for neutralizing anti-HIV monoclonal antibodies and is believed to have an interfacial topology in the membrane. To assess the possibility of a transmembrane topology of MPER, we examined the membrane interactions of a peptide corresponding to a 22-residue stretch of the MPER sequence (residues 662–683) using fluorescence spectroscopy and oriented circular dichroism. In addition to the previously reported interfacial location, we identify a stable transmembrane conformation of the peptide in synthetic lipid bilayers. All-atom molecular dynamics simulations of the MPER-derived peptide in a lipid bilayer demonstrate a stable helical structure with an average tilt of 24 degrees, with the five tryptophan residues sampling different environments inside the hydrocarbon core of the lipid bilayer, consistent with the observed spectral properties of intrinsic fluorescence. The degree of lipid bilayer penetration obtained by computer simulation was verified using depth-dependent fluorescence quenching of a selectively attached fluorescence probe. Overall, our data indicate that the MPER sequence can have at least two stable conformations in the lipid bilayer, interfacial and transmembrane, and suggest a possibility that external perturbations can switch the topology during physiological functioning. PMID:24507601

  17. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

    NASA Technical Reports Server (NTRS)

    He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  18. Structural plasticity in the topology of the membrane-interacting domain of HIV-1 gp41.

    PubMed

    Kyrychenko, Alexander; Freites, J Alfredo; He, Jing; Tobias, Douglas J; Wimley, William C; Ladokhin, Alexey S

    2014-02-04

    We use a number of computational and experimental approaches to investigate the membrane topology of the membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein. Several putative transmembrane regions are identified using hydrophobicity analysis based on the Wimley-White scales, including the membrane-proximal external region (MPER). The MPER region is an important target for neutralizing anti-HIV monoclonal antibodies and is believed to have an interfacial topology in the membrane. To assess the possibility of a transmembrane topology of MPER, we examined the membrane interactions of a peptide corresponding to a 22-residue stretch of the MPER sequence (residues 662-683) using fluorescence spectroscopy and oriented circular dichroism. In addition to the previously reported interfacial location, we identify a stable transmembrane conformation of the peptide in synthetic lipid bilayers. All-atom molecular dynamics simulations of the MPER-derived peptide in a lipid bilayer demonstrate a stable helical structure with an average tilt of 24 degrees, with the five tryptophan residues sampling different environments inside the hydrocarbon core of the lipid bilayer, consistent with the observed spectral properties of intrinsic fluorescence. The degree of lipid bilayer penetration obtained by computer simulation was verified using depth-dependent fluorescence quenching of a selectively attached fluorescence probe. Overall, our data indicate that the MPER sequence can have at least two stable conformations in the lipid bilayer, interfacial and transmembrane, and suggest a possibility that external perturbations can switch the topology during physiological functioning.

  19. F(ab')2 fragment of a gp41 NHR-trimer-induced IgM monoclonal antibody neutralizes HIV-1 infection and blocks viral fusion by targeting the conserved gp41 pocket.

    PubMed

    Lu, Lu; Wei, Meili; Chen, Yanxia; Xiong, Weiliang; Yu, Fei; Qi, Zhi; Jiang, Shibo; Pan, Chungen

    2013-11-01

    Using a recombinant protein N46FdFc that mimics the HIV-1 gp41 N-helix trimer to immunize mice, we identified the first IgM monoclonal antibody 18D3 that specifically bound to the conserved gp41 pocket. Its F(ab')2 fragment potently inhibited HIV-1 Env-mediated cell-cell fusion and neutralized infection by laboratory-adapted and primary HIV-1 isolates with different subtypes and tropism, including the T20-resistant variants. This F(ab')2 fragment can be used to develop a bispecific broad neutralizing monoclonal antibody or HIV-1 inactivator as a novel immunotherapeutic for treatment and prevention of HIV-1 infection.

  20. Conserved positive selection signals in gp41 across multiple subtypes and difference in selection signals detectable in gp41 sequences sampled during acute and chronic HIV-1 subtype C infection

    PubMed Central

    Bandawe, Gama P; Martin, Darren P; Treurnicht, Florette; Mlisana, Koleka; Karim, Salim S Abdool; Williamson, Carolyn

    2008-01-01

    Background The high diversity of HIV variants driving the global AIDS epidemic has caused many to doubt whether an effective vaccine against the virus is possible. However, by identifying the selective forces that are driving the ongoing diversification of HIV and characterising their genetic consequences, it may be possible to design vaccines that pre-empt some of the virus' more common evasion tactics. One component of such vaccines might be the envelope protein, gp41. Besides being targeted by both the humoral and cellular arms of the immune system this protein mediates fusion between viral and target cell membranes and is likely to be a primary determinant of HIV transmissibility. Results Using recombination aware analysis tools we compared site specific signals of selection in gp41 sequences from different HIV-1 M subtypes and circulating recombinant forms and identified twelve sites evolving under positive selection across multiple major HIV-1 lineages. To identify evidence of selection operating during transmission our analysis included two matched datasets sampled from patients with acute or chronic subtype C infections. We identified six gp41 sites apparently evolving under different selection pressures during acute and chronic HIV-1 infections. These sites mostly fell within functional gp41 domains, with one site located within the epitope recognised by the broadly neutralizing antibody, 4E10. Conclusion Whereas these six sites are potentially determinants of fitness and are therefore good candidate targets for subtype-C specific vaccines, the twelve sites evolving under diversifying selection across multiple subtypes might make good candidate targets for broadly protective vaccines. PMID:19025632

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

    PubMed Central

    Pejchal, Robert; Gach, Johannes S.; Brunel, Florence M.; Cardoso, Rosa M.; Stanfield, Robyn L.; Dawson, Philip E.; Burton, Dennis R.; Zwick, Michael B.; Wilson, Ian A.

    2009-01-01

    The membrane-proximal external region (MPER) of the human immunodeficiency virus (HIV) envelope glycoprotein (gp41) is critical for viral fusion and infectivity and is the target of three of the five known broadly neutralizing HIV type 1 (HIV-1) antibodies, 2F5, Z13, and 4E10. Here, we report the crystal structure of the Fab fragment of Z13e1, an affinity-enhanced variant of monoclonal antibody Z13, in complex with a 12-residue peptide corresponding to the core epitope (W670NWFDITN677) at 1.8-Å resolution. The bound peptide adopts an S-shaped conformation composed of two tandem, perpendicular helical turns. This conformation differs strikingly from the α-helical structure adopted by an overlapping MPER peptide bound to 4E10. Z13e1 binds to an elbow in the MPER at the membrane interface, making relatively few interactions with conserved aromatics (Trp672 and Phe673) that are critical for 4E10 recognition. The comparison of the Z13e1 and 4E10 epitope structures reveals a conformational switch such that neutralization can occur by the recognition of the different conformations and faces of the largely amphipathic MPER. The Z13e1 structure provides significant new insights into the dynamic nature of the MPER, which likely is critical for membrane fusion, and it has significant implications for mechanisms of HIV-1 neutralization by MPER antibodies and for the design of HIV-1 immunogens. PMID:19515770

  2. Evaluation of “Credit Card” Libraries for Inhibition of HIV-1 gp41 Fusogenic Core Formation

    PubMed Central

    Xu, Yang; Lu, Hong; Kennedy, Jack P.; Yan, Xuxia; McAllister, Laura; Yamamoto, Noboru; Moss, Jason A.; Boldt, Grant E.; Jiang, Shibo; Janda, Kim D.

    2008-01-01

    Protein-protein interactions are of critical importance in biological systems and small molecule modulators of such protein recognition and intervention processes are of particular interests. To investigate this area of research, we have synthesized small molecule libraries that can disrupt a number of biologically relevant protein-protein interactions. These library members are designed upon planar motifs, appended with a variety of chemical functions, which we have termed as “credit-card” structures. From two of our “credit-card” libraries, a series of molecules were uncovered which act as inhibitors against the HIV-1 gp41 fusogenic 6-helix bundle core formation, viral antigen p24 formation and cell-cell fusion at low micromolar concentrations. From the high-throughput screening assays we utilized, a selective index (SI) value of 4.2 was uncovered for compound 2261, which bodes well for future structure activity investigations and the design of more potent gp41 inhibitors. PMID:16827565

  3. Surface Glycoproteins of Exosomes Shed by Myeloid-Derived Suppressor Cells Contribute to Function.

    PubMed

    Chauhan, Sitara; Danielson, Steven; Clements, Virginia; Edwards, Nathan; Ostrand-Rosenberg, Suzanne; Fenselau, Catherine

    2017-01-06

    In this report, we use a proteomic strategy to identify glycoproteins on the surface of exosomes derived from myeloid-derived suppressor cells (MDSCs), and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSCs. We report successful modification of a surface chemistry method for use with exosomes and identify 21 surface N-glycoproteins on exosomes released by mouse mammary carcinoma-induced MDSCs. These glycoprotein identities and functionalities are compared with 93 N-linked glycoproteins identified on the surface of the parental cells. As with the lysate proteomes examined previously, the exosome surface N-glycoproteins are primarily a subset of the glycoproteins on the surface of the suppressor cells that released them, with related functions and related potential as therapeutic targets. The "don't eat me" molecule CD47 and its binding partners thrombospondin-1 (TSP1) and signal regulatory protein α (SIRPα) were among the surface N-glycoproteins detected. Functional bioassays using antibodies to these three molecules demonstrated that CD47, TSP1, and to a lesser extent SIRPα facilitate exosome-mediated MDSC chemotaxis and migration.

  4. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    SciTech Connect

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

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

    PubMed

    Ye, Ling; Wen, Zhiyuan; Dong, Ke; Wang, Xi; Bu, Zhigao; Zhang, Huizhong; Compans, Richard W; Yang, Chinglai

    2011-01-01

    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.

  6. pH-dependent vesicle fusion induced by the ectodomain of the human immunodeficiency virus membrane fusion protein gp41: Two kinetically distinct processes and fully-membrane-associated gp41 with predominant β sheet fusion peptide conformation.

    PubMed

    Ratnayake, Punsisi U; Sackett, Kelly; Nethercott, Matthew J; Weliky, David P

    2015-01-01

    The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region hypothesized to bind to the host cell membrane followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The present study focuses on the large gp41 ectodomain constructs "Hairpin" (HP) containing NHR+loop+CHR and "FP-Hairpin" (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH4 where the protein is positively-charged but do not induce fusion at pH7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. There are two kinetically distinct fusion processes at pH4: (1) a faster ~100 ms⁻¹ process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms⁻¹ process with extent strongly inversely correlated with this charge. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region between the two membranes. Previous solid-state NMR (SSNMR) of membrane-associated FP-HP has supported protein oligomers with FP's in an intermolecular antiparallel sheet. There was an additional population of molecules with α helical FPs and the samples likely contained a mixture of membrane-bound and -unbound proteins. For the present study, samples were prepared with fully membrane-bound FP-HP and subsequent SSNMR showed dominant β FP conformation at both low and neutral pH. SSNMR also showed close contact of the FP with the lipid headgroups at both low and neutral pH whereas the NHR+CHR regions had contact at low pH and were more distant at neutral p

  7. Amplification of the Gp41 gene for detection of mutations conferring resistance to HIV-1 fusion inhibitors on genotypic assay

    NASA Astrophysics Data System (ADS)

    Tanumihardja, J.; Bela, B.

    2017-08-01

    Fusion inhibitors have potential for future use in HIV control programs in Indonesia, so the capacity to test resistance to such drugs needs to be developed. Resistance-detection with a genotypic assay began with amplification of the target gene, gp41. Based on the sequence of the two most common HIV subtypes in Indonesia, AE and B, a primer pair was designed. Plasma samples containing both subtypes were extracted to obtain HIV RNA. Using PCR, the primer pair was used to produce the amplification product, the identity of which was checked based on length under electrophoresis. Eleven plasma samples were included in this study. One-step PCR using the primer pair was able to amplify gp41 from 54.5% of the samples, and an unspecific amplification product was seen in 1.1% of the samples. Amplification failed in 36.4% of the samples, which may be due to an inappropriate primer sequence. It was also found that the optimal annealing temperature for producing the single expected band was 57.2 °C. With one-step PCR, the designed primer pair amplified the HIV-1 gp41 gene from subtypes AE and B. However, further research should be done to determine the conditions that will increase the sensitivity and specificity of the amplification process.

  8. Downregulation of human immunodeficiency virus type 1 Gag expression by a gp41 cytoplasmic domain fusion protein

    SciTech Connect

    Chan, W.-E.; Chen, Steve S.-L. . E-mail: schen@ibms.sinica.edu.tw

    2006-05-10

    The cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) envelope (Env) transmembrane protein gp41 interacts with the viral matrix MA protein, which facilitates incorporation of the trimeric Env complex into the virus. It is thus feasible to design an anti-HIV strategy targeting this interaction. We herein describe that Gag expression can be downregulated by a cytoplasmic domain fusion protein of the Env transmembrane protein, {beta}-galactosidase ({beta}-gal)/706-856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli {beta}-gal. This mediator depleted intracellular Gag molecules in a dose-dependent manner. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag and {beta}-gal/706-856 had stable interactions and formed aggregated complexes in perinuclear, intracellular sites. Pulse-chase and cycloheximide chase analyses demonstrated that this mediator enhanced unmyristylated Gag degradation. The results demonstrate a novel mode of HIV-1 Gag downregulation by directing Gag to an intracellular site via the interaction of Gag with a gp41 cytoplasmic domain fusion protein.

  9. XTT formazan widely used to detect cell viability inhibits HIV type 1 infection in vitro by targeting gp41.

    PubMed

    Zhao, Qian; Ernst, Justin T; Hamilton, Andrew D; Debnath, Asim K; Jiang, Shibo

    2002-09-20

    XTT can be metabolically reduced by mitochondrial dehydrogenase in viable cells to a water-soluble formazan product. Thus XTT has been widely used to evaluate cell viability and to screen anti-HIV agents and the cytotoxicity of these agents. The present studies demonstrated that XTT formazan derived from XTT in cell culture significantly inhibits the fusion of HIV-1-infected cells with uninfected cells. Synthetic XTT formazan effectively inhibited the replication of laboratory-adapted and primary HIV-1 isolates and cell-to-cell fusion with low cytotoxicity. It blocks the six-helix bundle formation between peptides derived from the N- and C-terminal heptad repeat regions of the gp41 ectodomain (designated N- and C-peptides, respectively). Analysis by a computer-aided docking program indicates that XTT formazan may bind to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled coil of gp41. These results suggest that XTT formazan inhibits HIV-1 entry by targeting the alpha-helical coiled-coil domain of gp41. This small molecular nonpeptide antiviral compound can be used as a lead for designing more effective HIV-1 entry inhibitors targeting the fusion stage of HIV-1 infection. But because XTT formazan itself has anti-HIV-1 activity, caution should be exercised when XTT is used to evaluate HIV-1 infectivity.

  10. The amino-terminal fusion domain peptide of human immunodeficiency virus type 1 gp41 inserts into the sodium dodecyl sulfate micelle primarily as a helix with a conserved glycine at the micelle-water interface.

    PubMed Central

    Chang, D K; Cheng, S F; Chien, W J

    1997-01-01

    A peptide based on the N-terminal fusion domain of gp41 of human immunodeficiency virus type 1 (HIV-1) and its tryptophan analog were synthesized to examine the secondary structure in the micellar environment. Nuclear magnetic resonance (NMR), circular dichroism and electron paramagnetic resonance experiments indicated that the gp41 fusion peptide inserted into the micelle primarily as a helix (59%), with substantial beta-structure (26.7%). Deep penetration of the peptide into the apolar hydrocarbon core was supported by the results of fluorescence experiments in which the tryptophan analog exhibited a blue shift of about 30 nm in the presence of a sodium dodecyl sulfate micelle, in 1,2-dimyristoyl-rac-glycero-3-phosphocholine, and in 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine vesicular solutions. The results of spin label-attenuated 1H resonance experiments show that the region C-terminal to G16, which contains a turn structure, exhibited substantial interaction with the micelle, suggesting that it lies on the surface of micelle. Molecular simulation based on data from NMR experiments revealed a flexible hinge at residues 15 and 16 (alanine and glycine, respectively) from the N terminus of the peptide located at the micelle-solution interface. The highly conserved A15-G16 dipeptide may play a role in the function of fusion domain of HIV-1 envelope glycoprotein. PMID:9261381

  11. Determination of the equilibrium micelle-inserting position of the fusion peptide of gp41 of human immunodeficiency virus type 1 at amino acid resolution by exchange broadening of amide proton resonances.

    PubMed

    Chang, D K; Cheng, S F

    1998-11-01

    The exchange broadening of backbone amide proton resonances of a 23-mer fusion peptide of the transmembrane subunit of HIV-1 envelope glycoprotein gp41, gp41-FP, was investigated at pH 5 and 7 at room temperature in perdeuterated sodium dodecyl sulfate (SDS) micellar solution. Comparison of resonance peaks for these pHs revealed an insignificant change in exchange rate between pH 5 and 7 for amide protons of residues 4 through 14, while the exchange rate increase at neutral pH was more prominent for amide protons of the remaining residues, with peaks from some protons becoming undetectable. The relative insensitivity to pH of the exchange for the amide protons of residues 4 through 14 is attributable to the drastic reduction in [OH-] in the micellar interior, leading to a decreased exchange rate. The A15-G16 segment represents a transition between these two regimes. The data are thus consistent with the notion that the peptide inserts into the hydrophobic core of a membrane-like structure and the A15-G16 dipeptide is located at the micellar-aqueous boundary.

  12. Analysis of the Human Immunodeficiency Virus Type 1 gp41 Membrane Proximal External Region Arrayed on Hepatitis B Surface Antigen Particles

    PubMed Central

    Phogat, S; K, Svehla; M, Tang; A, Spadaccini; J, Muller; J, Mascola; Berkower; R, Wyatt

    2009-01-01

    Vaccine immunogens derived from the envelope glycoproteins of the human immunodeficiency virus type 1 (HIV-1) that elicit broad neutralizing antibodies remains an elusive goal. The highly conserved 30 amino acid membrane proximal external region (MPER) of HIV gp41 contains the hydrophobic epitopes for two rare HIV-1 broad cross-reactive neutralizing antibodies, 2F5 and 4E10. Both these antibodies possess relatively hydrophobic HCDR3 loops and demonstrate enhanced binding to their epitopes in the context of the native gp160 precursor envelope glycoprotein by the intimate juxtaposition of a lipid membrane. The Hepatitis B surface antigen (HBsAg) S1 protein forms nanoparticles that can be utilized both as an immunogenic array of the MPER and to provide the lipid environment needed for enhanced 2F5 and 4E10 binding. We show that recombinant HBsAg particles with MPER (HBsAg-MPER) appended at the C-terminus of the S1 protein are recognized by 2F5 and 4E10 with high affinity compared to positioning the MPER at the N-terminus or the extracellular loop (ECL) of S1. Addition of C-terminal hydrophobic residues derived from the HIV-1 Env transmembrane region further enhances recognition of the MPER by both 2F5 and 4E10. Delipidation of the HBsAg-MPER particles decreases 2F5 and 4E10 binding and subsequent reconstitution with synthetic lipids restores optimal binding. Inoculation of the particles into small animals raised cross-reactive antibodies that recognize both the MPER and HIV-1 gp160 envelope glycoproteins expressed on the cell surface; however, no neutralizing activity could be detected. Prime:boost immunization of the HBsAg-MPER particles in sequence with HIV envelope glycoprotein proteoliposomes (Env-PLs) did not raise neutralizing antibodies that could be mapped to the MPER region. However, the Env-PLs did raise anti-Env antibodies that had the ability to neutralize selected HIV-1 isolates. The first generation HBsAg-MPER particles represent a unique means to

  13. Human immunodeficiency virus type 1 gp 41 mutations in proviral DNA among antiretroviral treatment-naive individuals from India.

    PubMed

    Sen, Sourav; Tripathy, S P; Sahni, A K; Gupta, R M; Kapila, K; Chopra, G S; Chimanpure, Vaishali M; Patil, Ajit A; Paranjape, R S

    2009-05-01

    The HIV-1 gp41 has been identified as an important target for the immune response, for the development of antiviral and vaccine strategies, and for epidemiologic studies. This study describes the HIV-1 env gp41 region mutations, associated with enfuvirtide (ENF) resistance, in proviral DNA from PBMCs in antiretroviral treatment-naive individuals from Pune, India. Twenty-one antiretroviral drug-naive chronically HIV-1-infected individuals were enrolled. The study sequences belonged to subtype C (n = 17), subtype A1 (n = 2), and CRF_AE (n = 2). In subtype B-infected individuals, the various HR1 region substitutions in env gp41 that have been associated with ENF resistance include A30V, L33S/T/V, L34M, G36D/E/S/V, I37T/K/V, V38A/M/E/G, Q39R, Q40H, N42T/D, N43D/K/S, L44M, L45M, R46M, L54M, and Q56K/R as well as N126K and S138A in the HR2 region. The study sequences did not reveal any ENF resistance-associated mutations at env gp41 amino acid positions: 36 to 45. The presence of L54M and Q56K in combination is associated with 5-fold reduced sensitivity to inhibition by ENF. The mutation L54M was seen in seven subtype C and two CRF_AE study sequences. Q56K was observed in a subtype A1 sequence. All the study sequences harbored N42S, a natural polymorphism associated with increased susceptibility to ENF. Of the mutations V38A and N140I, known to provide immunologic gain, the latter was observed in four subtype C sequences. This is the first study from India highlighting the presence of certain mutations in Indian subtype C env gp41, which may play a role in the evolution of subtype-specific variations in the resistance to ENF and associated immune response.

  14. Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

    PubMed Central

    Ringe, Rajesh P.; Sanders, Rogier W.; Yasmeen, Anila; Kim, Helen J.; Lee, Jeong Hyun; Cupo, Albert; Korzun, Jacob; Derking, Ronald; van Montfort, Thijs; Julien, Jean-Philippe; Wilson, Ian A.; Klasse, Per Johan; Ward, Andrew B.; Moore, John P.

    2013-01-01

    We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp)140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120–gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a gp120–gp41 disulfide bond and an I559P gp41 change, together designated SOSIP), was assessed. Without SOSIP changes, cleaved trimers disintegrate into their gp120 and gp41-ectodomain (gp41ECTO) components; when only the disulfide bond is present, they dissociate into gp140 monomers. Uncleaved gp140s remain trimeric whether SOSIP substitutions are present or not. However, negative-stain electron microscopy reveals that only cleaved trimers form homogeneous structures resembling native Env spikes on virus particles. In contrast, uncleaved trimers are highly heterogeneous, adopting a variety of irregular shapes, many of which appear to be gp120 subunits dangling from a central core that is presumably a trimeric form of gp41ECTO. Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structure-dependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41ECTO epitopes that are occluded on cleaved trimers. These findings have adverse implications for using soluble, uncleaved trimers for structural studies, and the rationale for testing uncleaved trimers as vaccine candidates also needs to be reevaluated. PMID:24145402

  15. Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif

    PubMed Central

    Molinos-Albert, Luis M.; Bilbao, Eneritz; Agulló, Luis; Marfil, Silvia; García, Elisabet; Concepción, Maria Luisa Rodríguez de la; Izquierdo-Useros, Nuria; Vilaplana, Cristina; Nieto-Garai, Jon A.; Contreras, F.-Xabier; Floor, Martin; Cardona, Pere J.; Martinez-Picado, Javier; Clotet, Bonaventura; Villà-Freixa, Jordi; Lorizate, Maier; Carrillo, Jorge; Blanco, Julià

    2017-01-01

    The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants. PMID:28084464

  16. The crystal structure of HIV CRF07 B′/C gp41 reveals a hyper-mutant site in the middle of HR2 heptad repeat

    SciTech Connect

    Du, Jiansen; Xue, Hailing; Ma, Jing; Liu, Fang; Zhou, Jianhua; Shao, Yiming; Liu, Xinqi

    2013-11-15

    HIV CRF07 B′/C is a strain circulating mainly in northwest region of China. The gp41 region of CRF07 is derived from a clade C virus. In order to compare the difference of CRF07 gp41 with that of typical clade B virus, we solved the crystal structure of the core region of CRF07 gp41. Compared with clade B gp41, CRF07 gp41 evolved more basic and hydrophilic residues on its helix bundle surface. Based on sequence alignment, a hyper-mutant cluster located in the middle of HR2 heptads repeat was identified. The mutational study of these residues revealed that this site is important in HIV mediated cell–cell fusion and plays critical roles in conformational changes during viral invasion. - Highlights: • We solved the crystal structure of HIV CRF07 gp41 core region. • A hyper-mutant cluster in the middle of HR2 heptads repeat was identified. • The hyper-mutant site is important in HIV-cell fusion. • The model will help to understand the HIV fusion process.

  17. HIV-1 envelope gp41 antibodies can originate from terminal ileum B cells that share cross-reactivity with commensal bacteria.

    PubMed

    Trama, Ashley M; Moody, M Anthony; Alam, S Munir; Jaeger, Frederick H; Lockwood, Bradley; Parks, Robert; Lloyd, Krissey E; Stolarchuk, Christina; Scearce, Richard; Foulger, Andrew; Marshall, Dawn J; Whitesides, John F; Jeffries, Thomas L; Wiehe, Kevin; Morris, Lynn; Lambson, Bronwen; Soderberg, Kelly; Hwang, Kwan-Ki; Tomaras, Georgia D; Vandergrift, Nathan; Jackson, Katherine J L; Roskin, Krishna M; Boyd, Scott D; Kepler, Thomas B; Liao, Hua-Xin; Haynes, Barton F

    2014-08-13

    Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

  18. HIV-1 Envelope gp41 Antibodies Can Originate from Terminal Ileum B Cells that Share Cross-Reactivity with Commensal Bacteria

    PubMed Central

    Trama, Ashley M.; Moody, M. Anthony; Alam, S. Munir; Jaeger, Frederick H.; Lockwood, Bradley; Parks, Robert; Lloyd, Krissey E.; Stolarchuk, Christina; Scearce, Richard; Foulger, Andrew; Marshall, Dawn J.; Whitesides, John F.; Jeffries, Thomas L.; Wiehe, Kevin; Morris, Lynn; Lambson, Bronwen; Soderberg, Kelly; Hwang, Kwan-Ki; Tomaras, Georgia D.; Vandergrift, Nathan; Jackson, Katherine J.L.; Roskin, Krishna M.; Boyd, Scott D.; Kepler, Thomas B.; Liao, Hua-Xin; Haynes, Barton F.

    2014-01-01

    SUMMARY Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env. PMID:25121750

  19. Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif.

    PubMed

    Molinos-Albert, Luis M; Bilbao, Eneritz; Agulló, Luis; Marfil, Silvia; García, Elisabet; Concepción, Maria Luisa Rodríguez de la; Izquierdo-Useros, Nuria; Vilaplana, Cristina; Nieto-Garai, Jon A; Contreras, F-Xabier; Floor, Martin; Cardona, Pere J; Martinez-Picado, Javier; Clotet, Bonaventura; Villà-Freixa, Jordi; Lorizate, Maier; Carrillo, Jorge; Blanco, Julià

    2017-01-13

    The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.

  20. Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure

    SciTech Connect

    Banerjee, Saikat; Shi, Heliang; Habte, Habtom H.; Qin, Yali; Cho, Michael W.

    2016-03-15

    The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; {sup 671}NWFDITNWLWYIK{sup 683}) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-∆10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies. - Highlights: • Four gp41 MPER-based immunogens that resemble fusion intermediates were generated. • C-terminal region of MPER that contains 4E10/10E8 epitopes was highly immunogenic. • Altering 6HB structure can influence immunogenic properties of the MPER. • Induced antibodies targeted multiple residues critical for 4E10/10E8 binding. • Development of immunogens based on fusion intermediates is a promising strategy.

  1. Membrane structure correlates to function of LLP2 on the cytoplasmic tail of HIV-1 gp41 protein.

    PubMed

    Boscia, Alexander L; Akabori, Kiyotaka; Benamram, Zachary; Michel, Jonathan A; Jablin, Michael S; Steckbeck, Jonathan D; Montelaro, Ronald C; Nagle, John F; Tristram-Nagle, Stephanie

    2013-08-06

    Mutation studies previously showed that the lentivirus lytic peptide (LLP2) sequence of the cytoplasmic C-terminal tail of the HIV-1 gp41 envelope protein inhibited viral-initiated T-cell death and T-cell syncytium formation, at which time in the HIV life cycle the gp41 protein is embedded in the T-cell membrane. In striking contrast, the mutants did not affect virion infectivity, during which time the gp41 protein is embedded in the HIV envelope membrane. To examine the role of LLP2/membrane interactions, we applied synchrotron x-radiation to determine structure of hydrated membranes. We focused on WT LLP2 peptide (+3 charge) and MX2 mutant (-1 charge) with membrane mimics for the T-cell and the HIV-1 membranes. To investigate the influence of electrostatics, cholesterol content, and peptide palmitoylation, we also studied three other LLP2 variants and HIV-1 mimics without negatively charged lipids or cholesterol as well as extracted HIV-1 lipids. All LLP2 peptides bound strongly to T-cell membrane mimics, as indicated by changes in membrane structure and bending. In contrast, none of the weakly bound LLP2 variants changed the HIV-1 membrane mimic structure or properties. This correlates well with, and provides a biophysical basis for, previously published results that reported lack of a mutant effect in HIV virion infectivity in contrast to an inhibitory effect in T-cell syncytium formation. It shows that interaction of LLP2 with the T-cell membrane modulates biological function.

  2. Recognition by Human Monoclonal Antibodies of Free and Complexed Peptides Representing the Prefusogenic and Fusogenic Forms of Human Immunodeficiency Virus Type 1 gp41

    PubMed Central

    Gorny, Miroslaw K.; Zolla-Pazner, Susan

    2000-01-01

    Human immunodeficiency virus type 1 (HIV-1) entry into target cells appears to be triggered when two heptad repeat regions in the ectodomain of gp41 associate, converting the prefusogenic form of gp41 to a fusogenic form. Peptides from these two heptad repeat regions, designated N51 and C43, form a coiled coil consisting of an α-helical trimer of heterodimers which approximates the core of the fusogenic form of gp41. To understand the antigenic structures of gp41 in these two configurations, and to examine the specificity of anti-gp41 antibodies produced by HIV-1-infected individuals, human anti-gp41 monoclonal antibodies (MAbs) were tested for their reactivity against N51, C43, and the complex formed by these peptides. Of 11 MAbs, 7 reacted with the complex but with neither of the parent peptides. These MAbs reacted optimally with the N51-C43 complex prepared at a 1:1 ratio and appeared to recognize the fusogenic form of gp41 in which the two heptad repeat regions are associated to form the coiled coil. The existence of antibodies from HIV-infected humans that exclusively recognize the N51-C43 complex constitutes the first proof that the coiled-coil conformation of gp41 exists in vivo and is immunogenic. Two of the 11 MAbs were specific for the hydrophilic loop region of gp41 and failed to react with either peptide alone or with the peptide complex, while the remaining 2 MAbs reacted with peptide C43. One of these two latter MAbs, 98-6, also reacted well with the equimolar N51-C43 complex, while reactivity with C43 by the other MAb, 2F5, was inhibited by even small amounts of N51, suggesting that the interaction of these peptides occludes or disrupts the epitope recognized by MAb 2F5. MAbs 98-6 and 2F5 are also unusual among the MAbs tested in their ability to neutralize multiple primary HIV isolates, although 2F5 displays more broad and potent activity. The data suggest that anti-gp41 neutralizing activity is associated with specificity for a region in C43 which

  3. Recognition by human monoclonal antibodies of free and complexed peptides representing the prefusogenic and fusogenic forms of human immunodeficiency virus type 1 gp41.

    PubMed

    Gorny, M K; Zolla-Pazner, S

    2000-07-01

    Human immunodeficiency virus type 1 (HIV-1) entry into target cells appears to be triggered when two heptad repeat regions in the ectodomain of gp41 associate, converting the prefusogenic form of gp41 to a fusogenic form. Peptides from these two heptad repeat regions, designated N51 and C43, form a coiled coil consisting of an alpha-helical trimer of heterodimers which approximates the core of the fusogenic form of gp41. To understand the antigenic structures of gp41 in these two configurations, and to examine the specificity of anti-gp41 antibodies produced by HIV-1-infected individuals, human anti-gp41 monoclonal antibodies (MAbs) were tested for their reactivity against N51, C43, and the complex formed by these peptides. Of 11 MAbs, 7 reacted with the complex but with neither of the parent peptides. These MAbs reacted optimally with the N51-C43 complex prepared at a 1:1 ratio and appeared to recognize the fusogenic form of gp41 in which the two heptad repeat regions are associated to form the coiled coil. The existence of antibodies from HIV-infected humans that exclusively recognize the N51-C43 complex constitutes the first proof that the coiled-coil conformation of gp41 exists in vivo and is immunogenic. Two of the 11 MAbs were specific for the hydrophilic loop region of gp41 and failed to react with either peptide alone or with the peptide complex, while the remaining 2 MAbs reacted with peptide C43. One of these two latter MAbs, 98-6, also reacted well with the equimolar N51-C43 complex, while reactivity with C43 by the other MAb, 2F5, was inhibited by even small amounts of N51, suggesting that the interaction of these peptides occludes or disrupts the epitope recognized by MAb 2F5. MAbs 98-6 and 2F5 are also unusual among the MAbs tested in their ability to neutralize multiple primary HIV isolates, although 2F5 displays more broad and potent activity. The data suggest that anti-gp41 neutralizing activity is associated with specificity for a region in C43

  4. pH-Dependent Vesicle Fusion Induced by the Ectodomain of the Human Immunodeficiency Virus Membrane Fusion Protein gp41: Two Kinetically Distinct Processes and Fully-Membrane-Associated gp41 with Predominant β Sheet Fusion Peptide Conformation

    PubMed Central

    Ratnayake, Punsisi U.; Sackett, Kelly; Nethercott, Matthew J.; Weliky, David P.

    2014-01-01

    The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The FP is critical for fusion and is hypothesized to bind to the host cell membrane. Large ectodomain constructs either with or without the FP are highly aggregated at physiologic pH but soluble in the pH 3–4 range with hyperthermostable hairpin structure with antiparallel NHR and CHR helices. The present study focuses on the large gp41 ectodomain constructs “Hairpin” (HP) containing NHR+loop+CHR and “FP-Hairpin” (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH 4 where the protein is positively-charged but do not induce fusion at pH 7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. The functional role of the hydrophobic FP is supported by increases in the rate and extent of fusion for FP-HP relative to HP. There are two kinetically distinct fusion processes at pH 4: (1) a faster ~100 ms−1 process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms−1 process with extent strongly inversely correlated with this charge. The faster charge-dependent process is likely related to the electrostatic energy released upon initial monomer protein binding to the vesicle. After dissipation of this energy, the subsequent slower process is likely due to the equilibrium membrane-associated structure of the protein. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region

  5. Sub-inhibitory concentrations of human α-defensin potentiate neutralizing antibodies against HIV-1 gp41 pre-hairpin intermediates in the presence of serum.

    PubMed

    Demirkhanyan, Lusine; Marin, Mariana; Lu, Wuyuan; Melikyan, Gregory B

    2013-01-01

    Human defensins are at the forefront of the host responses to HIV and other pathogens in mucosal tissues. However, their ability to inactivate HIV in the bloodstream has been questioned due to the antagonistic effect of serum. In this study, we have examined the effect of sub-inhibitory concentrations of human α-defensin HNP-1 on the kinetics of early steps of fusion between HIV-1 and target cells in the presence of serum. Direct measurements of HIV-cell fusion using an enzymatic assay revealed that, in spite of the modest effect on the extent of fusion, HNP-1 prolonged the exposure of functionally important transitional epitopes of HIV-1 gp41 on the cell surface. The increased lifetime of gp41 intermediates in the presence of defensin was caused by a delay in the post-coreceptor binding steps of HIV-1 entry that correlated with the marked enhancement of the virus' sensitivity to neutralizing anti-gp41 antibodies. By contrast, the activity of antibodies to gp120 was not affected. HNP-1 appeared to specifically potentiate antibodies and peptides targeting the first heptad repeat domain of gp41, while its effect on inhibitors and antibodies to other gp41 domains was less prominent. Sub-inhibitory concentrations of HNP-1 also promoted inhibition of HIV-1 entry into peripheral blood mononuclear cells by antibodies and, more importantly, by HIV-1 immune serum. Our findings demonstrate that: (i) sub-inhibitory doses of HNP-1 potently enhance the activity of a number of anti-gp41 antibodies and peptide inhibitors, apparently by prolonging the lifetime of gp41 intermediates; and (ii) the efficiency of HIV-1 fusion inhibitors and neutralizing antibodies is kinetically restricted. This study thus reveals an important role of α-defensin in enhancing adaptive immune responses to HIV-1 infection and suggests future strategies to augment these responses.

  6. Immunogenic properties of a trimeric gp41-based immunogen containing an exposed membrane-proximal external region

    PubMed Central

    Habte, Habtom H.; Banerjee, Saikat; Shi, Heliang; Qin, Yali; Cho, Michael W

    2015-01-01

    The membrane-proximal external region (MPER) of HIV-1 gp41 is an attractive target for vaccine development. Thus, better understanding of its immunogenic properties in various structural contexts is important. We previously described the crystal structure of a trimeric protein complex named gp41-HR1-54Q, which consists of the heptad repeat regions 1 and 2 and the MPER. The protein was efficiently recognized by broadly neutralizing antibodies. Here, we describe its immunogenic properties in rabbits. The protein was highly immunogenic, especially the C-terminal end of the MPER containing 4E10 and 10E8 epitopes (671NWFDITNWLWYIK683). Although antibodies exhibited strong competition activity against 4E10 and 10E8, neutralizing activity was not detected. Detailed mapping analyses indicated that amino acid residues critical for recognition resided on faces of the alpha helix that are either opposite of or perpendicular to the epitopes recognized by 4E10 and 10E8. These results provide critical information for designing the next generation of MPER-based immunogens. PMID:26454663

  7. Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure

    PubMed Central

    Banerjee, Saikat; Shi, Heliang; Habte, Habtom H.; Qin, Yali; Cho, Michael W.

    2016-01-01

    The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; 671NWFDITNWLWYIK683) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-Δ10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies. PMID:26803471

  8. NMR second site screening for structure determination of ligands bound in the hydrophobic pocket of HIV-1 gp41.

    PubMed

    Balogh, Edina; Wu, Dong; Zhou, Guangyan; Gochin, Miriam

    2009-03-04

    The development of nonpeptide fusion inhibitors through rational drug design has been hampered by the limited accessibility of the gp41 coiled coil target, which is highly hydrophobic, and the absence of structural data defining details of small molecule interactions. Here we describe a new approach for obtaining structural information on small molecules bound in the hydrophobic pocket of gp41, using a paramagnetic probe peptide which binds adjacent to the pocket along an extended coiled coil. Ligand binding in the pocket leads to paramagnetic relaxation effects or pseudocontact shifts of ligand protons. These effects are distance and/or orientation dependent, permitting determination of ligand pose in the pocket. The method is demonstrated with a fast-exchanging ligand. Multiple measurements at different coiled coil and probe peptide ratios enabled accurate determination of the NMR parameters. Use of a labeled probe peptide stabilizes an otherwise aggregation-prone coiled coil and also enables modulation of the paramagnetic effect to study ligands of various affinities. Ultimately, this technique can provide essential information for structure-based design of nonpeptide fusion inhibitors.

  9. Design and characterization of an engineered gp41 protein from human immunodeficiency virus-1 as a tool for drug discovery

    NASA Astrophysics Data System (ADS)

    Stewart, Kent D.; Steffy, Kevin; Harris, Kevin; Harlan, John E.; Stoll, Vincent S.; Huth, Jeffrey R.; Walter, Karl A.; Gramling-Evans, Emily; Mendoza, Renaldo R.; Severin, Jean M.; Richardson, Paul L.; Barrett, Leo W.; Matayoshi, Edmund D.; Swift, Kerry M.; Betz, Stephen F.; Muchmore, Steve W.; Kempf, Dale J.; Molla, Akhter

    2007-01-01

    Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the "Trp-Trp-Ile" binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.

  10. Serologic reactivity of a synthetic peptide from human immunodeficiency virus type 1 gp41 with sera from a Mexican population.

    PubMed Central

    Gevorkian, G; Soler, C; Viveros, M; Padilla, A; Govezensky, T; Larralde, C

    1996-01-01

    The reactivities of 1,172 serum samples obtained from asymptomatic human immunodeficiency virus type 1 (HIV-1)-positive and HIV-1-negative individuals residing in Mexico to a synthetic disulfide-looped peptide from the HIV-1 gp41 (amino acids 602 to 616 [IWGCSGKLICTTAVP] were examined by an enzyme-linked immunoadsorbent assay (ELISA) procedure. Antibodies to the synthetic peptide were detected in 261 of 268 serum samples from HIV-positive individuals (sensitivity, 97.4%). The peptide also reacted with 12 of 904 serum samples from control HIV-negative individuals (specificity, 98.7%). Western blots (immunoblots) of four of the seven serum samples that produced false-negative results in the ELISA showed that three of them reacted weakly with gp41 and strongly with gp120, p55, and/or p24. Potential diagnostic difficulties raised by the reported C1q binding capacity of this peptide were also evaluated: few and weak false-positive results were found among sera from patients with rheumatoid arthritis (1 of 31) and neurocysticercosis (2 of 111). In fact, strong reactivity with the peptide spotted an undetected HIV infection underlying clinical neurocysticercosis. PMID:8914754

  11. Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure.

    PubMed

    Banerjee, Saikat; Shi, Heliang; Habte, Habtom H; Qin, Yali; Cho, Michael W

    2016-03-01

    The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; (671)NWFDITNWLWYIK(683)) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-∆10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies.

  12. Immunogenic properties of a trimeric gp41-based immunogen containing an exposed membrane-proximal external region.

    PubMed

    Habte, Habtom H; Banerjee, Saikat; Shi, Heliang; Qin, Yali; Cho, Michael W

    2015-12-01

    The membrane-proximal external region (MPER) of HIV-1 gp41 is an attractive target for vaccine development. Thus, better understanding of its immunogenic properties in various structural contexts is important. We previously described the crystal structure of a trimeric protein complex named gp41-HR1-54Q, which consists of the heptad repeat regions 1 and 2 and the MPER. The protein was efficiently recognized by broadly neutralizing antibodies. Here, we describe its immunogenic properties in rabbits. The protein was highly immunogenic, especially the C-terminal end of the MPER containing 4E10 and 10E8 epitopes ((671)NWFDITNWLWYIK(683)). Although antibodies exhibited strong competition activity against 4E10 and 10E8, neutralizing activity was not detected. Detailed mapping analyses indicated that amino acid residues critical for recognition resided on faces of the alpha helix that are either opposite of or perpendicular to the epitopes recognized by 4E10 and 10E8. These results provide critical information for designing the next generation of MPER-based immunogens.

  13. Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges.

    PubMed

    Bomsel, Morgane; Tudor, Daniela; Drillet, Anne-Sophie; Alfsen, Annette; Ganor, Yonatan; Roger, Marie-Gaëlle; Mouz, Nicolas; Amacker, Mario; Chalifour, Anick; Diomede, Lorenzo; Devillier, Gilles; Cong, Zhe; Wei, Qiang; Gao, Hong; Qin, Chuan; Yang, Gui-Bo; Zurbriggen, Rinaldo; Lopalco, Lucia; Fleury, Sylvain

    2011-02-25

    Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS.

  14. Structure of the HIV-1 gp41 Membrane-Proximal Ectodomain Region in a Putative Prefusion Conformation

    SciTech Connect

    Liu, J.; Deng, Y; Dey, A; Moore, J; Lu, M

    2009-01-01

    The conserved membrane-proximal external region (MPER) of the HIV-1 gp41 envelope protein is the established target for very rare but broadly neutralizing monoclonal antibodies (NAbs) elicited during natural human infection. Nevertheless, attempts to generate an HIV-1 neutralizing antibody response with immunogens bearing MPER epitopes have met with limited success. Here we show that the MPER peptide (residues 662-683) forms a labile ?-helical trimer in aqueous solution and report the crystal structure of this autonomous folding subdomain stabilized by addition of a C-terminal isoleucine zipper motif. The structure reveals a parallel triple-stranded coiled coil in which the neutralization epitope residues are buried within the interface between the associating MPER helices. Accordingly, both the 2F5 and 4E10 NAbs recognize the isolated MPER peptide but fail to bind the trimeric MPER subdomain. We propose that the trimeric MPER structure represents the prefusion conformation of gp41, preceding the putative prehairpin intermediate and the postfusion trimer-of-hairpins structure. As such, the MPER trimer should inform the design of new HIV-1 immunogens to elicit broadly neutralizing antibodies.

  15. Characterization of Gp41 polymorphisms in the fusion peptide domain and T-20 (Enfuvirtide) resistance-associated regions in Korean HIV-1 isolates.

    PubMed

    Jang, Dai-Ho; Yoon, Cheol-Hee; Choi, Byeong-Sun; Chung, Yoon-Seok; Kim, Hye-Young; Chi, Sung-Gil; Kim, Sung Soon

    2014-03-01

    HIV-1 gp41 is an envelope protein that plays an essential role in virus entry. The mutation of gp41 affects HIV-1 entry and susceptibility to the fusion inhibitor T-20. Therefore, we analyzed the natural polymorphism of gp41 of 163 HIV-1 isolates from T-20-naïve Koreans infected with HIV-1. This study of gp41 polymorphisms showed that insertions in the fourth threonine (74.8%) and L7M substitutions (85.3%) were more frequent in the fusion peptide motif in Korean HIV-1 isolates compared with those from other countries. Minor T-20 resistance mutations such as L45M (1.2%), N126K (1.2%), and E137K (6.7%) were detected, but the critical T-20 resistance mutations were not detected in the gp41 HR1 and HR2 regions. In addition, the N42S mutation (12.9%) associated with T-20 hypersusceptibility was detected at a high frequency. These results may serve as useful data for studies considering T-20 for use in the development of a more effective anti-retroviral treatment in Korea.

  16. Structural and Functional Properties of Peptides Based on the N-terminus of HIV-1 gp41 and the C-terminus of the Amyloid-Beta Protein

    PubMed Central

    Gordon, Larry M.; Nisthal, Alex; Lee, Andy B.; Eskandari, Sepehr; Ruchala, Piotr; Jung, Chun-Ling; Waring, Alan J.; Mobley, Patrick W.

    2008-01-01

    Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26–42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer’s. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26–42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26–42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26–42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’). PMID:18515070

  17. In silico vaccine design based on molecular simulations of rhinovirus chimeras presenting HIV-1 gp41 epitopes.

    PubMed

    Lapelosa, Mauro; Gallicchio, Emilio; Arnold, Gail Ferstandig; Arnold, Eddy; Levy, Ronald M

    2009-01-16

    A cluster of promising epitopes for the development of human immunodeficiency virus (HIV) vaccines is located in the membrane-proximal external region (MPER) of the gp41 subunit of the HIV envelope spike structure. The crystal structure of the peptide corresponding to the so-called ELDKWA epitope (HIV-1 HxB2 gp41 residues 662-668), in complex with the corresponding broadly neutralizing human monoclonal antibody 2F5, provides a target for structure-based vaccine design strategies aimed at finding macromolecular carriers that are able to present this MPER-derived epitope with optimal antigenic activity. To this end, a series of replica exchange molecular dynamics computer simulations was conducted to characterize the distributions of conformations of ELDKWA-based epitopes inserted into a rhinovirus carrier and to identify those with the highest fraction of conformations that are able to bind 2F5. The length, hydrophobic character, and precise site of insertion were found to be critical for achieving structural similarity to the target crystal structure. A construct with a high degree of complementarity to the corresponding determinant region of 2F5 was obtained. This construct was employed to build a high-resolution structural model of the complex between the 2F5 antibody and the chimeric human rhinovirus type 14:HIV-1 ELDKWA virus particle. Additional simulations, which were conducted to study the conformational propensities of the ELDKWA region in solution, confirm the hypothesis that the ELDKWA region of gp41 is highly flexible and capable of assuming helical conformations (as in the postfusion helical bundle structure) and beta-turn conformations (as in the complex with the 2F5 antibody). These results also suggest that the ELDKWA epitope can be involved in intramolecular--and likely intermolecular--hydrophobic interactions. This tendency offers an explanation for the observation that mutations decreasing the hydrophobic character of the MPER in many cases result

  18. Anti-HIV Double Variable Domain Immunoglobulins Binding Both gp41 and gp120 for Targeted Delivery of Immunoconjugates

    PubMed Central

    Craig, Ryan B.; Summa, Christopher M.; Corti, Miriam; Pincus, Seth H.

    2012-01-01

    Background Anti-HIV immunoconjugates targeted to the HIV envelope protein may be used to eradicate the latent reservoir of HIV infection using activate-and-purge protocols. Previous studies have identified the two target epitopes most effective for the delivery of cytotoxic immunoconjugates the CD4-binding site of gp120, and the hairpin loop of gp41. Here we construct and test tetravalent double variable domain immunoglobulin molecules (DVD-Igs) that bind to both epitopes. Methods Synthetic genes that encode DVD-Igs utilizing V-domains derived from human anti-gp120 and anti-gp41 Abs were designed and expressed in 293F cells. A series of constructs tested different inter-V-linker domains and orientations of the two V domains. Antibodies were tested for binding to recombinant Ag and native Env expressed on infected cells, for neutralization of infectious HIV, and for their ability to deliver cytotoxic immunoconjugates to infected cells. Findings The outer V-domain was the major determinant of binding and functional activity of the DVD-Ig. Function of the inner V-domain and bifunctional binding required at least 15 AA in the inter-V-domain linker. A molecular model showing the spatial orientation of the two epitopes is consistent with this observation. Linkers that incorporated helical domains (A[EAAAK]nA) resulted in more effective DVD-Igs than those based solely on flexible domains ([GGGGS]n). In general, the DVD-Igs outperformed the less effective parental antibody and equaled the activity of the more effective. The ability of the DVD-Igs to deliver cytotoxic immunoconjugates in the absence of soluble CD4 was improved over that of either parent. Conclusions DVD-Igs can be designed that bind to both gp120 and gp41 on the HIV envelope. DVD-Igs are effective in delivering cytotoxic immunoconjugates. The optimal design of these DVD-Igs, in which both domains are fully functional, has not yet been achieved. PMID:23056448

  19. GLUE that sticks to HIV: a helix-grafted GLUE protein that selectively binds the HIV gp41 N-terminal helical region.

    PubMed

    Walker, Susanne N; Tennyson, Rachel L; Chapman, Alex M; Kennan, Alan J; McNaughton, Brian R

    2015-01-19

    Methods for the stabilization of well-defined helical peptide drugs and basic research tools have received considerable attention in the last decade. Here, we report the stable and functional display of an HIV gp41 C-peptide helix mimic on a GRAM-Like Ubiquitin-binding in EAP45 (GLUE) protein. C-peptide helix-grafted GLUE selectively binds a mimic of the N-terminal helical region of gp41, a well-established HIV drug target, in a complex cellular environment. Additionally, the helix-grafted GLUE is folded in solution, stable in human serum, and soluble in aqueous solutions, and thus overcomes challenges faced by a multitude of peptide drugs, including those derived from HIV gp41 C-peptide.

  20. Passively transmitted gp41 antibodies in babies born from HIV-1 subtype C-seropositive women: correlation between fine specificity and protection.

    PubMed

    Diomede, L; Nyoka, S; Pastori, C; Scotti, L; Zambon, A; Sherman, G; Gray, C M; Sarzotti-Kelsoe, M; Lopalco, L

    2012-04-01

    HIV-exposed, uninfected (EUN) babies born to HIV-infected mothers are examples of natural resistance to HIV infection. In this study, we evaluated the titer and neutralizing potential of gp41-specific maternal antibodies and their correlation with HIV transmission in HIV-infected mother-child pairs. Specific gp41-binding and -neutralizing antibodies were determined in a cohort of 74 first-time mother-child pairs, of whom 40 mothers were infected with HIV subtype C. Within the infected mother cohort, 16 babies were born infected and 24 were PCR negative and uninfected at birth (i.e., exposed but uninfected). Thirty-four HIV-uninfected and HIV-unexposed mother-child pairs were included as controls. All HIV-positive mothers and their newborns showed high IgG titers to linear epitopes within the HR1 region and to the membrane-proximal (MPER) domain of gp41; most sera also recognized the disulfide loop immunodominant epitope (IDE). Antibody titers to the gp41 epitopes were significantly lower in nontransmitting mothers (P < 0.01) and in the EUN babies (P < 0.005) than in HIV-positive mother-child pairs. Three domains of gp41, HR1, IDE, and MPER, elicited antibodies that were effectively transmitted to EUN babies. Moreover, in EUN babies, epitopes overlapping the 2F5 epitope (ELDKWAS), but not the 4E10 epitope, were neutralization targets in two out of four viruses tested. Our findings highlight important epitopes in gp41 that appear to be associated with exposure without infection and would be important to consider for vaccine design.

  1. Functional role of the zipper motif region of human immunodeficiency virus type 1 transmembrane protein gp41.

    PubMed Central

    Chen, S S

    1994-01-01

    To study the functional role of the zipper motif region, located in the N-terminal region of the envelope transmembrane protein of human immunodeficiency virus type 1, a series of vaccinia virus-expressed mutant proteins containing a proline substitution in this region were characterized. All of the mutant proteins showed partial or no inhibition in gp160 cleavage, demonstrated impaired ability of gp120 to associate with gp41, and were unable to mediate syncytium formation with CD4+ cells. Moreover, mutants 580 and 587 secreted excessive gp120 into the medium compared with the wild type. Mutations in this region affected the conformation of the local or proximal sequence but did not alter the conformation conferred by a distal site. These studies reveal the crucial role of the C-terminal segment of the zipper motif region in envelope heterodimeric association and suggest that this sequence forms a gp120 contact site. Images PMID:7509005

  2. Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

    NASA Technical Reports Server (NTRS)

    Casale, Elena; He, Xiao-Min; Snyder, Robert S.; Carter, Daniel C.; Wenisch, Elisabeth; Jungbauer, Alois; Tauer, Christa; Ruker, Florian; Righetti, Pier Giorgio

    1990-01-01

    A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable platelike crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A, and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

  3. Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

    NASA Technical Reports Server (NTRS)

    Casale, Elena; He, Xiao-Min; Snyder, Robert S.; Carter, Daniel C.; Wenisch, Elisabeth; Jungbauer, Alois; Tauer, Christa; Ruker, Florian; Righetti, Pier Giorgio

    1990-01-01

    A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable platelike crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A, and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

  4. ADS-J1 inhibits HIV-1 infection and membrane fusion by targeting the highly conserved pocket in the gp41 NHR-trimer.

    PubMed

    Yu, Fei; Lu, Lu; Liu, Qi; Yu, Xiaowen; Wang, Lili; He, Elaine; Zou, Peng; Du, Lanying; Sanders, Rogier W; Liu, Shuwen; Jiang, Shibo

    2014-05-01

    We previously identified a potent small-molecule human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, termed ADS-J1, and hypothesized that it mainly targeted the hydrophobic pocket in the gp41 N-terminal heptad repeat (NHR) trimer. However, this hypothesis has been challenged by the fact that ADS-J1 cannot induce drug-resistance mutation in the gp41 pocket region. Therefore, we show herein that HIV-1 mutants resistant to T2635, a peptide derived from the gp41 C-terminal heptad repeat (CHR) region with pocket-binding domain (PBD), were also resistant to ADS-J1. We also show that pseudoviruses with mutations at positions 64 and 67 in the gp41 pocket region were highly resistant to ADS-J1 and C34, another CHR-peptide with PBD, but relatively sensitive to T20, a CHR-peptide without PBD. ADS-J1 could effectively bind to N36Fd, a mimic of the gp41 NHR-trimer with pocket exposed, and block binding of C34 to N36Fd trimer to form six-helix bundle (6-HB). However, ADS-J1 was less effective in binding to N36Fd trimer with mutations in the gp41 pocket region, such as N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd, and N36(Q66R)Fd, as well as less effective in blocking 6-HB formation between C34 and these mutant N36Fd trimers. These results confirm that ADS-J1 mainly targets the pocket region in the HIV-1 gp41 NHR trimer and suggest that it could be used as a lead for developing small-molecule HIV fusion inhibitors and as a molecule probe for studying the mechanisms of gp41-mediated membrane fusion. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Design of an engineered N-terminal HIV-1 gp41 trimer with enhanced stability and potency

    PubMed Central

    Dwyer, John J.; Wilson, Karen L.; Martin, Kimberly; Seedorff, Jennifer E.; Hasan, Aisha; Medinas, Robyn J.; Davison, Donna K.; Feese, Michael D.; Richter, Hans-Thomas; Kim, Hidong; Matthews, Thomas J.; Delmedico, Mary K.

    2008-01-01

    HIV fusion is mediated by a conformational transition in which the C-terminal region (HR2) of gp41 interacts with the N-terminal region (HR1) to form a six-helix bundle. Peptides derived from the HR1 form a well-characterized, trimeric coiled-coil bundle in the presence of HR2 peptides, but there is little structural information on the isolated HR1 trimer. Using protein design, we have designed synthetic HR1 peptides that form soluble, thermostable HR1 trimers. In vitro binding of HR2 peptides to the engineered trimer suggests that the design strategy has not significantly impacted the ability to form the six-helix bundle. The peptides have enhanced antiviral activity compared to wild type, with up to 30-fold greater potency against certain viral isolates. In vitro passaging was used to generate HR1-resistant virus and the observed resistance mutations map to the HR2 region of gp41, demonstrating that the peptides block the fusion process by binding to the viral HR2 domain. Interestingly, the activity of the HR2 fusion inhibitor, enfuvirtide (ENF), against these resistant viruses is maintained or improved up to fivefold. The 1.5 Å crystal structure of one of these designs has been determined, and we show that the isolated HR1 is very similar to the conformation of the HR1 in the six-helix bundle. These results provide an initial model of the pre-fusogenic state, are attractive starting points for identifying novel fusion inhibitors, and offer new opportunities for developing HIV therapeutics based on HR1 peptides. PMID:18359857

  6. Characterization of the HIV N-terminal fusion peptide-containing region in context of key gp41 fusion conformations.

    PubMed

    Sackett, Kelly; Wexler-Cohen, Yael; Shai, Yechiel

    2006-08-04

    Central to our understanding of human immunodeficiency virus-induced fusion is the high resolution structure of fragments of the gp41 fusion protein folded in a low energy core conformation. However, regions fundamental to fusion, like the fusion peptide (FP), have yet to be characterized in the context of the cognate protein regardless of its conformation. Based on conformation-specific monoclonal antibody recognition, we identified the polar region consecutive to the N36 fragment as a stabilizer of trimeric coiled-coil assembly, thereby enhancing inhibitory potency. This tertiary organization is retained in the context of the hydrophobic FP (N70 fragment). Our data indicate that the N70 fragment recapitulates the expected organization of this region in the viral fusion intermediate (N-terminal half of the pre-hairpin intermediate (N-PHI)), which happens to be the prime target for fusion inhibitors. Regarding the low energy conformation, we show for the first time core formation in the context of the FP (N70 core). The alpha-helical and coiled-coil stabilizing polar region confers substantial thermal stability to the core, whereas the hydrophobic FP does not add further stability. For the two key fusion conformations, N-PHI and N70 core, we find that the FP adopts a nonhelical structure and directs higher order assembly (assembly of coiled coils in N-PHI and assembly of bundles in the N70 core). This supra-molecular organization of coiled coils or folded cores is seen only in the context of the FP. This study is the first to characterize the FP region in the context of the folded core and provides a basic understanding of the role of the elusive FP for key gp41 fusion conformations.

  7. Folded monomers and hexamers of the ectodomain of the HIV gp41 membrane fusion protein: potential roles in fusion and synergy between the fusion peptide, hairpin, and membrane-proximal external region.

    PubMed

    Banerjee, Koyeli; Weliky, David P

    2014-11-25

    HIV is an enveloped virus and fusion between the HIV and host cell membranes is catalyzed by the ectodomain of the HIV gp41 membrane protein. Both the N-terminal fusion peptide (FP) and C-terminal membrane-proximal external region (MPER) are critical for fusion and are postulated to bind to the host cell and HIV membranes, respectively. Prior to fusion, the gp41 on the virion is a trimer in noncovalent complex with larger gp120 subunits. The gp120 bind host cell receptors and move away or dissociate from gp41 which subsequently catalyzes fusion. In the present work, large gp41 ectodomain constructs were produced and biophysically and structurally characterized. One significant finding is observation of synergy between the FP, hairpin, and MPER in vesicle fusion. The ectodomain-induced fusion can be very efficient with only ∼15 gp41 per vesicle, which is comparable to the number of gp41 on a virion. Conditions are found with predominant monomer or hexamer but not trimer and these may be oligomeric states during fusion. Monomer gp41 ectodomain is hyperthermostable and has helical hairpin structure. A new HIV fusion model is presented where (1) hemifusion is catalyzed by folding of gp41 ectodomain monomers into hairpins and (2) subsequent fusion steps are catalyzed by assembly into a hexamer with FPs in an antiparallel β sheet. There is also significant interest in the gp41 MPER because it is the epitope of several broadly neutralizing antibodies. Two of these antibodies bind our gp41 ectodomain constructs and support investigation of the gp41 ectodomain as an immunogen in HIV vaccine development.

  8. The HIV gp41 pocket binding domain enables C-terminal heptad repeat transition from mediating membrane fusion to immune modulation.

    PubMed

    Klug, Yoel A; Kapach, Gal; Rotem, Etai; Dubreuil, Benjamin; Shai, Yechiel

    2016-04-01

    For successful infection and propagation viruses must overcome many obstacles such as the immune system and entry into their host cells. HIV utilizes its trimeric envelope protein gp160, specifically the gp41 subunit, to enter its host cell. During this process, a gp41-central coiled coil is formed from three N- and three C-terminal heptad repeats, termed the six-helix bundle (SHB), which drives membrane fusion. Recently, T-cell suppression has been reported as an additional function for several regions of gp41 by interfering with the T-cell receptor (TCR) signalling cascade. One of these regions encompasses the conserved pocket binding domain (PBD) that is situated in the C-terminal heptad repeat (CHR) and stabilizes SHB formation. This could indicate that the PBD plays a role in T-cell suppression in addition to its role in membrane fusion. To investigate this dual function, we used two independent cell cultures coupled with biophysical techniques. The data reveal that the PBD mediates T-cell suppression by stabilizing a TCR-binding conformation in the membrane. Moreover, we show that the clinically used HIV fusion inhibitor T-20 did not show suppressive abilities, in contrast with the potent fusion inhibitor C34. In addition, by focusing on SHB conformation after its assembly, we shed light on a mechanism by which gp41's function alternates from membrane fusion facilitation to suppression of TCR activation.

  9. Significant differences in cell-cell fusion and viral entry between strains revealed by scanning mutagenesis of the C-heptad repeat of HIV gp41.

    PubMed

    Diaz-Aguilar, Barbara; Dewispelaere, Karen; Yi, Hyun Ah; Jacobs, Amy

    2013-05-21

    The transmembrane subunit, gp41, of the HIV envelope mediates the viral fusion step of entry into the host cell. The protein consists of an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The extracellular domain contains a fusion peptide, an N-terminal heptad repeat, a loop region, a C-terminal heptad repeat (CHR), and a membrane-proximal external region. For this study, we examined each amino acid in the CHR (residues 623-659) by alanine scanning mutagenesis in two HIV strains: one CCR5-utilizing strain (JRFL) and one CXCR4-utilizing strain (HXB2). We studied the functional importance of each amino acid residue by measuring mutational effects in both cell-cell fusion and viral entry and assessing envelope expression and gp120-gp41 proteolytic processing. The transmembrane subunit of the HIV envelope, gp41, is very sensitive to subtle changes, like alanine substitution, which severely affect envelope function at multiple sites. Two important general findings are apparent when the entire data set from this study is taken into account. (1) Strain HXB2 is much more stable to mutagenesis than strain JRFL, and (2) viral entry is much more stable to mutagenesis than cell-cell fusion. These findings strengthen our notion that gp41 is a vulnerable target for therapeutic and prophylactic intervention. Further structural studies aimed at gaining a full understanding of the intermediate states that drive HIV membrane fusion are imperative.

  10. Deletion of fusion peptide or destabilization of fusion core of HIV gp41 enhances antigenicity and immunogenicity of 4E10 epitope

    SciTech Connect

    Li Jing; Chen Xi; Jiang Shibo Chen Yinghua

    2008-11-07

    The human monoclonal antibody 4E10 against the membrane-proximal external region (MPER) of HIV-1 gp41 demonstrates broad neutralizing activity across various strains, and makes its epitope an attractive target for HIV-1 vaccine development. Although the contiguous epitope of 4E10 has been identified, attempts to re-elicit 4E10-like antibodies have failed, possibly due to the lack of proper conformation of the 4E10 epitope. Here we used pIg-tail expression system to construct a panel of eukaryotic cell-surface expression plasmids encoding the extracellular domain of gp41 with deletion of fusion peptide and/or introduction of L568P mutation that may disrupt the gp41 six-helix bundle core conformation as DNA vaccines for immunization of mice. We found that these changes resulted in significant increase of the antigenicity and immunogenicity of 4E10 epitope. This information is thus useful for rational design of vaccines targeting the HIV-1 gp41 MPER.

  11. Allosteric modulation of the HIV-1 gp120-gp41 association site by adjacent gp120 variable region 1 (V1) N-glycans linked to neutralization sensitivity.

    PubMed

    Drummer, Heidi E; Hill, Melissa K; Maerz, Anne L; Wood, Stephanie; Ramsland, Paul A; Mak, Johnson; Poumbourios, Pantelis

    2013-01-01

    The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and C-terminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn¹³⁶ in V1 (T138N mutation) in conjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N¹³⁹INN sequence, which ablates the overlapping Asn¹⁴¹-Asn¹⁴²-Ser-Ser potential N-linked glycosylation sequons in V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu⁵⁹³, Trp⁵⁹⁶ and Lys⁶⁰¹. The 136 and/or 142 glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a

  12. Molecular dynamics studies of the inhibitor C34 binding to the wild-type and mutant HIV-1 gp41: inhibitory and drug resistant mechanism.

    PubMed

    Ma, Xueting; Tan, Jianjun; Su, Min; Li, Chunhua; Zhang, Xiaoyi; Wang, Cunxin

    2014-01-01

    Mutations on NHR (N-terminal heptad repeat) associated with resistance to fusion inhibitor were observed. In addition, mutations on CHR (C-terminal heptad repeat) accompanied NHR mutations of gp41 are noted in many cases, like N43D/S138A double mutation. In this work, we explored the drug resistant mechanism of N43D mutation and the role of S138A second mutation in drug resistance. The binding modes of the wild type gp41 and the two mutants, N43D and N43D/S138A, with the HIV-1 fusion inhibitor C34, a 34-residue peptide mimicking CHR of gp41, were carried out by using molecular dynamics simulations. Based on the MD simulations, N43D mutation affects not only the stability of C34 binding, but also the binding energy of the inhibitor C34. Because N43D mutation may also affect the stable conformation of 6-HB, we introduced S138A second mutation into CHR of gp41 and determined the impact of this mutation. Through the comparative analysis of MD results of the N43D mutant and the N43D/S138A mutant, we found that CHR with S138A mutation shown more favorable affinity to NHR. Compelling differences in structures have been observed for these two mutants, particularly in the binding modes and in the hydrophobic interactions of the CHR (C34) located near the hydrophobic groove of the NHR. Because the conformational stability of 6-HB is important to HIV-1 infection, we suggested a hypothetical mechanism for the drug resistance: N43D single mutation not only impact the binding of inhibitor, but also affect the affinity between NHR and CHR of gp41, thus may reduce the rate of membrane fusion; compensatory mutation S138A would induce greater hydrophobic interactions between NHR and CHR, and render the CHR more compatible to NHR than inhibitors.

  13. Estimating the probability of polyreactive antibodies 4E10 and 2F5 disabling a gp41 trimer after T cell-HIV adhesion.

    PubMed

    Hu, Bin; Liao, Hua-Xin; Alam, S Munir; Goldstein, Byron

    2014-01-01

    A few broadly neutralizing antibodies, isolated from HIV-1 infected individuals, recognize epitopes in the membrane proximal external region (MPER) of gp41 that are transiently exposed during viral entry. The best characterized, 4E10 and 2F5, are polyreactive, binding to the viral membrane and their epitopes in the MPER. We present a model to calculate, for any antibody concentration, the probability that during the pre-hairpin intermediate, the transient period when the epitopes are first exposed, a bound antibody will disable a trivalent gp41 before fusion is complete. When 4E10 or 2F5 bind to the MPER, a conformational change is induced that results in a stably bound complex. The model predicts that for these antibodies to be effective at neutralization, the time to disable an epitope must be shorter than the time the antibody remains bound in this conformation, about five minutes or less for 4E10 and 2F5. We investigate the role of avidity in neutralization and show that 2F5 IgG, but not 4E10, is much more effective at neutralization than its Fab fragment. We attribute this to 2F5 interacting more stably than 4E10 with the viral membrane. We use the model to elucidate the parameters that determine the ability of these antibodies to disable epitopes and propose an extension of the model to analyze neutralization data. The extended model predicts the dependencies of IC50 for neutralization on the rate constants that characterize antibody binding, the rate of fusion of gp41, and the number of gp41 bridging the virus and target cell at the start of the pre-hairpin intermediate. Analysis of neutralization experiments indicate that only a small number of gp41 bridges must be disabled to prevent fusion. However, the model cannot determine the exact number from neutralization experiments alone.

  14. Estimating the Probability of Polyreactive Antibodies 4E10 and 2F5 Disabling a gp41 Trimer after T Cell-HIV Adhesion

    PubMed Central

    Hu, Bin; Liao, Hua-Xin; Alam, S. Munir; Goldstein, Byron

    2014-01-01

    A few broadly neutralizing antibodies, isolated from HIV-1 infected individuals, recognize epitopes in the membrane proximal external region (MPER) of gp41 that are transiently exposed during viral entry. The best characterized, 4E10 and 2F5, are polyreactive, binding to the viral membrane and their epitopes in the MPER. We present a model to calculate, for any antibody concentration, the probability that during the pre-hairpin intermediate, the transient period when the epitopes are first exposed, a bound antibody will disable a trivalent gp41 before fusion is complete. When 4E10 or 2F5 bind to the MPER, a conformational change is induced that results in a stably bound complex. The model predicts that for these antibodies to be effective at neutralization, the time to disable an epitope must be shorter than the time the antibody remains bound in this conformation, about five minutes or less for 4E10 and 2F5. We investigate the role of avidity in neutralization and show that 2F5 IgG, but not 4E10, is much more effective at neutralization than its Fab fragment. We attribute this to 2F5 interacting more stably than 4E10 with the viral membrane. We use the model to elucidate the parameters that determine the ability of these antibodies to disable epitopes and propose an extension of the model to analyze neutralization data. The extended model predicts the dependencies of for neutralization on the rate constants that characterize antibody binding, the rate of fusion of gp41, and the number of gp41 bridging the virus and target cell at the start of the pre-hairpin intermediate. Analysis of neutralization experiments indicate that only a small number of gp41 bridges must be disabled to prevent fusion. However, the model cannot determine the exact number from neutralization experiments alone. PMID:24499928

  15. Role of lipid structure in the humoral immune response in mice to covalent lipid-peptides from the membrane proximal region of HIV-1 gp41

    PubMed Central

    Watson, Douglas S.; Szoka, Francis C.

    2009-01-01

    The membrane proximal region (MPR) of HIV-1 gp41 is a desirable target for development of a vaccine that elicits neutralizing antibodies since the patient-derived monoclonal antibodies, 2F5 and 4E10, bind to the MPR and neutralize primary HIV isolates. The 2F5 and 4E10 antibodies cross-react with lipids and structural studies suggest that MPR immunogens may be presented in a membrane environment. We hypothesized that covalent attachment of lipid anchors would enhance the humoral immune response to MPR-derived peptides presented in liposomal bilayers. In a comparison of eight lipids conjugated to an extended 2F5 epitope peptide, a sterol, cholesterol hemisuccinate (CHEMS), was found to promote the strongest anti-peptide IgG titers (6.4 × 104) in sera of BALB/C mice. Two lipid anchors, palmitic acid and phosphatidylcholine, failed to elicit a detectable serum anti-peptide IgG response. Association with the liposomal vehicle contributed to the ability of a lipopeptide to elicit anti-peptide antibodies, but no other single factor, such as position of the lipid anchor, peptide helical content, lipopeptide partition coefficient, or presence of phosphate on the anchor clearly determined lipopeptide potency. Conjugation to CHEMS also rendered a 4E10 epitope peptide immunogenic (5.6 × 102 IgG titer in serum). Finally, attachment of CHEMS to a peptide spanning both the 2F5 and 4E10 epitopes elicited serum IgG antibodies that bound to each of the individual epitopes as well as to recombinant gp140. Further research into the mechanism of how structure influences the immune response to the MPR may lead to immunogens that could be useful in prime-boost regimens for focusing the immune response in an HIV vaccine. PMID:19520200

  16. HIV gp41-Mediated Membrane Fusion Occurs at Edges of Cholesterol-Rich Lipid Domains

    PubMed Central

    Yang, Sung-Tae; Kiessling, Volker; Simmons, James A.; White, Judith M.; Tamm, Lukas K.

    2015-01-01

    Lipid rafts in plasma membranes have emerged as possible platforms for entry of HIV and other viruses into cells. However, how lipid phase heterogeneity contributes to viral entry is little known due to the fine-grained and still poorly understood complexity of biological membranes. We used model systems mimicking HIV envelopes and T-cell membranes and showed that raft-like (Lo phase) lipid domains are necessary and sufficient for efficient membrane targeting and fusion. Interestingly, membrane binding and fusion was low in homogeneous Ld and Lo phase membranes, indicating that lipid phase heterogeneity is essential. The HIV fusion peptide preferentially targeted to Lo/Ld boundary regions and promoted full fusion at the interface between ordered and disordered lipids. Ld phase vesicles proceeded only to hemifusion. Thus, we propose that the edges, but not the areas of raft-like ordered lipid domains are vital for HIV entry and membrane fusion. PMID:25915200

  17. Structure and Immunogenicity of a Peptide Vaccine, Including the Complete HIV-1 gp41 2F5 Epitope

    PubMed Central

    Serrano, Soraya; Araujo, Aitziber; Apellániz, Beatriz; Bryson, Steve; Carravilla, Pablo; de la Arada, Igor; Huarte, Nerea; Rujas, Edurne; Pai, Emil F.; Arrondo, José L. R.; Domene, Carmen; Jiménez, María Angeles; Nieva, José L.

    2014-01-01

    The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund's adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development. PMID:24429284

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

    SciTech Connect

    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Sanders, Rogier W.; Johan Klasse, Per; Moore, John P.

    2012-07-05

    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.

  19. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

    NASA Technical Reports Server (NTRS)

    He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  20. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

    NASA Technical Reports Server (NTRS)

    He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  1. The crystal structure of the streptococcal collagen-like protein 2 globular domain from invasive M3-type group A Streptococcus shows significant similarity to immunomodulatory HIV protein gp41.

    PubMed

    Squeglia, Flavia; Bachert, Beth; De Simone, Alfonso; Lukomski, Slawomir; Berisio, Rita

    2014-02-21

    The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.

  2. P20A inhibits HIV-1 fusion through its electrostatic interaction with the distal region of the gp41 fusion core.

    PubMed

    Fu, Shushu; Tong, Pei; Tan, Yue; Zhu, Yun; Chen, Ying-Hua

    2015-09-01

    We previously identified an HIV-1 fusion inhibitor P20A targeting HIV-1 gp41 6-HB fusion core. Using alanine scanning mutagenesis, we investigated the effect of 6-HB surface residue mutations on the binding affinity between P20A and 6-HB. Substitution of positively or negatively charged residues in the distal region of 6-HB with alanines resulted in significant decrease or increase of its binding affinity to P20A, respectively. The 6-HB with E630K, D632K, or E634K mutation exhibited enhanced binding affinity with P20A, suggesting that P20A blocks HIV-1 fusion through electrostatic interaction with the positively charged residues in the distal region of the gp41 fusion core.

  3. Insights into the Conformation of the Membrane Proximal Regions Critical to the Trimerization of the HIV-1 gp41 Ectodomain Bound to Dodecyl Phosphocholine Micelles

    PubMed Central

    Louis, John M.; Baber, James L.; Ghirlando, Rodolfo; Aniana, Annie; Bax, Ad; Roche, Julien

    2016-01-01

    The transitioning of the ectodomain of gp41 from a pre-hairpin to a six-helix bundle conformation is a crucial aspect of virus-cell fusion. To gain insight into the intermediary steps of the fusion process we have studied the pH and dodecyl phosphocholine (DPC) micelle dependent trimer association of gp41 by systematic deletion analysis of an optimized construct termed 17–172 (residues 528 to 683 of Env) that spans the fusion peptide proximal region (FPPR) to the membrane proximal external region (MPER) of gp41, by sedimentation velocity and double electron-electron resonance (DEER) EPR spectroscopy. Trimerization at pH 7 requires the presence of both the FPPR and MPER regions. However, at pH 4, the protein completely dissociates to monomers. DEER measurements reveal a partial fraying of the C-terminal MPER residues in the 17–172 trimer while the other regions, including the FPPR, remain compact. In accordance, truncating nine C-terminal MPER residues (675–683) in the 17–172 construct does not shift the trimer-monomer equilibrium significantly. Thus, in the context of the gp41 ectodomain spanning residues 17–172, trimerization is clearly dependent on FPPR and MPER regions even when the terminal residues of MPER unravel. The antibody Z13e1, which spans both the 2F5 and 4E10 epitopes in MPER, binds to 17–172 with a Kd of 1 ± 0.12 μM. Accordingly, individual antibodies 2F5 and 4E10 also recognize the 17–172 trimer/DPC complex. We propose that binding of the C-terminal residues of MPER to the surface of the DPC micelles models a correct positioning of the trimeric transmembrane domain anchored in the viral membrane. PMID:27513582

  4. Sublingual Priming with a HIV gp41-Based Subunit Vaccine Elicits Mucosal Antibodies and Persistent B Memory Responses in Non-Human Primates

    PubMed Central

    Bekri, Selma; Bourdely, Pierre; Luci, Carmelo; Dereuddre-Bosquet, Nathalie; Su, Bin; Martinon, Frédéric; Braud, Véronique M.; Luque, Irene; Mateo, Pedro L.; Crespillo, Sara; Conejero-Lara, Francisco; Moog, Christiane; Le Grand, Roger; Anjuère, Fabienne

    2017-01-01

    Persistent B cell responses in mucosal tissues are crucial to control infection against sexually transmitted pathogens like human immunodeficiency virus 1 (HIV-1). The genital tract is a major site of infection by HIV. Sublingual (SL) immunization in mice was previously shown to generate HIV-specific B cell immunity that disseminates to the genital tract. We report here the immunogenicity in female cynomolgus macaques of a SL vaccine based on a modified gp41 polypeptide coupled to the cholera toxin B subunit designed to expose hidden epitopes and to improve mucosal retention. Combined SL/intramuscular (IM) immunization with such mucoadhesive gp41-based vaccine elicited mucosal HIV-specific IgG and IgA antibodies more efficiently than IM immunization alone. This strategy increased the number and duration of gp41-specific IgA secreting cells. Importantly, combined immunization improved the generation of functional antibodies 3 months after vaccination as detected in HIV-neutralizing assays. Therefore, SL immunization represents a promising vaccine strategy to block HIV-1 transmission. PMID:28203239

  5. The membrane proximal external regions of gp41 from HIV-1 strains HXB2 and JRFL have different sensitivities to alanine mutation.

    PubMed

    Yi, Hyun Ah; Diaz-Rohrer, Barbara; Saminathan, Priyanka; Jacobs, Amy

    2015-03-03

    The transmembrane subunit (gp41) of the HIV envelope protein complex (Env) mediates the viral fusion step of HIV entry. The membrane proximal external region (MPER), one of the functional domains of gp41, has been the focus of a great deal of research because it is a target for neutralizing antibodies. In this study, we examined 23 amino acid residues in the MPER (660-683) in both a CXCR4 coreceptor-utilizing strain (HXB2) and a CCR5-utilizing strain (JRFL) by alanine scanning mutagenesis. Despite the high degree of gp41 sequence conservation, the effects of alanine mutation in the MPER were different between the two strains. Most mutations in HXB2 had fusogenicity and protein expression levels not less than 50% of that of the wild type in the case of cell-cell fusion. However, ∼30% of the mutants in HXB2 showed a severe defect in fusogenicity in viral entry. Mutations in the MPER of strain JRFL had more dramatic effects than that in HXB2 in cell-cell fusion and viral entry. The fact that there are large differences in the effects of mutation between two strains suggests the potential for the interaction of the MPER with nonconserved sequences such as the fusion peptide and/or other NHR domains as well as potential long-range structural effects on the conformational changes that occur with the Env complex during membrane fusion.

  6. Sublingual Priming with a HIV gp41-Based Subunit Vaccine Elicits Mucosal Antibodies and Persistent B Memory Responses in Non-Human Primates.

    PubMed

    Bekri, Selma; Bourdely, Pierre; Luci, Carmelo; Dereuddre-Bosquet, Nathalie; Su, Bin; Martinon, Frédéric; Braud, Véronique M; Luque, Irene; Mateo, Pedro L; Crespillo, Sara; Conejero-Lara, Francisco; Moog, Christiane; Le Grand, Roger; Anjuère, Fabienne

    2017-01-01

    Persistent B cell responses in mucosal tissues are crucial to control infection against sexually transmitted pathogens like human immunodeficiency virus 1 (HIV-1). The genital tract is a major site of infection by HIV. Sublingual (SL) immunization in mice was previously shown to generate HIV-specific B cell immunity that disseminates to the genital tract. We report here the immunogenicity in female cynomolgus macaques of a SL vaccine based on a modified gp41 polypeptide coupled to the cholera toxin B subunit designed to expose hidden epitopes and to improve mucosal retention. Combined SL/intramuscular (IM) immunization with such mucoadhesive gp41-based vaccine elicited mucosal HIV-specific IgG and IgA antibodies more efficiently than IM immunization alone. This strategy increased the number and duration of gp41-specific IgA secreting cells. Importantly, combined immunization improved the generation of functional antibodies 3 months after vaccination as detected in HIV-neutralizing assays. Therefore, SL immunization represents a promising vaccine strategy to block HIV-1 transmission.

  7. HIV Type 1 Molecular Epidemiology in pol and gp41 Genes Among Naive Patients from Mato Grosso do Sul State, Central Western Brazil

    PubMed Central

    da Silveira, Alexsander Augusto; Cardoso, Ludimila Paula Vaz; Francisco, Roberta Barbosa Lopes

    2012-01-01

    Abstract Antiretroviral naive patients (n=49) were recruited in central western Brazil (Campo Grande City/Mato Grosso do Sul State, located across the Bolivia and Paraguay borders). HIV-1 protease (PR), reverse transcriptase (RT), and env gp41 HR1 fragments were sequenced. Genetic diversity was analyzed by REGA/phylogenetic analyses. Intersubtype recombinants were identified by SimPlot/phylogenetic trees. PR/RT resistance was analyzed by Calibrated Population Resistance/Stanford databases. T-20 resistance in gp41 was assessed by Stanford, Los Alamos, and other sources. Of HIV-1 subtypes 65.3% were BPRBRT, 10.2% were CPRCRT, and 8.2% were F1PRF1RT. Intersubtype recombinants were 16.3%: four B/F1 and four B/C (two were “CRF31_BC-like”). The Pol-RT V75M mutation was detected in two homosexual partners; one patient had the T215S revertant mutation. T-20/gp41 resistance mutations were L44M (n=2) and V38A (n=1). The high percentage of non-B isolates (∼35%) highlights the importance of molecular surveillance studies in settings distant from the origin of the epidemic. Our data help elaborate the molecular epidemiological map of HIV-1 in Brazil. PMID:21790471

  8. Compensatory Changes in the Cytoplasmic Tail of gp41 Confer Resistance to Tetherin/BST-2 in a Pathogenic Nef-deleted SIV

    PubMed Central

    Serra-Moreno, Ruth; Jia, Bin; Breed, Matthew; Alvarez, Xavier; Evans, David T.

    2011-01-01

    SUMMARY Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. Whereas HIV-1 Vpu and HIV-2 Env counteract human tetherin, most SIVs use Nef to antagonize the tetherin proteins of their non-human primate hosts. Here we show that compensatory changes in the cytoplasmic domain of SIV gp41, acquired by a nef-deleted virus that regained a pathogenic phenotype in infected rhesus macaques, restore resistance to tetherin. These changes facilitate virus release in the presence of rhesus tetherin, but not human tetherin, and enhance virus replication in interferon-treated primary lymphocytes. The substitutions in gp41 result in a selective physical association with rhesus tetherin, and the internalization and sequestration of rhesus tetherin by a mechanism that depends on a conserved endocytosis motif in gp41. These results are consistent with HIV-2 Env antagonism of human tetherin, and suggest that the ability to oppose tetherin is important for lentiviral pathogenesis. PMID:21238946

  9. Mutation of critical serine residues in HIV-1 matrix result in an envelope incorporation defect which can be rescued by truncation of the gp41 cytoplasmic tail

    SciTech Connect

    Bhatia, Ajay K.; Kaushik, Rajnish; Campbell, Nancy A.; Pontow, Suzanne E.; Ratner, Lee

    2009-02-05

    The human immunodeficiency virus type 1 (HIV-1) matrix (MA) domain is involved in both early and late events of the viral life cycle. Simultaneous mutation of critical serine residues in MA has been shown previously to dramatically reduce phosphorylation of MA. However, the role of phosphorylation in viral replication remains unclear. Viruses harboring serine to alanine substitutions at positions 9, 67, 72, and 77 are severely impaired in their ability to infect target cells. In addition, the serine mutant viruses are defective in their ability to fuse with target cell membranes. Interestingly, both the fusion defect and the infectivity defect can be rescued by truncation of the long cytoplasmic tail of gp41 envelope protein (gp41CT). Sucrose density gradient analysis also reveals that these mutant viruses have reduced levels of gp120 envelope protein incorporated into the virions as compared to wild type virus. Truncation of the gp41CT rescues the envelope incorporation defect. Here we propose a model in which mutation of specific serine residues prevents MA interaction with lipid rafts during HIV-1 assembly and thereby impairs recruitment of envelope to the sites of viral budding.

  10. A neutralizing monoclonal antibody previously mapped exclusively on human immunodeficiency virus type 1 gp41 recognizes an epitope in p17 sharing the core sequence IEEE.

    PubMed Central

    Buratti, E; Tisminetzky, S G; D'Agaro, P; Baralle, F E

    1997-01-01

    We report here that a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing monoclonal antibody (MAb 1575) mapped to the conserved putative intracellular region from amino acid residues 735 to 752 (735-752 region) of gp41 also recognizes a region in an extracellular portion of p17. Both epitopes have a core recognition sequence (IEEE) in a nonhomologous context. The IEEE motif found in HIV-1 p17 is located in a region known as HGP-30 (residues 86 to 115) which has been previously associated with virus neutralization, cytotoxic T lymphocyte activity, and mother-to-child transmission. An analysis of available gp41 and p17 sequences demonstrates that in these regions both IEEE sequences are highly conserved in different HIV-1 clades. The presence of the IEEE epitope in p17 allows us to explain some unexpected neutralizing characteristics of MAb 1575. In addition, the gp41 735-752 region has been previously reported both in intra- and extracellular locations. Our results suggest that the extracellular location was the result of cross-reactivity with p17. PMID:9032383

  11. Functional and structural characterization of HIV-1 gp41 ectodomain regions in phospholipid membranes suggests that the fusion-active conformation is extended.

    PubMed

    Korazim, Ofir; Sackett, Kelly; Shai, Yechiel

    2006-12-15

    HIV-1 entry into its host cell involves a sequential interaction whereby gp41 is in direct contact with the plasma membrane. Understanding the effect of membrane composition on the fusion mechanism can shed light on the unsolved phases of this complex mechanism. Here, we studied N36, a peptide derived from the N-heptad-repeat (NHR) of the gp41 ectodomain, its six helix bundle (SHB) forming counterpart C34, together with the N-terminal 70-mer wild-type peptide (N70), and additional gp41 ectodomain-derived peptides in the presence of two membranes, modeling inner and outer leaflets of the plasma membrane. Information on the structure of these peptides, their affinity towards phospholipids and their ability to induce vesicle fusion was gathered by a variety of fluorescence, spectroscopic and microscopy methods. We found that N36, having strong affinity towards phospholipids, prominently shifts conformation from alpha-helix in an outer leaflet-like zwitterionic membrane to beta-sheet in a membrane mimicking the negatively charged inner leaflet environment, leading to pronounced fusion-activity. Real-time atomic force microscopy (AFM) was used to study the peptides' effect on the membrane morphology, revealing severe bilayer perturbation and extensive pore formation. We also found, that the N36/C34 core is destabilized by electronegative, but not zwitterionic phospholipids. Taken together, our data suggest that the fusion-active pore forming conformation of gp41 is extended, upstream of the SHB. In this manner, folding of the ectodomain into a SHB might also serve as a negative regulator of fusion by impeding gp41 fusion-active surfaces, thus preventing irreversible damage to the cell membrane. This assumption is supported by the finding that pre-incubation of large unilamellar vesicles (LUV) with C-heptad repeat (CHR)-derived fusion inhibitors reduces the fusogenic activity of N-terminal peptides in a dose-dependant manner, and suggests that CHR-derived fusion

  12. Molecular Dynamics Studies of the Inhibitor C34 Binding to the Wild-Type and Mutant HIV-1 gp41: Inhibitory and Drug Resistant Mechanism

    PubMed Central

    Ma, Xueting; Tan, Jianjun; Su, Min; Li, Chunhua; Zhang, Xiaoyi; Wang, Cunxin

    2014-01-01

    Mutations on NHR (N-terminal heptad repeat) associated with resistance to fusion inhibitor were observed. In addition, mutations on CHR (C-terminal heptad repeat) accompanied NHR mutations of gp41 are noted in many cases, like N43D/S138A double mutation. In this work, we explored the drug resistant mechanism of N43D mutation and the role of S138A second mutation in drug resistance. The binding modes of the wild type gp41 and the two mutants, N43D and N43D/S138A, with the HIV-1 fusion inhibitor C34, a 34-residue peptide mimicking CHR of gp41, were carried out by using molecular dynamics simulations. Based on the MD simulations, N43D mutation affects not only the stability of C34 binding, but also the binding energy of the inhibitor C34. Because N43D mutation may also affect the stable conformation of 6-HB, we introduced S138A second mutation into CHR of gp41 and determined the impact of this mutation. Through the comparative analysis of MD results of the N43D mutant and the N43D/S138A mutant, we found that CHR with S138A mutation shown more favorable affinity to NHR. Compelling differences in structures have been observed for these two mutants, particularly in the binding modes and in the hydrophobic interactions of the CHR (C34) located near the hydrophobic groove of the NHR. Because the conformational stability of 6-HB is important to HIV-1 infection, we suggested a hypothetical mechanism for the drug resistance: N43D single mutation not only impact the binding of inhibitor, but also affect the affinity between NHR and CHR of gp41, thus may reduce the rate of membrane fusion; compensatory mutation S138A would induce greater hydrophobic interactions between NHR and CHR, and render the CHR more compatible to NHR than inhibitors. PMID:25393106

  13. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    PubMed

    Louis, John M; Aniana, Annie; Lohith, Katheryn; Sayer, Jane M; Roche, Julien; Bewley, Carole A; Clore, G Marius

    2014-01-01

    We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  14. The Ebola Virus Glycoprotein Contributes to but Is Not Sufficient for Virulence In Vivo

    PubMed Central

    Groseth, Allison; Marzi, Andrea; Hoenen, Thomas; Herwig, Astrid; Gardner, Don; Becker, Stephan; Ebihara, Hideki; Feldmann, Heinz

    2012-01-01

    Among the Ebola viruses most species cause severe hemorrhagic fever in humans; however, Reston ebolavirus (REBOV) has not been associated with human disease despite numerous documented infections. While the molecular basis for this difference remains unclear, in vitro evidence has suggested a role for the glycoprotein (GP) as a major filovirus pathogenicity factor, but direct evidence for such a role in the context of virus infection has been notably lacking. In order to assess the role of GP in EBOV virulence, we have developed a novel reverse genetics system for REBOV, which we report here. Together with a previously published full-length clone for Zaire ebolavirus (ZEBOV), this provides a unique possibility to directly investigate the role of an entire filovirus protein in pathogenesis. To this end we have generated recombinant ZEBOV (rZEBOV) and REBOV (rREBOV), as well as chimeric viruses in which the glycoproteins from these two virus species have been exchanged (rZEBOV-RGP and rREBOV-ZGP). All of these viruses could be rescued and the chimeras replicated with kinetics similar to their parent virus in tissue culture, indicating that the exchange of GP in these chimeric viruses is well tolerated. However, in a mouse model of infection rZEBOV-RGP demonstrated markedly decreased lethality and prolonged time to death when compared to rZEBOV, confirming that GP does indeed contribute to the full expression of virulence by ZEBOV. In contrast, rREBOV-ZGP did not show any signs of virulence, and was in fact slightly attenuated compared to rREBOV, demonstrating that GP alone is not sufficient to confer a lethal phenotype or exacerbate disease in this model. Thus, while these findings provide direct evidence that GP contributes to filovirus virulence in vivo, they also clearly indicate that other factors are needed for the acquisition of full virulence. PMID:22876185

  15. Lung and salivary scavenger receptor glycoprotein-340 contribute to the host defense against influenza A viruses.

    PubMed

    Hartshorn, Kevan L; White, Mitchell R; Mogues, Tirsit; Ligtenberg, Toon; Crouch, Erika; Holmskov, Uffe

    2003-11-01

    The lung scavenger receptor-rich protein glycoprotein-340 (gp-340) is present in bronchoalveolar lavage (BAL) fluids and saliva and mediates specific adhesion to and aggregation of bacteria. It also binds to surfactant proteins A and D (SP-A and -D). Prior studies demonstrated that SP-A and SP-D contribute to innate defense against influenza A virus (IAV). We now show that lung and salivary gp-340 inhibit the hemagglutination activity and infectivity of IAV and agglutinate the virions through a mechanism distinct from that of SP-D. As in the case of SP-A, the antiviral effects of gp-340 are mediated by noncalcium-dependent interactions between the virus and sialic acid-bearing carbohydrates on gp-340. Gp-340 inhibits IAV strains that are resistant to SP-D. Concentrations of gp-340 present in saliva and BAL fluid of healthy donors are sufficient to bind to IAV and inhibit viral infectivity. On the basis of competition experiments using competing saccharide ligands, it appears that SP-D does not entirely mediate that anti-IAV activity of BAL fluid and contributes little to that of saliva. Furthermore, removal of gp-340 from BAL fluid and saliva significantly reduced anti-IAV activity. Hence, gp-340 contributes to defense against IAV and may be particularly relevant to defense against SP-D-resistant viral strains.

  16. Prevalence of Transmitted Drug-Resistance Mutations and Polymorphisms in HIV-1 Reverse Transcriptase, Protease, and gp41 Sequences Among Recent Seroconverters in Southern Poland

    PubMed Central

    Smoleń-Dzirba, Joanna; Rosińska, Magdalena; Kruszyński, Piotr; Bratosiewicz-Wąsik, Jolanta; Wojtyczka, Robert; Janiec, Janusz; Szetela, Bartosz; Beniowski, Marek; Bociąga-Jasik, Monika; Jabłonowska, Elżbieta; Wąsik, Tomasz J.

    2017-01-01

    Background Monitoring of drug resistance-related mutations among patients with recent HIV-1 infection offers an opportunity to describe current patterns of transmitted drug resistance (TDR) mutations. Material/Methods Of 298 individuals newly diagnosed from March 2008 to February 2014 in southern Poland, 47 were deemed to have recent HIV-1 infection by the limiting antigen avidity immunoassay. Proviral DNA was amplified and sequenced in the reverse transcriptase, protease, and gp41 coding regions. Mutations were interpreted according to the Stanford Database algorithm and/or the International Antiviral Society USA guidelines. TDR mutations were defined according to the WHO surveillance list. Results Among 47 patients with recent HIV-1 infection only 1 (2%) had evidence of TDR mutation. No major resistance mutations were found, but the frequency of strains with ≥1 accessory resistance-associated mutations was high, at 98%. Accessory mutations were present in 11% of reverse transcriptase, 96% of protease, and 27% of gp41 sequences. Mean number of accessory resistance mutations in the reverse transcriptase and protease sequences was higher in viruses with no compensatory mutations in the gp41 HR2 domain than in strains with such mutations (p=0.031). Conclusions Despite the low prevalence of strains with TDR mutations, the frequency of accessory mutations was considerable, which may reflect the history of drug pressure among transmitters or natural viral genetic diversity, and may be relevant for future clinical outcomes. The accumulation of the accessory resistance mutations within the pol gene may restrict the occurrence of compensatory mutations related to enfuvirtide resistance or vice versa. PMID:28167814

  17. Functional characteristics of the natural polymorphisms of HIV-1 gp41 in HIV-1 isolates from enfuvirtide-naïve Korean patients.

    PubMed

    Shin, YoungHyun; Yoon, Cheol-Hee; Yang, Hyo-Jin; Lim, Hoyong; Choi, Byeong-Sun; Kim, Sung Soon; Kang, Chun

    2016-06-01

    HIV-1 gp41 plays a key role in viral entry. The insertion of Thr at position 4 and Met/Val/Phe substitutions at position 7 are frequently observed in the fusion peptide (FP) motif of gp41 without major enfuvirtide resistance associated with mutation in heptad repeats 1/2 (HR1/2) of HIV-1 isolates from Korean patients. Here, the influence of these mutations on their biological function was evaluated by employing HIV-1 variants with mutant FPs as shown previously and with recombinant HIV-1 using the env genes of 20 HIV-1 isolates from Korean patients. In an infectivity assay, all FP mutants showed lower infectivity than the wild-type NL4-3. In particular, the substitutions at position 7 led to much greater reductions in infectivity than the insertions at position 4. Nevertheless, the replication kinetics of most mutants were similar to those of the wild type, except that the FP mutants with an Ile insertion at position 4 and a Phe substitution at position 7 showed reduced replication. Moreover, most point mutants showed lower IC50 values for enfuvirtide than the wild type, whereas the L7M substitution resulted in a slightly increased IC50 value. The infectivity using the HIV-1 env recombinant viruses decreased in 14 cases but increased slightly in six cases compared with the wild type. Most recombinants were more susceptible to enfuvirtide than the wild type, except for three recombinants that showed slight resistance. Our findings may help to explain the potential mechanisms corresponding to the natural polymorphism of gp41 and to predict the efficiency of enfuvirtide in treatment of HIV-1-infected patients in Korea.

  18. Prevalence of Transmitted Drug-Resistance Mutations and Polymorphisms in HIV-1 Reverse Transcriptase, Protease, and gp41 Sequences Among Recent Seroconverters in Southern Poland.

    PubMed

    Smoleń-Dzirba, Joanna; Rosińska, Magdalena; Kruszyński, Piotr; Bratosiewicz-Wąsik, Jolanta; Wojtyczka, Robert; Janiec, Janusz; Szetela, Bartosz; Beniowski, Marek; Bociąga-Jasik, Monika; Jabłonowska, Elżbieta; Wąsik, Tomasz J; The Cascade Collaboration In EuroCoord, And

    2017-02-07

    BACKGROUND Monitoring of drug resistance-related mutations among patients with recent HIV-1 infection offers an opportunity to describe current patterns of transmitted drug resistance (TDR) mutations. MATERIAL AND METHODS Of 298 individuals newly diagnosed from March 2008 to February 2014 in southern Poland, 47 were deemed to have recent HIV-1 infection by the limiting antigen avidity immunoassay. Proviral DNA was amplified and sequenced in the reverse transcriptase, protease, and gp41 coding regions. Mutations were interpreted according to the Stanford Database algorithm and/or the International Antiviral Society USA guidelines. TDR mutations were defined according to the WHO surveillance list. RESULTS Among 47 patients with recent HIV-1 infection only 1 (2%) had evidence of TDR mutation. No major resistance mutations were found, but the frequency of strains with ≥1 accessory resistance-associated mutations was high, at 98%. Accessory mutations were present in 11% of reverse transcriptase, 96% of protease, and 27% of gp41 sequences. Mean number of accessory resistance mutations in the reverse transcriptase and protease sequences was higher in viruses with no compensatory mutations in the gp41 HR2 domain than in strains with such mutations (p=0.031). CONCLUSIONS Despite the low prevalence of strains with TDR mutations, the frequency of accessory mutations was considerable, which may reflect the history of drug pressure among transmitters or natural viral genetic diversity, and may be relevant for future clinical outcomes. The accumulation of the accessory resistance mutations within the pol gene may restrict the occurrence of compensatory mutations related to enfuvirtide resistance or vice versa.

  19. Thymic pathogenicity of an HIV-1 envelope is associated with increased CXCR4 binding efficiency and V5-gp41-dependent activity, but not V1/V2-associated CD4 binding efficiency and viral entry.

    PubMed

    Meissner, Eric G; Coffield, Vernon M; Su, Lishan

    2005-06-05

    We previously described a thymus-tropic HIV-1 envelope (R3A Env) from a rapid progressor obtained at the time of transmission. An HIV-1 molecular recombinant with the R3A Env supported extensive replication and pathogenesis in the thymus and did not require Nef. Another Env from the same patient did not display the same thymus-tropic pathogenesis (R3B Env). Here, we show that relative to R3B Env, R3A Env enhances viral entry of T cells, increases fusion-induced cytopathicity, and shows elevated binding efficiency for both CD4 and CXCR4, but not CCR5, in vitro. We created chimeric envelopes to determine the region(s) responsible for each in vitro phenotype and for thymic pathogenesis. Surprisingly, while V1/V2 contributed to enhanced viral entry, CD4 binding efficiency, and cytopathicity in vitro, it made no contribution to thymic pathogenesis. Rather, CXCR4 binding efficiency and V5-gp41-associated activity appear to independently contribute to thymic pathogenesis of the R3A Env. These data highlight the contribution of unique HIV pathogenic factors in the thymic microenvironment and suggest that novel mechanisms may be involved in Env pathogenic activity in vivo.

  20. Thymic pathogenicity of an HIV-1 envelope is associated with increased CXCR4 binding efficiency and V5-gp41-dependent activity, but not V1/V2-associated CD4 binding efficiency and viral entry

    SciTech Connect

    Meissner, Eric G.; Coffield, Vernon M.; Su Lishan . E-mail: lsu@med.unc.edu

    2005-06-05

    We previously described a thymus-tropic HIV-1 envelope (R3A Env) from a rapid progressor obtained at the time of transmission. An HIV-1 molecular recombinant with the R3A Env supported extensive replication and pathogenesis in the thymus and did not require Nef. Another Env from the same patient did not display the same thymus-tropic pathogenesis (R3B Env). Here, we show that relative to R3B Env, R3A Env enhances viral entry of T cells, increases fusion-induced cytopathicity, and shows elevated binding efficiency for both CD4 and CXCR4, but not CCR5, in vitro. We created chimeric envelopes to determine the region(s) responsible for each in vitro phenotype and for thymic pathogenesis. Surprisingly, while V1/V2 contributed to enhanced viral entry, CD4 binding efficiency, and cytopathicity in vitro, it made no contribution to thymic pathogenesis. Rather, CXCR4 binding efficiency and V5-gp41-associated activity appear to independently contribute to thymic pathogenesis of the R3A Env. These data highlight the contribution of unique HIV pathogenic factors in the thymic microenvironment and suggest that novel mechanisms may be involved in Env pathogenic activity in vivo.

  1. Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41.

    PubMed

    Gustchina, Elena; Li, Mi; Ghirlando, Rodolfo; Schuck, Peter; Louis, John M; Pierson, Jason; Rao, Prashant; Subramaniam, Sriram; Gustchina, Alla; Clore, G Marius; Wlodawer, Alexander

    2013-01-01

    A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.

  2. Polymorphisms in the HIV-1 gp41 env gene, natural resistance to enfuvirtide (T-20) and pol resistance among pregnant Brazilian women.

    PubMed

    Reis, Mônica Nogueira da Guarda; de Alcântara, Keila Correa; Cardoso, Ludimila Paula Vaz; Stefani, Mariane Martins Araújo

    2014-01-01

    The selective pressure of antiretroviral drugs (ARVs) targeting HIV-1 pol can promote drug resistance mutations in other genomic regions, such as env. Drug resistance among women should be monitored to avoid horizontal and mother-to-child transmission. To describe natural resistance to T-20 (enfuvirtide), gp41 env polymorphisms, mutations in pol and HIV-1 subtypes, 124 pregnant women were recruited. For 98 patients, the gp41 env, protease (PR) and reverse transcriptase (RT) fragments were sequenced. The patients were ARV naïve (n = 30), taking mother-to-child transmission prophylaxis (n = 50), or being treated with highly active ARV therapy/HAART (n = 18). The Stanford and IAS/USA databases and other sources were used to analyze PR/RT, gp41 env resistance mutations. The HIV-1 genetic diversity was analyzed by REGA/phylogenetic analyses. The patients' median age was 25 years (range, 16-42), 18.4% had AIDS. The frequency of natural resistance to T-20 (N42D, L44M, and R46M-low-impact mutations) was 6.1% (6/98); 20.4% (20/98) had compensatory mutations in HR2. The prevalence of transmitted drug resistance in the pol was 13.3% (4/30), and the prevalence of secondary drug resistance was 33.3% (6/18). Two patients were infected with multidrug resistant/MDR viruses. The analysis of HIV-1 subtypes (PR/RT/gp41) revealed that 61.2% (60/98) were subtype B, 12.2% (12/98) were subtype C, 4.1% (4/98) were subtype F1, and 22.4% (22/98) were possible recombinants (BF1 = 20.4%; BC = 2%). Natural resistance to T-20 was not associated with pol resistance or previous ARV use. The high rate of secondary resistance, including MDR, indicates that the number of women that may need T-20 salvage therapy may be higher than anticipated.

  3. Selected amino acid changes in HIV-1 subtype-C gp41 are associated with specific gp120(V3) signatures in the regulation of co-receptor usage.

    PubMed

    Dimonte, Salvatore; Babakir-Mina, Muhammed; Mercurio, Fabio; Di Pinto, Domenico; Ceccherini-Silberstein, Francesca; Svicher, Valentina; Perno, Carlo-Federico

    2012-09-01

    The majority of studies have characterized the tropism of HIV-1 subtype-B isolates, but little is known about the determinants of tropism in other subtypes. So, the goal of the present study was to genetically characterize the envelope of viral proteins in terms of co-receptor usage by analyzing 356 full-length env sequences derived from HIV-1 subtype-C infected individuals. The co-receptor usage of V3 sequences was inferred by using the Geno2Pheno and PSSM algorithms, and also analyzed to the "11/25 rule". All reported env sequences were also analyzed with regard to N-linked glycosylation sites, net charge and hydrophilicity, as well as the binomial correlation phi coefficient to assess covariation among gp120(V3) and gp41 signatures and the average linkage hierarchical agglomerative clustering were also performed. Among env sequences present in Los Alamos Database, 255 and 101 sequences predicted as CCR5 and CXCR4 were selected, respectively. The classical V3 signatures at positions 11 and 25, and other specific V3 and gp41 amino acid changes were found statistically associated with different co-receptor usage. Furthermore, several statistically significant associations between V3 and gp41 signatures were also observed. The dendrogram topology showed a cluster associated with CCR5-usage composed by five gp41 mutated positions, A22V, R133M, E136G, N140L, and N166Q that clustered with T2V(V3) and G24T(V3) (bootstrap=1). Conversely, a heterogeneous cluster with CXCR4-usage, involving S11GR(V3), 13-14insIG/LG(V3), P16RQ(V3), Q18KR(V3), F20ILV(V3), D25KRQ(V3), Q32KR(V3) along with A30T(gp41), S107N(gp41), D148E(gp41), A189S(gp41) was identified (bootstrap=0.86). Our results show that as observed for HIV-1 subtype-B, also in subtype-C specific and different gp41 and gp120V3 amino acid changes are associated individually or together with CXCR4 and/or CCR5 usage. These findings strengthen previous observations that determinants of tropism may also reside in the gp41

  4. Leucine-rich α2-glycoprotein overexpression in the brain contributes to memory impairment.

    PubMed

    Akiba, Chihiro; Nakajima, Madoka; Miyajima, Masakazu; Ogino, Ikuko; Miura, Masami; Inoue, Ritsuko; Nakamura, Eri; Kanai, Fumio; Tada, Norihiro; Kunichika, Miyuki; Yoshida, Mitsutaka; Nishimura, Kinya; Kondo, Akihide; Sugano, Hidenori; Arai, Hajime

    2017-08-24

    We previously reported increase in leucine-rich α2-glycoprotein (LRG) concentration in cerebrospinal fluid is associated with cognitive decline in humans. To investigate relationship between LRG expression in the brain and memory impairment, we analyzed transgenic mice overexpressing LRG in the brain (LRG-Tg) focusing on hippocampus. Immunostaining and Western blotting revealed age-related increase in LRG expression in hippocampal neurons in 8-, 24-, and 48-week-old controls and LRG-Tg. Y-maze and Morris water maze tests indicated retained spatial memory in 8- and 24-week-old LRG-Tg, while deteriorated in 48-week-old LRG-Tg compared with age-matched controls. Field excitatory postsynaptic potentials declined with age in LRG-Tg compared with controls at 8, 24, and 48 weeks. Paired-pulse ratio decreased with age in LRG-Tg, while increased in controls. As a result, long-term potentiation was retained in 8- and 24-week-old LRG-Tg, whereas diminished in 48-week-old LRG-Tg compared with age-matched controls. Electron microscopy observations revealed fewer synaptic vesicles and junctions in LRG-Tg compared with age-matched controls, which became significant with age. Hippocampal LRG overexpression contributes to synaptic dysfunction, which leads to memory impairment with advance of age. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Solid-state NMR spectroscopy of the HIV gp41 membrane fusion protein supports intermolecular antiparallel β sheet fusion peptide structure in the final six-helix bundle state.

    PubMed

    Sackett, Kelly; Nethercott, Matthew J; Zheng, Zhaoxiong; Weliky, David P

    2014-03-06

    The HIV gp41 protein catalyzes fusion between viral and target cell membranes. Although the ~20-residue N-terminal fusion peptide (FP) region is critical for fusion, the structure of this region is not well characterized in large gp41 constructs that model the gp41 state at different times during fusion. This paper describes solid-state NMR (SSNMR) studies of FP structure in a membrane-associated construct (FP-Hairpin), which likely models the final fusion state thought to be thermostable trimers with six-helix bundle structure in the region C-terminal of the FP. The SSNMR data show that there are populations of FP-Hairpin with either α helical or β sheet FP conformation. For the β sheet population, measurements of intermolecular (13)C-(13)C proximities in the FP are consistent with a significant fraction of intermolecular antiparallel β sheet FP structure with adjacent strand crossing near L7 and F8. There appears to be negligible in-register parallel structure. These findings support assembly of membrane-associated gp41 trimers through interleaving of N-terminal FPs from different trimers. Similar SSNMR data are obtained for FP-Hairpin and a construct containing the 70 N-terminal residues of gp41 (N70), which is a model for part of the putative pre-hairpin intermediate state of gp41. FP assembly may therefore occur at an early fusion stage. On a more fundamental level, similar SSNMR data are obtained for FP-Hairpin and a construct containing the 34 N-terminal gp41 residues (FP34) and support the hypothesis that the FP is an autonomous folding domain.

  6. Sequence analysis of selected regions of the env (V3 loop and gp41) and gag (p7) reading frames of Ethiopian human immunodeficiency virus type 1 strains.

    PubMed

    Ayehunie, S; Johansson, B; Sönnerborg, A; Zewdie, D W; Britton, S; Strannegård, O

    1993-01-01

    Amplified polymerase chain reaction (PCR) products, corresponding to the V3 loop and gp41 of the env, and p7 of the gag region, from proviral DNA of several Ethiopian and Swedish HIV-1 strains were sequenced. Of the six amino acids (GPGRAF) that constitute the principal neutralizing determinant (PND) within the V3 loop, the Ethiopian isolates all showed two amino acid changes (GPGQTF). Four to five other substitutions were found in the amino acids flanking the PND. Substitution of alanine (A) for threonine (T) should result in a change in the predicted secondary structure, i.e., disappearance of a coil structure. Percentage similarity data on a stretch of 22 amino acids within the V3 loop showed a concordance of the Ethiopian HIV-1 isolates with the sequences of published macrophage-T-cell tropic HIV isolates. Additionally derived protein sequences in two other regions showed two common substitutions in p7 and one to two substitutions in gp41 compared to a recent consensus sequence. These changes are hitherto unique for the Ethiopian strains, and suggest the presence of a clustering of a divergent HIV-1 strain in Addis Ababa, Ethiopia.

  7. Comparative mucosal immunogenicity of HIV gp41 membrane-proximal external region (MPER) containing single and multiple repeats of ELDKWA sequence with defensin peptides.

    PubMed

    Mohan, Teena; Verma, Priyanka; Rao, D N

    2014-04-01

    The MPER of gp41 of HIV-1 has received great attention and is widely recognized as a promising target for the development of AIDS vaccine. We investigated the ability of trirepeat of ELDKWA sequence of gp41 antigen with defensins in liposome using multiple-shot immunization strategy in the mice model. The designed was used to enhance the immunogenicity and exposure of MPER in its native conformation for the induction of MPER-specific HIV-1 neutralizing antibodies. To characterize, we estimated the antibody levels (IgG/IgA) in serum as well as in lung, intestinal, vaginal and rectal washes till day 120 in outbred and inbred (H-2(b) and H-2(d)) mice using liposome as delivery vehicle. The representative sera and washes were also tested for in vitro neutralization with CCR5-tropic Indian HIV-1 primary isolates. We observed that the modified HIV antigen containing trirepeat of ELDKWA with defensins was showing significantly (p<0.001) higher IgG/IgA antibody titre (102,400-204,800) in sera as well as in different mucosal washes (1600-6400) than standard HIV-1 antigen. Furthermore, sera from the modified HIV-1 antigen with defensins found to exhibit higher neutralizing activities (ranging from 59.3% to 84.6%) than the standard HIV-1 antigen. These results show that the induction of MPER-specific HIV-1 neutralizing antibodies could be achieved through a rationally designed vaccine strategy.

  8. Resistance of a human immunodeficiency virus type 1 isolate to a small molecule CCR5 inhibitor can involve sequence changes in both gp120 and gp41

    SciTech Connect

    Anastassopoulou, Cleo G. Ketas, Thomas J.; Depetris, Rafael S.; Thomas, Antonia M.; Klasse, Per Johan; Moore, John P.

    2011-04-25

    Here, we describe the genetic pathways taken by a human immunodeficiency virus type 1 (HIV-1) isolate, D101.12, to become resistant to the small molecule CCR5 inhibitor, vicriviroc (VCV), in vitro. Resistant D101.12 variants contained at least one substitution in the gp120 V3 region (H308P), plus one of two patterns of gp41 sequence changes involving the fusion peptide (FP) and a downstream residue: G514V+V535M or M518V+F519L+V535M. Studies of Env-chimeric and point-substituted viruses in peripheral blood mononuclear cells (PBMC) and TZM-bl cells showed that resistance can arise from the cooperative action of gp120 and gp41 changes, while retaining CCR5 usage. Modeling the VCV inhibition data from the two cell types suggests that D101.12 discriminates between high- and low-VCV affinity forms of CCR5 less than D1/85.16, a resistant virus with three FP substitutions.

  9. Novel recombinant engineered gp41 N-terminal heptad repeat trimers and their potential as anti-HIV-1 therapeutics or microbicides.

    PubMed

    Chen, Xi; Lu, Lu; Qi, Zhi; Lu, Hong; Wang, Ji; Yu, Xiaoxia; Chen, Yinghua; Jiang, Shibo

    2010-08-13

    Peptides derived from N-terminal heptad repeat (NHR) of the HIV-1 gp41 are generally poor inhibitors of HIV-1 entry, because they tend to aggregate and do not form a trimeric coiled-coil. In this study, we have fused portions of gp41 NHR, e.g. N36 or N28, to the T4 fibritin trimerization domain, Foldon (Fd), thus constructing novel NHR trimers, designated N36Fd or N28Fd, which could be expressed in Escherichia coli cells. The purified N36Fd and N28Fd exhibited SDS-resistant trimeric coiled-coil conformation with improved alpha-helicity compared with the corresponding N-peptides. They could interact with a C-peptide (e.g. C34) to form stable six-helix bundle and possessed potent anti-HIV-1 activity against a broad spectrum of HIV-1 strains. N28Fd was effective against T20-resistant HIV-1 variants and more resistant to proteinase K compared with T20 (enfuvirtide), a C-peptide-based HIV fusion inhibitor. Therefore, N28Fd trimer has great potentials for further development as an affordable therapeutic or microbicide for treatment and prevention of HIV-1 infection.

  10. Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti–HIV-1 drugs

    PubMed Central

    Crespillo, Sara; Cámara-Artigas, Ana; Casares, Salvador; Morel, Bertrand; Cobos, Eva S.; Mateo, Pedro L.; Mouz, Nicolas; Martin, Christophe E.; Roger, Marie G.; El Habib, Raphaelle; Su, Bin; Moog, Christiane; Conejero-Lara, Francisco

    2014-01-01

    During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates. PMID:25489108

  11. Antigenic characterization of the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor incorporated into nanodiscs

    PubMed Central

    Witt, Kristen C.; Castillo-Menendez, Luis; Ding, Haitao; Espy, Nicole; Zhang, Shijian; Kappes, John C.; Sodroski, Joseph

    2017-01-01

    The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development. Here we investigate variables associated with reconstitution of the HIV-1 Env precursor into nanodiscs, nanoscale lipid bilayer discs enclosed by membrane scaffolding proteins. We identified detergents, as well as lipids similar in composition to the viral lipidome, that allowed efficient formation of Env-nanodiscs (Env-NDs). Env-NDs were created with the full-length Env precursor and with an Env precursor with the majority of the cytoplasmic tail intact. The self-association of Env-NDs was decreased by glutaraldehyde crosslinking. The Env-NDs exhibited an antigenic profile expected for the HIV-1 Env precursor. Env-NDs were recognized by broadly neutralizing antibodies. Of note, neutralizing antibody epitopes in the gp41 membrane-proximal external region and in the gp120:gp41 interface were well exposed on Env-NDs compared with Env expressed on cell surfaces. Most Env epitopes recognized by non-neutralizing antibodies were masked on the Env-NDs. This antigenic profile was stable for several days, exhibiting a considerably longer half-life than that of Env solubilized in detergents. Negative selection with weak neutralizing antibodies could be used to improve the antigenic profile of the Env-NDs. Finally, we show that lipid adjuvants can be incorporated into Env-NDs. These results indicate that Env-NDs represent a potentially useful platform for investigating the structural, functional and antigenic properties of the HIV-1 Env trimer in a membrane context

  12. Contribution of Disulfide Bridging to Epitope Expression of the Dengue Type 2 Virus Envelope Glycoprotein

    PubMed Central

    Roehrig, John T.; Volpe, Katharine E.; Squires, Jennifer; Hunt, Ann R.; Davis, Brent S.; Chang, Gwong-Jen J.

    2004-01-01

    The individual contributions of each of the six conserved disulfide (SS) bonds in the dengue 2 virus envelope (E) glycoprotein (strain 16681) to epitope expression was determined by measuring the reactivities of a panel of well-defined monoclonal antibodies (MAbs) with LLC-MK2 cells that had been transiently transformed with plasmid vectors expressing E proteins that were mutant in their SS bonds. Three domain I (DI) epitopes (C1, C3, and C4) were affected by elimination of any SS bond and were essentially the only epitopes affected by elimination of the amino-proximal SS1 formed between Cys 3 and Cys 30. The remaining DI epitope (C2) was sensitive to only SS3-bond (Cys 74-Cys 105) and SS6-bond (Cys 302-Cys 333) elimination. Of the four DII epitopes examined, reactivities of three anti-epitope MAbs (A1, A2, and A5) were reduced by elimination of SS2 (Cys 61-Cys 121), SS3, SS4 (Cys 94-Cys 116), SS5 (Cys 185-Cys 285), or SS6. The other DII epitope examined (A3) was sensitive only to SS2- and SS3-bond elimination. The three DIII epitopes tested (B2, B3, and B4) were most sensitive to elimination of SS6. The flavivirus group epitope (A1) was less sensitive to elimination of SS3 and SS6. This result may indicate that the region proximal to the E-protein fusion motif (amino acids 98 to 110) may have important linear components. If this observation can be confirmed, peptide mimics from this region of E protein might be able to interfere with flavivirus replication. PMID:14963174

  13. Structural characterization of the feline-immunodeficiency-virus envelope glycoprotein 36 ectodomain for the development of new antivirals

    PubMed Central

    2005-01-01

    In the fight against the human HIV, new targets are being explored, such as the proteins involved in the process of fusion of the virus with the host cell. Recently, the first generation of fusion inhibitors (enfuvirtide), targeting gp41 (virus envelope glycoprotein 41), has become commercially available. However, this promising class of drugs has to be improved in respect of their efficacy and bioavailability. Considering the strong homologies between HIV and FIV (feline immunodeficiency virus), as well as the highly conserved structure of the transmembrane envelope protein among species, FIV represents a relevant model of pre-screening studies for HIV. Taking into account (i) sequence homologies between the ectodomain of HIV gp41 and FIV gp36 (envelope glycoprotein 36), (ii) structural data available for gp41 and (iii) the fact that synthetic peptides derived from gp36 are effective inhibitors of FIV infection, we designed several peptides derived from gp36 sequence. We checked that these peptides had the same structural features as the corresponding peptides from gp41 HIV by CD, analytical ultracentrifugation and 1H–2H (hydrogen–deuterium) exchange combined with MS. By combining this latter technique with surface-plasmon-resonance assays, we identified the amino acid residues of the C-terminal region of the ectodomain of gp36 that are critical for interaction with the N-terminal region. This gave clues for therapy and vaccines against FIV, thus providing helpful data for treatments against HIV. PMID:15799719

  14. 3D NMR Experiments for Measuring 15N Relaxation Data of Large Proteins: Application to the 44 kDa Ectodomain of SIV gp41

    NASA Astrophysics Data System (ADS)

    Caffrey, Michael; Kaufman, Joshua; Stahl, Stephen J.; Wingfield, Paul T.; Gronenborn, Angela M.; Clore, G. Marius

    1998-12-01

    A suite of 3D NMR experiments for measuring15N-{1H} NOE,15NT1, and15NT1ρvalues in large proteins, uniformly labeled with15N and13C, is presented. These experiments are designed for proteins that exhibit extensive spectral overlap in the 2D1H-15N HSQC spectrum. The pulse sequences are readily applicable to perdeuterated samples, which increases the spectral resolution and signal-to-noise ratio, thereby permitting the characterization of protein dynamics to be extended to larger protein systems. Application of the pulse sequences is demonstrated on a perdeuterated13C/15N-labeled sample of the 44 kDa ectodomain of SIV gp41.

  15. Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion

    PubMed Central

    Gong, Zhen; Martin-Garcia, Jose M.; Daskalova, Sasha M.; Craciunescu, Felicia M.; Song, Lusheng; Dörner, Katerina; Hansen, Debra T.; Yang, Jay-How; LaBaer, Joshua; Hogue, Brenda G.; Mor, Tsafrir S.; Fromme, Petra

    2015-01-01

    The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41. PMID:26295457

  16. Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion.

    PubMed

    Gong, Zhen; Martin-Garcia, Jose M; Daskalova, Sasha M; Craciunescu, Felicia M; Song, Lusheng; Dörner, Katerina; Hansen, Debra T; Yang, Jay-How; LaBaer, Joshua; Hogue, Brenda G; Mor, Tsafrir S; Fromme, Petra

    2015-01-01

    The membrane proximal region (MPR, residues 649-683) and transmembrane domain (TMD, residues 684-705) of the gp41 subunit of HIV-1's envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662-683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649-705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.

  17. Transcytosis-blocking abs elicited by an oligomeric immunogen based on the membrane proximal region of HIV-1 gp41 target non-neutralizing epitopes.

    PubMed

    Matoba, Nobuyuki; Griffin, Tagan A; Mittman, Michele; Doran, Jeffrey D; Alfsen, Annette; Montefiori, David C; Hanson, Carl V; Bomsel, Morgane; Mor, Tsafrir S

    2008-05-01

    CTB-MPR(649-684), a translational fusion protein consisting of cholera toxin B subunit (CTB) and residues 649 684 of gp41 membrane proximal region (MPR), is a candidate vaccine aimed at blocking early steps of HIV-1 mucosal transmission. Bacterially produced CTB MPR(649-684) was purified to homogeneity by two affinity chromatography steps. Similar to gp41 and derivatives thereof, the MPR domain can specifically and reversibly self-associate. The affinities of the broadly-neutralizing monoclonal Abs 4E10 and 2F5 to CTB MPR(649-684) were equivalent to their nanomolar affinities toward an MPR peptide. The fusion protein's affinity to GM1 ganglioside was comparable to that of native CTB. Rabbits immunized with CTB-MPR(649-684) raised only a modest level of anti-MPR(649-684) Abs. However, a prime-boost immunization with CTB-MPR(649-684) and a second MPR(649-684)-based immunogen elicited a more productive anti-MPR(649-684) antibody response. These Abs strongly blocked the epithelial transcytosis of a primary subtype B HIV-1 isolate in a human tight epithelial model, expanding our previously reported results using a clade D virus. The Abs recognized epitopes at the N-terminal portion of the MPR peptide, away from the 2F5 and 4E10 epitopes and were not effective in neutralizing infection of CD4+ cells. These results indicate distinct vulnerabilities of two separate interactions of HIV-1 with human cells - Abs against the C-terminal portion of the MPR can neutralize CD4+-dependent infection, while Abs targeting the MPR's N-terminal portion can effectively block galactosyl ceramide dependent transcytosis. We propose that Abs induced by MPR(649-684)-based immunogens may provide broad protective value independent of infection neutralization.

  18. Identification of a Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Variant Resistant to Cold Inactivation▿ †

    PubMed Central

    Kassa, Aemro; Finzi, Andrés; Pancera, Marie; Courter, Joel R.; Smith, Amos B.; Sodroski, Joseph

    2009-01-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein trimer consists of gp120 and gp41 subunits and undergoes a series of conformational changes upon binding to the receptors, CD4 and CCR5/CXCR4, that promote virus entry. Surprisingly, we found that the envelope glycoproteins of some HIV-1 strains are functionally inactivated by prolonged incubation on ice. Serial exposure of HIV-1 to extremes of temperature, followed by expansion of replication-competent viruses, allowed selection of a temperature-resistant virus. The envelope glycoproteins of this virus resisted cold inactivation due to a single passage-associated change, H66N, in the gp120 exterior envelope glycoprotein. Histidine 66 is located within the gp41-interactive inner domain of gp120 and, in other studies, has been shown to decrease the sampling of the CD4-bound conformation by unliganded gp120. Substituting asparagine or other amino acid residues for histidine 66 in cold-sensitive HIV-1 envelope glycoproteins resulted in cold-stable phenotypes. Cold inactivation of the HIV-1 envelope glycoproteins occurred even at high pH, indicating that protonation of histidine 66 is not necessary for this process. Increased exposure of epitopes in the ectodomain of the gp41 transmembrane envelope glycoprotein accompanied cold inactivation, but shedding of gp120 did not. An amino acid change in gp120 (S375W) that promotes the CD4-bound state or treatment with soluble CD4 or a small-molecule CD4 mimic resulted in increased cold sensitivity. These results indicate that the CD4-bound intermediate of the HIV-1 envelope glycoproteins is cold labile; avoiding the CD4-bound state increases temperature stability. PMID:19211747

  19. Identification of a human immunodeficiency virus type 1 envelope glycoprotein variant resistant to cold inactivation.

    PubMed

    Kassa, Aemro; Finzi, Andrés; Pancera, Marie; Courter, Joel R; Smith, Amos B; Sodroski, Joseph

    2009-05-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein trimer consists of gp120 and gp41 subunits and undergoes a series of conformational changes upon binding to the receptors, CD4 and CCR5/CXCR4, that promote virus entry. Surprisingly, we found that the envelope glycoproteins of some HIV-1 strains are functionally inactivated by prolonged incubation on ice. Serial exposure of HIV-1 to extremes of temperature, followed by expansion of replication-competent viruses, allowed selection of a temperature-resistant virus. The envelope glycoproteins of this virus resisted cold inactivation due to a single passage-associated change, H66N, in the gp120 exterior envelope glycoprotein. Histidine 66 is located within the gp41-interactive inner domain of gp120 and, in other studies, has been shown to decrease the sampling of the CD4-bound conformation by unliganded gp120. Substituting asparagine or other amino acid residues for histidine 66 in cold-sensitive HIV-1 envelope glycoproteins resulted in cold-stable phenotypes. Cold inactivation of the HIV-1 envelope glycoproteins occurred even at high pH, indicating that protonation of histidine 66 is not necessary for this process. Increased exposure of epitopes in the ectodomain of the gp41 transmembrane envelope glycoprotein accompanied cold inactivation, but shedding of gp120 did not. An amino acid change in gp120 (S375W) that promotes the CD4-bound state or treatment with soluble CD4 or a small-molecule CD4 mimic resulted in increased cold sensitivity. These results indicate that the CD4-bound intermediate of the HIV-1 envelope glycoproteins is cold labile; avoiding the CD4-bound state increases temperature stability.

  20. Evaluation of a novel multi-immunogen vaccine strategy for targeting 4E10/10E8 neutralizing epitopes on HIV-1 gp41 membrane proximal external region.

    PubMed

    Banerjee, Saikat; Shi, Heliang; Banasik, Marisa; Moon, Hojin; Lees, William; Qin, Yali; Harley, Andrew; Shepherd, Adrian; Cho, Michael W

    2017-02-23

    The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by broadly neutralizing antibodies (bnAbs) 4E10 and 10E8. In this proof-of-concept study, we evaluated a novel multi-immunogen vaccine strategy referred to as Incremental, Phased Antigenic Stimulation for Rapid Antibody Maturation (IPAS-RAM) to induce 4E10/10E8-like bnAbs. Rabbits were immunized sequentially, but in a phased manner, with three immunogens that are progressively more native (gp41-28×3, gp41-54CT, and rVV-gp160DH12). Although nAbs were not induced, epitope-mapping analyses indicated that IPAS-RAM vaccination was better able to target antibodies towards the 4E10/10E8 epitopes than homologous prime-boost immunization using gp41-28×3 alone. MPER-specific rabbit monoclonal antibodies were generated, including 9F6. Although it lacked neutralizing activity, the target epitope profile of 9F6 closely resembled those of 4E10 and 10E8 ((671)NWFDITNWLWYIK(683)). B-cell repertoire analyses suggested the importance of co-immunizations for maturation of 9F6, which warrants further evaluation of our IPAS-RAM vaccine strategy using an improved priming immunogen.

  1. A viral regulator of glycoprotein complexes contributes to human cytomegalovirus cell tropism.

    PubMed

    Li, Gang; Nguyen, Christopher C; Ryckman, Brent J; Britt, William J; Kamil, Jeremy P

    2015-04-07

    Viral glycoproteins mediate entry of enveloped viruses into cells and thus play crucial roles in infection. In herpesviruses, a complex of two viral glycoproteins, gH and gL (gH/gL), regulates membrane fusion events and influences virion cell tropism. Human cytomegalovirus (HCMV) gH/gL can be incorporated into two different protein complexes: a glycoprotein O (gO)-containing complex known as gH/gL/gO, and a complex containing UL128, UL130, and UL131 known as gH/gL/UL128-131. Variability in the relative abundance of the complexes in the virion envelope correlates with differences in cell tropism exhibited between strains of HCMV. Nonetheless, the mechanisms underlying such variability have remained unclear. We have identified a viral protein encoded by the UL148 ORF (UL148) that influences the ratio of gH/gL/gO to gH/gL/UL128-131 and the cell tropism of HCMV virions. A mutant disrupted for UL148 showed defects in gH/gL/gO maturation and enhanced infectivity for epithelial cells. Accordingly, reintroduction of UL148 into an HCMV strain that lacked the gene resulted in decreased levels of gH/gL/UL128-131 on virions and, correspondingly, decreased infectivity for epithelial cells. UL148 localized to the endoplasmic reticulum, but not to the cytoplasmic sites of virion envelopment. Coimmunoprecipitation results indicated that gH, gL, UL130, and UL131 associate with UL148, but that gO and UL128 do not. Taken together, the findings suggest that UL148 modulates HCMV tropism by regulating the composition of alternative gH/gL complexes.

  2. Common tolerance mechanisms, but distinct cross-reactivities associated with gp41 and lipids, limit production of HIV-1 broad neutralizing antibodies 2F5 and 4E10.

    PubMed

    Chen, Yao; Zhang, Jinsong; Hwang, Kwan-Ki; Bouton-Verville, Hilary; Xia, Shi-Mao; Newman, Amanda; Ouyang, Ying-Bin; Haynes, Barton F; Verkoczy, Laurent

    2013-08-01

    Developing an HIV-1 vaccine has been hampered by the inability of immunogens to induce broadly neutralizing Abs (BnAbs) that protect against infection. Previously, we used knockin (KI) mice expressing a prototypical gp41-specific BnAb, 2F5, to demonstrate that immunological tolerance triggered by self-reactivity of the 2F5 H chain impedes BnAb induction. In this study, we generate KI models expressing H chains from two other HIV-1 Abs, 4E10 (another self-/polyreactive, anti-gp41 BnAb) and 48d (an anti-CD4 inducible, nonpolyreactive Ab), and find a similar developmental blockade consistent with central B cell deletion in 4E10, but not in 48d VH KI mice. Furthermore, in KI strains expressing the complete 2F5 and 4E10 Abs as BCRs, we find that residual splenic B cells arrest at distinct developmental stages, yet exhibit uniformly low BCR densities, elevated basal activation, and profoundly muted responses to BCR ligation and, when captured as hybridoma mAb lines, maintain their dual (gp41/lipid) affinities and capacities to neutralize HIV-1, establishing a key role for anergy in suppressing residual 2F5- or 4E10-expressing B cells. Importantly, serum IgGs from naive 2F5 and 4E10 KI strains selectively eliminate gp41 and lipid binding, respectively, suggesting B cells expressing 2F5 or 4E10 as BCRs exhibit specificity for a distinct spectrum of host Ags, including selective interactions by 2F5 BCR(+) B cells (i.e., and not 4E10 BCR(+) B cells) with those mimicked by its gp41 neutralization epitope.

  3. HIV Entry and Envelope Glycoprotein-mediated Fusion*

    PubMed Central

    Blumenthal, Robert; Durell, Stewart; Viard, Mathias

    2012-01-01

    HIV entry involves binding of the trimeric viral envelope glycoprotein (Env) gp120/gp41 to cell surface receptors, which triggers conformational changes in Env that drive the membrane fusion reaction. The conformational landscape that the lipids and Env navigate en route to fusion has been examined by biophysical measurements on the microscale, whereas electron tomography, x-rays, and NMR have provided insights into the process on the nanoscale and atomic scale. However, the coupling between the lipid and protein pathways that give rise to fusion has not been resolved. Here, we discuss the known and unknown about the overall HIV Env-mediated fusion process. PMID:23043104

  4. Rational improvement of gp41-targeting HIV-1 fusion inhibitors: an innovatively designed Ile-Asp-Leu tail with alternative conformations

    NASA Astrophysics Data System (ADS)

    Zhu, Yun; Su, Shan; Qin, Lili; Wang, Qian; Shi, Lei; Ma, Zhenxuan; Tang, Jianchao; Jiang, Shibo; Lu, Lu; Ye, Sheng; Zhang, Rongguang

    2016-09-01

    Peptides derived from the C-terminal heptad repeat (CHR) of HIV gp41 have been developed as effective fusion inhibitors against HIV-1, but facing the challenges of enhancing potency and stability. Here, we report a rationally designed novel HIV-1 fusion inhibitor derived from CHR-derived peptide (Trp628~Gln653, named CP), but with an innovative Ile-Asp-Leu tail (IDL) that dramatically increased the inhibitory activity by up to 100 folds. We also determined the crystal structures of artificial fusion peptides N36- and N43-L6-CP-IDL. Although the overall structures of both fusion peptides share the canonical six-helix bundle (6-HB) configuration, their IDL tails adopt two different conformations: a one-turn helix with the N36, and a hook-like structure with the longer N43. Structural comparison showed that the hook-like IDL tail possesses a larger interaction interface with NHR than the helical one. Further molecular dynamics simulations of the two 6-HBs and isolated CP-IDL peptides suggested that hook-like form of IDL tail can be stabilized by its binding to NHR trimer. Therefore, CP-IDL has potential for further development as a new HIV fusion inhibitor, and this strategy could be widely used in developing artificial fusion inhibitors against HIV and other enveloped viruses.

  5. Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative

    DOE PAGES

    Heller, William T.; Rai, Durgesh K.

    2017-01-16

    HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. In this paper, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observedmore » to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Finally, of the two different bilayer structures, the one corresponding to the smaller area fraction, being ~8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion.« less

  6. Unique functional properties of conserved arginine residues in the lentivirus lytic peptide domains of the C-terminal tail of HIV-1 gp41.

    PubMed

    Kuhlmann, Anne-Sophie; Steckbeck, Jonathan D; Sturgeon, Timothy J; Craigo, Jodi K; Montelaro, Ronald C

    2014-03-14

    A previous study from our laboratory reported a preferential conservation of arginine relative to lysine in the C-terminal tail (CTT) of HIV-1 envelope (Env). Despite substantial overall sequence variation in the CTT, specific arginines are highly conserved in the lentivirus lytic peptide (LLP) motifs and are scarcely substituted by lysines, in contrast to gp120 and the ectodomain of gp41. However, to date, no explanation has been provided to explain the selective incorporation and conservation of arginines over lysines in these motifs. Herein, we address the functions in virus replication of the most conserved arginines by performing conservative mutations of arginine to lysine in the LLP1 and LLP2 motifs. The presence of lysine in place of arginine in the LLP1 motif resulted in significant impairment of Env expression and consequently virus replication kinetics, Env fusogenicity, and incorporation. By contrast, lysine exchanges in LLP2 only affected the level of Env incorporation and fusogenicity. Our findings demonstrate that the conservative lysine substitutions significantly affect Env functional properties indicating a unique functional role for the highly conserved arginines in the LLP motifs. These results provide for the first time a functional explanation to the preferred incorporation of arginine, relative to lysine, in the CTT of HIV-1 Env. We propose that these arginines may provide unique functions for Env interaction with viral or cellular cofactors that then influence overall Env functional properties.

  7. N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies.

    PubMed

    Li, Dezhi; Liu, Jie; Zhang, Li; Xu, Tianshu; Chen, Junheng; Wang, Liping; Zhao, Qi

    2015-12-01

    The Human immunodeficiency virus type 1 (HIV-1) gp41 membrane proximal external region (MPER) is targeted by broadly neutralizing antibodies (e.g. 2F5, 4E10, Z13e and m66.6), which makes this region a promising target for vaccine design. One strategy to elicit neutralizing antibodies against the MPER epitope is to design peptide immunogens mimicking neutralization structures. To probe 2F5-like neutralizing antibodies, two yeast-displayed antibody libraries from peripheral blood mononuclear cells from a HIV-1 patient were screened against the 2F5 epitope peptide SP62. Two 2F5-like antibodies were identified that specifically recognized SP62. However, these antibodies only weakly neutralized HIV-1 primary isolates. The epitopes recognized by these two 2F5-like antibodies include not only the 2F5 epitope (amino acids (aa) 662-667 in the MPER) but also several other residues (aa 652-655) locating at the N-terminus in SP62. Experimental results suggest that residues of SP62 adjacent to the 2F5 epitope influence the response of broadly neutralizing 2F5-like antibodies in vaccination. Our findings may aid the design of vaccine immunogens and development of therapeutics against HIV-1 infection.

  8. Identification of a new epitope for HIV-neutralizing antibodies in the gp41 membrane proximal external region by an Env-tailored phage display library.

    PubMed

    Zhou, Mingkui; Meyer, Torsten; Koch, Stefanie; Koch, Joachim; von Briesen, Hagen; Benito, José M; Soriano, Vincent; Haberl, Annette; Bickel, Markus; Dübel, Stefan; Hust, Michael; Dietrich, Ursula

    2013-02-01

    HIV controllers are a valuable source for the identification of HIV-neutralizing antibodies, as chronic infection over decades allows extensive affinity maturation of antibodies for improved Ag recognition. We analyzed a small cohort of elite controllers (ECs) for HIV-neutralizing antibodies using a panel of standardized HIV-1 pseudovirions on TZM-bl cells. An HIV-1 Env-tailored phage display library was generated to select epitopes targeted by neutralizing antibodies in the EC26 plasma sample showing the broadest neutralizing activity. Selected Env fragments were mostly allocated to the membrane proximal external region of gp41. After preabsorbing the EC26 plasma with the selected phage EC26-2A4, we achieved 50% depletion of its neutralizing activity. Furthermore, antibodies affinity-purified with the EC26-2A4 epitope from EC26 plasma showed neutralizing activity, proving that the selected phage indeed contains an epitope targeted by neutralizing plasma antibodies. Epitope fine mapping of the purified plasma antibodies on peptide arrays identified a new epitope overlapping, but clearly distinct, from the prominent 2F5 epitope. Of note, the purified antibodies did not show autoreactivity with cardiolipin, whereas low reactivity with phosphatidylserine comparable to mAb 2F5 was observed. Thus, this new epitope represents a promising candidate for further analysis in view of HIV vaccine development. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. NK cytotoxicity against CD4+ T cells during HIV-1 infection: A gp41 peptide induces the expression of an NKp44 ligand

    PubMed Central

    Vieillard, Vincent; Strominger, Jack L.; Debré, Patrice

    2005-01-01

    HIV infection leads to a state of chronic immune activation and progressive deterioration in immune function, manifested most recognizably by the progressive depletion of CD4+ T cells. A substantial percentage of natural killer (NK) cells from patients with HIV infection are activated and express the natural cytotoxicity receptor (NCR) NKp44. Here we show that a cellular ligand for NKp44 (NKp44L) is expressed during HIV-1 infection and is correlated with both the progression of CD4+ T cell depletion and the increase of viral load. CD4+ T cells expressing this ligand are highly sensitive to the NK lysis activity mediated by NKp44+ NK cells. The expression of NKp44L is induced by the linear motif NH2-SWSNKS-COOH of the HIV-1 envelope gp41 protein. This highly conserved motif appears critical to the sharp increase in NK lysis of CD4+ T cells from HIV-infected patients. These studies strongly suggest that induction of NKp44L plays a key role in the lysis of CD4+ T cells by activated NK cells in HIV infection and consequently provide a framework for considering how HIV-1 may use NK cell immune surveillance to trigger CD4+ T cells. Understanding this mechanism may help to develop future therapeutic strategies and vaccines against HIV-1 infection. PMID:16046540

  10. Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative.

    PubMed

    Heller, William T; Rai, Durgesh K

    2017-03-01

    HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. Here, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observed to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Of the two different bilayer structures, the one corresponding to the smaller area fraction, being ∼8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Rational improvement of gp41-targeting HIV-1 fusion inhibitors: an innovatively designed Ile-Asp-Leu tail with alternative conformations

    PubMed Central

    Zhu, Yun; Su, Shan; Qin, Lili; Wang, Qian; Shi, Lei; Ma, Zhenxuan; Tang, Jianchao; Jiang, Shibo; Lu, Lu; Ye, Sheng; Zhang, Rongguang

    2016-01-01

    Peptides derived from the C-terminal heptad repeat (CHR) of HIV gp41 have been developed as effective fusion inhibitors against HIV-1, but facing the challenges of enhancing potency and stability. Here, we report a rationally designed novel HIV-1 fusion inhibitor derived from CHR-derived peptide (Trp628~Gln653, named CP), but with an innovative Ile-Asp-Leu tail (IDL) that dramatically increased the inhibitory activity by up to 100 folds. We also determined the crystal structures of artificial fusion peptides N36- and N43-L6-CP-IDL. Although the overall structures of both fusion peptides share the canonical six-helix bundle (6-HB) configuration, their IDL tails adopt two different conformations: a one-turn helix with the N36, and a hook-like structure with the longer N43. Structural comparison showed that the hook-like IDL tail possesses a larger interaction interface with NHR than the helical one. Further molecular dynamics simulations of the two 6-HBs and isolated CP-IDL peptides suggested that hook-like form of IDL tail can be stabilized by its binding to NHR trimer. Therefore, CP-IDL has potential for further development as a new HIV fusion inhibitor, and this strategy could be widely used in developing artificial fusion inhibitors against HIV and other enveloped viruses. PMID:27666394

  12. Structure and immunogenicity of a peptide vaccine, including the complete HIV-1 gp41 2F5 epitope: implications for antibody recognition mechanism and immunogen design.

    PubMed

    Serrano, Soraya; Araujo, Aitziber; Apellániz, Beatriz; Bryson, Steve; Carravilla, Pablo; de la Arada, Igor; Huarte, Nerea; Rujas, Edurne; Pai, Emil F; Arrondo, José L R; Domene, Carmen; Jiménez, María Angeles; Nieva, José L

    2014-03-07

    The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund's adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development.

  13. Antibody elicited against the gp41 N-heptad repeat (NHR) coiled-coil can neutralize HIV-1 with modest potency but non-neutralizing antibodies also bind to NHR mimetics.

    PubMed

    Nelson, Josh D; Kinkead, Heather; Brunel, Florence M; Leaman, Dan; Jensen, Richard; Louis, John M; Maruyama, Toshiaki; Bewley, Carole A; Bowdish, Katherine; Clore, G Marius; Dawson, Philip E; Frederickson, Shana; Mage, Rose G; Richman, Douglas D; Burton, Dennis R; Zwick, Michael B

    2008-07-20

    Following CD4 receptor binding to the HIV-1 envelope spike (Env), the conserved N-heptad repeat (NHR) region of gp41 forms a coiled-coil that is a precursor to the fusion reaction. Although it has been a target of drug and vaccine design, there are few monoclonal antibody (mAb) tools with which to probe the antigenicity and immunogenicity specifically of the NHR coiled-coil. Here, we have rescued HIV-1-neutralizing anti-NHR mAbs from immune phage display libraries that were prepared (i) from b9 rabbits immunized with a previously described mimetic of the NHR coiled-coil, N35(CCG)-N13, and (ii) from an HIV-1 infected individual. We describe a rabbit single-chain Fv fragment (scFv), 8K8, and a human Fab, DN9, which specifically recognize NHR coiled-coils that are unoccupied by peptide corresponding to the C-heptad repeat or CHR region of gp41 (e.g. C34). The epitopes of 8K8 and DN9 were found to partially overlap with that of a previously described anti-NHR mAb, IgG D5; however, 8K8 and DN9 were much more specific than D5 for unoccupied NHR trimers. The mAbs, including a whole IgG 8K8 molecule, neutralized primary HIV-1 of clades B and C in a pseudotyped virus assay with comparable, albeit relatively modest potency. Finally, a human Fab T3 and a rabbit serum (both non-neutralizing) were able to block binding of D5 and 8K8 to a gp41 NHR mimetic, respectively, but not the neutralizing activity of these mAbs. We conclude from these results that NHR coiled-coil analogs of HIV-1 gp41 elicit many Abs during natural infection and through immunization, but that due to limited accessibility to the corresponding region on fusogenic gp41 few can neutralize. Caution is therefore required in targeting the NHR for vaccine design. Nevertheless, the mAb panel may be useful as tools for elucidating access restrictions to the NHR of gp41 and in designing potential improvements to mimetics of receptor-activated Env.

  14. Antibody elicited against the gp41 N-heptad repeat (NHR) coiled-coil can neutralize HIV-1 with modest potency but non-neutralizing antibodies also bind to NHR mimetics

    SciTech Connect

    Nelson, Josh D.; Kinkead, Heather; Brunel, Florence M.; Leaman, Dan; Jensen, Richard; Louis, John M.; Maruyama, Toshiaki; Bewley, Carole A.; Bowdish, Katherine; Clore, G. Marius; Dawson, Philip E.; Frederickson, Shana; Mage, Rose G.; Richman, Douglas D.; Burton, Dennis R.; Zwick, Michael B.

    2008-07-20

    Following CD4 receptor binding to the HIV-1 envelope spike (Env), the conserved N-heptad repeat (NHR) region of gp41 forms a coiled-coil that is a precursor to the fusion reaction. Although it has been a target of drug and vaccine design, there are few monoclonal antibody (mAb) tools with which to probe the antigenicity and immunogenicity specifically of the NHR coiled-coil. Here, we have rescued HIV-1-neutralizing anti-NHR mAbs from immune phage display libraries that were prepared (i) from b9 rabbits immunized with a previously described mimetic of the NHR coiled-coil, N35{sub CCG}-N13, and (ii) from an HIV-1 infected individual. We describe a rabbit single-chain Fv fragment (scFv), 8K8, and a human Fab, DN9, which specifically recognize NHR coiled-coils that are unoccupied by peptide corresponding to the C-heptad repeat or CHR region of gp41 (e.g. C34). The epitopes of 8K8 and DN9 were found to partially overlap with that of a previously described anti-NHR mAb, IgG D5; however, 8K8 and DN9 were much more specific than D5 for unoccupied NHR trimers. The mAbs, including a whole IgG 8K8 molecule, neutralized primary HIV-1 of clades B and C in a pseudotyped virus assay with comparable, albeit relatively modest potency. Finally, a human Fab T3 and a rabbit serum (both non-neutralizing) were able to block binding of D5 and 8K8 to a gp41 NHR mimetic, respectively, but not the neutralizing activity of these mAbs. We conclude from these results that NHR coiled-coil analogs of HIV-1 gp41 elicit many Abs during natural infection and through immunization, but that due to limited accessibility to the corresponding region on fusogenic gp41 few can neutralize. Caution is therefore required in targeting the NHR for vaccine design. Nevertheless, the mAb panel may be useful as tools for elucidating access restrictions to the NHR of gp41 and in designing potential improvements to mimetics of receptor-activated Env.

  15. P-Glycoprotein in skin contributes to transdermal absorption of topical corticosteroids.

    PubMed

    Hashimoto, Naoto; Nakamichi, Noritaka; Yamazaki, Erina; Oikawa, Masashi; Masuo, Yusuke; Schinkel, Alfred H; Kato, Yukio

    2017-04-15

    ATP binding cassette transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), are expressed in skin, but their involvement in transdermal absorption of clinically used drugs remains unknown. Here, we examined their role in transdermal absorption of corticosteroids. Skin and plasma concentrations of dexamethasone after dermal application were reduced in P-gp and BCRP triple-knockout (Mdr1a/1b/Bcrp(-/-)) mice. The skin concentration in Mdr1a/1b/Bcrp(-/-) mice was reduced in the dermis, but not in the epidermis, indicating that functional expression of these transporters in skin is compartmentalized. Involvement of these transporters in dermal transport of dexamethasone was also supported by the observation of a higher epidermal concentration in Mdr1a/1b/Bcrp(-/-) than wild-type mice during intravenous infusion. Transdermal absorption after dermal application of prednisolone, but not methylprednisolone or ethinyl estradiol, was also lower in Mdr1a/1b/Bcrp(-/-) than in wild-type mice. Transport studies in epithelial cell lines transfected with P-gp or BCRP showed that dexamethasone and prednisolone are substrates of P-gp, but are minimally transported by BCRP. Thus, our findings suggest that P-gp is involved in transdermal absorption of at least some corticosteroids in vivo. P-gp might be available as a target for inhibition in order to deliver topically applied drugs and cosmetics in a manner that minimizes systemic exposure.

  16. Aedes aegypti Rhesus glycoproteins contribute to ammonia excretion by larval anal papillae.

    PubMed

    Durant, Andrea C; Chasiotis, Helen; Misyura, Lidiya; Donini, Andrew

    2017-02-15

    In larval Aedes aegypti, transcripts of the Rhesus-like glycoproteins AeRh50-1 and AeRh50-2 have been detected in the anal papillae, sites of ammonia (NH3/NH4(+)) excretion; however, these putative ammonia transporters have not been previously localized or functionally characterized. In this study, we show that the AeRh50s co-immunolocalize with apical V-type H(+)-ATPase as well as with basal Na(+)/K(+)-ATPase in the epithelium of anal papillae. The double-stranded RNA-mediated knockdown of AeRh50-1 and AeRh50-2 resulted in a significant reduction in AeRh50 protein abundance in the anal papillae, and this was coupled to decreased ammonia excretion. The knockdown of AeRh50-1 resulted in decreased hemolymph [NH4(+)] and pH whereas knockdown of AeRh50-2 had no effect on these parameters. We conclude that the AeRh50s are important contributors to ammonia excretion at the anal papillae of larval A. aegypti, which may be the basis for their ability to inhabit areas with high ammonia levels. © 2017. Published by The Company of Biologists Ltd.

  17. Coreceptor Tropism Can Be Influenced by Amino Acid Substitutions in the gp41 Transmembrane Subunit of Human Immunodeficiency Virus Type 1 Envelope Protein▿

    PubMed Central

    Huang, Wei; Toma, Jonathan; Fransen, Signe; Stawiski, Eric; Reeves, Jacqueline D.; Whitcomb, Jeannette M.; Parkin, Neil; Petropoulos, Christos J.

    2008-01-01

    Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants. The introduction of distinct V3 sequences from CXCR4-using clones into an R5-tropic env backbone conferred the inefficient use of CXCR4 in some but not all cases. Conversely, in many cases, X4- and dual-tropic env backbones containing the V3 sequences of R5-tropic clones retained the ability to use CXCR4, suggesting that sequences outside of the V3 regions of these CXCR4-using clones were responsible for CXCR4 use. The determinants of CXCR4 use in a set of dual-tropic env sequences with V3 sequences identical to those of R5-tropic clones mapped to the gp41 transmembrane (TM) subunit. In one case, a single-amino-acid substitution in the fusion peptide of TM was able to confer CXCR4 use; however, TM substitutions associated with CXCR4 use varied among different env sequences. These results demonstrate that sequences in TM can modulate coreceptor specificity and that env sequences other than that of V3 may facilitate efficient CXCR4-mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use. PMID:18353956

  18. A fully human antibody to gp41 selectively eliminates HIV-infected cells that transmigrated across a model human blood brain barrier

    PubMed Central

    McFARREN, Alicia; LOPEZ, Lillie; WILLIAMS, Dionna W.; VEENSTRA, Mike; BRYAN, Ruth A.; GOLDSMITH, Aliza; MORGENSTERN, Alfred; BRUCHERTSEIFER, Frank; ZOLLA-PAZNER, Susan; GORNY, Miroslaw K.; EUGENIN, Eliseo A.; BERMAN, Joan W.; DADACHOVA, Ekaterina

    2015-01-01

    Objective Many HIV patients on cART exhibit HIV-associated neurocognitive disorders because the brain becomes a viral reservoir. There is a need for therapeutics that can enter the CNS and eradicate the virus. Design Radiolabeled human mAb 2556 to HIV gp41 selectively kills HIV-infected cells in vivo and in vitro. Here we tested the ability of 213Bi-2556 to cross a tissue culture model of the human BBB and kill HIV-infected PBMCs and monocytes on the CNS side of the barrier. Methods 2556 mAb isoelectric point (pI) was determined with IEF. The ability of radiolabeled 2556 to penetrate through the barrier was studied by adding it to the upper chamber of the barriers and its penetration into the CNS side was followed for 5 hrs. To assess the ability of 213Bi-2556 to kill the HIV-infected cells on the CNS side of barrier, the HIV-infected and uninfected PBMCs and monocytes were allowed to transmigrate across the barriers overnight followed by application of 213Bi-2556 or control mAb 213Bi-1418 to the top of the barrier. Killing of cells was measured by TUNEL and Trypan blue assays. The barriers were examined by confocal microscopy for overt damage. Results The pI of 213Bi-2556 was 9.6 enabling its penetration through the barrier by transcytosis. 213Bi-2556 killed significantly more transmigrated HIV-infected cells in comparison to 213Bi-1418 and uninfected cells. No overt damage to barriers was observed. Conclusions We demonstrated that 213Bi-2556 mAb crossed an in vitro human BBB and specifically killed transmigrated HIV-infected PBMCs and monocytes without overt damage to the barrier. PMID:26595540

  19. Structural Basis of HIV-1 Neutralization by Affinity Matured Fabs Directed against the Internal Trimeric Coiled-Coil of gp41

    SciTech Connect

    Gustchina, Elena; Li, Mi; Louis, John M.; Anderson, D.Eric; Lloyd, John; Frisch, Christian; Bewley, Carole A.; Gustchina, Alla; Wlodawer, Alexander; Clore, G.Marius

    2010-12-03

    The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naive human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.

  20. HIV-1 variants with a single-point mutation in the gp41 pocket region exhibiting different susceptibility to HIV fusion inhibitors with pocket- or membrane-binding domain.

    PubMed

    Lu, Lu; Tong, Pei; Yu, Xiaowen; Pan, Chungen; Zou, Peng; Chen, Ying-Hua; Jiang, Shibo

    2012-12-01

    Enfuvirtide (T20), the first FDA-approved peptide HIV fusion/entry inhibitor derived from the HIV-1 gp41 C-terminal heptad-repeat (CHR) domain, is believed to share a target with C34, another well-characterized CHR-peptide, by interacting with the gp41 N-terminal heptad-repeat (NHR) to form six-helix bundle core. However, our previous studies showed that T20 mainly interacts with the N-terminal region of the NHR (N-NHR) and lipid membranes, while C34 mainly binds to the NHR C-terminal pocket region. But so far, no one has shown that C34 can induce drug-resistance mutation in the gp41 pocket region. In this study, we constructed pseudoviruses in which the Ala at the position of 67 in the gp41 pocket region was substituted with Asp, Gly or Ser, respectively, and found that these mutations rendered the viruses highly resistant to C34, but sensitive to T20. The NHR-peptide N36 with mutations of A67 exhibited reduced anti-HIV-1 activity and decreased α-helicity. The stability of six-helix bundle formed by C34 and N36 with A67 mutations was significantly lower than that formed by C34 and N36 with wild-type sequence. The combination of C34 and T20 resulted in potent synergistic anti-HIV-1 effect against the viruses with mutations in either N- or C-terminal region in NHR. These results suggest that C34 with a pocket-binding domain and T20 containing the N-NHR- and membrane-binding domains inhibit HIV-1 fusion by interacting with different target sites and the combinatorial use of C34 and T20 is expected to be effective against HIV-1 variants resistant to HIV fusion inhibitors.

  1. Truncation of the membrane-spanning domain of human immunodeficiency virus type 1 envelope glycoprotein defines elements required for fusion, incorporation, and infectivity.

    PubMed

    Yue, Ling; Shang, Liang; Hunter, Eric

    2009-11-01

    The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a "snorkeling" model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.

  2. Opioid analgesics and P-glycoprotein efflux transporters: a potential systems-level contribution to analgesic tolerance.

    PubMed

    Mercer, Susan L; Coop, Andrew

    2011-01-01

    Chronic clinical pain remains poorly treated. Despite attempts to develop novel analgesic agents, opioids remain the standard analgesics of choice in the clinical management of chronic and severe pain. However, mu opioid analgesics have undesired side effects including, but not limited to, respiratory depression, physical dependence and tolerance. A growing body of evidence suggests that P-glycoprotein (P-gp), an efflux transporter, may contribute a systems-level approach to the development of opioid tolerance. Herein, we describe current in vitro and in vivo methodology available to analyze interactions between opioids and P-gp and critically analyze P-gp data associated with six commonly used mu opioids to include morphine, methadone, loperamide, meperidine, oxycodone, and fentanyl. Recent studies focused on the development of opioids lacking P-gp substrate activity are explored, concentrating on structure-activity relationships to develop an optimal opioid analgesic lacking this systems-level contribution to tolerance development. Continued work in this area will potentially allow for delineation of the mechanism responsible for opioid-related P-gp up-regulation and provide further support for evidence based medicine supporting clinical opioid rotation.

  3. Opioid Analgesics and P-glycoprotein Efflux Transporters: A Potential Systems-Level Contribution to Analgesic Tolerance

    PubMed Central

    Mercer, Susan L.; Coop, Andrew

    2012-01-01

    Chronic clinical pain remains poorly treated. Despite attempts to develop novel analgesic agents, opioids remain the standard analgesics of choice in the clinical management of chronic and severe pain. However, mu opioid analgesics have undesired side effects including, but not limited to, respiratory depression, physical dependence and tolerance. A growing body of evidence suggests that P-glycoprotein (P-gp), an efflux transporter, may contribute a systems-level approach to the development of opioid tolerance. Herein, we describe current in vitro and in vivo methodology available to analyze interactions between opioids and P-gp and critically analyze P-gp data associated with six commonly used mu opioids to include morphine, methadone, loperamide, meperidine, oxycodone, and fentanyl. Recent studies focused on the development of opioids lacking P-gp substrate activity are explored, concentrating on structure-activity relationship development to develop an optimal opioid analgesic lacking this systems-level contribution to tolerance development. Continued work in this area will potentially allow for delineation of the mechanism responsible for opioid-related P-gp up-regulation and provide further support for evidence based medicine supporting clinical opioid rotation. PMID:21050174

  4. Generation of Long-Lived Bone Marrow Plasma Cells Secreting Antibodies Specific for the HIV-1 gp41 Membrane-Proximal External Region in the Absence of Polyreactivity

    PubMed Central

    Donius, Luke R.; Cheng, Yuxing; Choi, Jaewon; Sun, Zhen-Yu J.; Hanson, Melissa; Zhang, Michael; Gierahn, Todd M.; Marquez, Susanna; Uduman, Mohammed; Kleinstein, Steven H.; Irvine, Darrell; Love, J. Christopher; Reinherz, Ellis L.

    2016-01-01

    ABSTRACT An effective preventive vaccine is highly sought after in order to stem the current HIV-1 pandemic. Both conservation of contiguous gp41 membrane-proximal external region (MPER) amino acid sequences across HIV-1 clades and the ability of anti-MPER broadly neutralizing antibodies (BNAbs) to block viral hemifusion/fusion establish the MPER as a prime vaccination target. In earlier studies, we described the development of an MPER vaccine formulation that takes advantage of liposomes to array the MPER on a lipid bilayer surface, paralleling its native configuration on the virus membrane while also incorporating molecular adjuvant and CD4 T cell epitope cargo. Here we demonstrate that several immunizations with MPER/liposomes induce high levels of bone marrow long-lived plasma cell (LLPC) antibody production. Single-cell immunoglobulin gene retrieval analysis shows that these plasma cells are derived from a germ line repertoire of B cells with a diverse representation of immunoglobulin genes, exhibiting antigen-driven positive selection. Characterization of LLPC recombinant monoclonal antibodies (rMAbs) indicates that antigen recognition is achieved through convergence on a common epitopic focus by utilizing various complementarity-determining region H3 (CDRH3) lengths. Importantly, the vast majority of rMAbs produced from these cells lack polyreactivity yet manifest antigen specificity in the context of lipids, shaping MPER-specific paratopes through selective pressure. Taken together, these findings demonstrate that the MPER is a vaccine target with minimal risk of generating off-target autoimmunity. IMPORTANCE A useful vaccine must generate desired long-term, antigen-specific antibody responses devoid of polyreactivity or autoreactivity. The common polyreactive features of some HIV-1 BNAbs have raised concern about elicitation of anti-MPER antibodies. Utilizing single-LLPC repertoire analysis and biophysical characterization of anti-MPER rMAbs, we show that

  5. Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus

    PubMed Central

    Haggarty, Beth S.; Duong, Jennifer; Jordon, Andrea P. O.; Romano, Josephine; DeClercq, Joshua J.; Gregory, Philip D.; Riley, James L.; Holmes, Michael C.

    2016-01-01

    HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans. PMID:27855210

  6. Degradation of P-glycoprotein by pristimerin contributes to overcoming ABCB1-mediated chemotherapeutic drug resistance in vitro

    PubMed Central

    Yan, Yan-Yan; Wang, Fang; Zhao, Xiao-Qin; Wang, Xiao-Kun; Chen, Yi-Fan; Liu, Hong; Xie, Yong; Fu, Li-Wu

    2016-01-01

    ABCB1 (P-glycoprotein, ABCB1/MDR1) is one of the major members of the ABC transporters linked to MDR in cancer cells. In this study, we observed that pristimerin, a natural triterpenoid, potently decreased P-gp in a dose-dependent manner in both drug-resistant KBv200 and stable transfected HEK293/ABCB1 cell lines. Moreover, pristimerin also inhibited cell proliferation and induced apoptosis in both cell lines. Intriguingly, reverse transcription-PCR, real-time PCR and protein turn-over assay revealed that the decrease of P-gp was independent of mRNA level but primarily owing to its protein stability. Furthermore, immunofluorescence study with anti-P-gp antibody showed that pristimerin disturbed the subcellular distribution of P-gp with decreased location in the plasma membrane. Taken together, these data suggest that subcellular distribution of P-gp and subsequent downregulation by pristimerin contribute to overcoming ABCB1-mediated chemotherapeutic drug resistance. Our findings suggested inducing the decrease of P-gp membrane protein could be a new promising alternative therapeutic strategy in ABCB1-mediated MDR. PMID:27840996

  7. Mitochondrial P-glycoprotein ATPase contributes to insecticide resistance in the cotton bollworm, Helicoverpa armigera (Noctuidae: Lepidoptera).

    PubMed

    Akbar, S Md; Aurade, Ravindra M; Sharma, H C; Sreeramulu, K

    2014-09-01

    Cotton bollworm, Helicoverpa armigera, is one of the most damaging polyphagous pests worldwide, which has developed high levels of resistance to commonly applied insecticides. Mitochondrial P-glycoprotein (Pgp) was detected in the insecticide-resistant strain of H. armigera using C219 antibodies, and its possible role was demonstrated in the efflux of xenobiotic compounds using spectrofluorometer. The TMR accumulated in mitochondria in the absence of ATP, and effluxed out in presence of ATP; the process of efflux was inhibited in the presence of ortho-vandate, an inhibitor of Pgp, in insecticide-resistant larvae of H. armigera. The mitochondria isolated from insecticide-resistant larvae were resistant to insecticide-induced inhibition of oxygen consumption and cytochrome c release. Membrane potential decreased in a dose-dependent manner in the presence of higher concentration of insecticides (>50 µM) in mitochondria of insecticide-resistant larvae. In conclusion, mitochondrial Pgp ATPase detected in the insecticide-resistant larvae influenced the efflux of xenobiotic compounds. Pgp might be involved in protecting the mitochondrial DNA and the components of the electron transport chain from damage due to insecticides, and contributing to the resistance to the deleterious effects of insecticides on the growth of insecticide-resistant H. armigera larvae.

  8. A broad HIV-1 inhibitor blocks envelope glycoprotein transitions critical for entry.

    PubMed

    Herschhorn, Alon; Gu, Christopher; Espy, Nicole; Richard, Jonathan; Finzi, Andrés; Sodroski, Joseph G

    2014-10-01

    Binding to the primary receptor, CD4, triggers conformational changes in the metastable HIV-1 envelope glycoprotein (Env) trimer ((gp120-gp41)3) that are important for virus entry into host cells. These changes include an 'opening' of the trimer, creation of a binding site for the CCR5 co-receptor and formation and/or exposure of a gp41 coiled coil. Here we identify a new compound, 18A (1), that specifically inhibits the entry of a wide range of HIV-1 isolates. 18A does not interfere with CD4 or CCR5 binding, but it inhibits the CD4-induced disruption of quaternary structures at the trimer apex and the exposure of the gp41 HR1 coiled coil. Analysis of HIV-1 variants with increased or reduced sensitivity to 18A suggests that the inhibitor can distinguish distinct conformational states of gp120 in the unliganded Env trimer. The broad-range activity and observed hypersensitivity of resistant mutants to antibody neutralization support further investigation of 18A.

  9. T21/DP107, A synthetic leucine zipper-like domain of the HIV-1 envelope gp41, attracts and activates human phagocytes by using G-protein-coupled formyl peptide receptors.

    PubMed

    Su, S B; Gao, J l; Gong, W h; Dunlop, N M; Murphy, P M; Oppenheim, J J; Wang, J M

    1999-05-15

    A leucine zipper-like domain, T21/DP107, located in the amino terminus of the ectodomain of gp41, is crucial to the formation of fusogenic configuration of the HIV-1 envelope protein gp41. We report that the synthetic T21/DP107 segment is a potent stimulant of migration and calcium mobilization in human monocytes and neutrophils. The activity of T21/DP107 on phagocytes was pertussis toxin-sensitive, suggesting this peptide uses Gi-coupled seven-transmembrane receptor(s). Since the bacterial chemotactic peptide fMLP partially desensitized the calcium-mobilizing activity of T21/DP107 in phagocytes, we postulated that T21/DP107 might preferentially use a lower affinity fMLP receptor. By using cells transfected to express cloned prototype chemotactic N-formyl peptide receptor (FPR) or its variant, FPR-like 1 (FPRL1), we demonstrate that T21/DP107 activates both receptors but has a much higher efficacy for FPRL1. In addition, T21/DP107 at nM concentrations induced migration of FPRL1-transfected human embryonic kidney 293 cells. In contrast, fMLP did not induce significant chemotaxis of the same cells at a concentration as high as 50 microM. Although a lipid metabolite, lipoxin A4, was a high-affinity ligand for FPRL1, it was not reported to induce Ca2+ mobilization or chemotaxis in FPRL1-transfected cells. Therefore, T21/DP107 is a first chemotactic peptide agonist identified thus far for FPRL1. Our results suggest that this peptide domain of the HIV-1 gp41 may have the potential to activate host innate immune response by interacting with FPR and FPRL1 on phagocytes.

  10. Evaluating the Immunogenicity of a Disulfide-Stabilized, Cleaved, Trimeric Form of the Envelope Glycoprotein Complex of Human Immunodeficiency Virus Type 1

    PubMed Central

    Beddows, Simon; Schülke, Norbert; Kirschner, Marc; Barnes, Kelly; Franti, Michael; Michael, Elizabeth; Ketas, Thomas; Sanders, Rogier W.; Maddon, Paul J.; Olson, William C.; Moore, John P.

    2005-01-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) complex comprises three gp120 exterior glycoproteins each noncovalently linked to a gp41 transmembrane glycoprotein. Monomeric gp120 proteins can elicit antibodies capable of neutralizing atypically sensitive test viruses in vitro, but these antibodies are ineffective against representative primary isolates and the gp120 vaccines failed to provide protection against HIV-1 transmission in vivo. Alternative approaches to raising neutralizing antibodies are therefore being pursued. Here we report on the antibody responses generated in rabbits against a soluble, cleaved, trimeric form of HIV-1JR-FL Env. In this construct, the gp120 and gp41 moieties are covalently linked by an intermolecular disulfide bond (SOS gp140), and an I559P substitution has been added to stabilize gp41-gp41 interactions (SOSIP gp140). We investigated the value of DNA priming and compared the use of membrane-bound and soluble priming antigens and of repeat boosting with soluble and particulate protein antigen. Compared to monomeric gp120, SOSIP gp140 trimers elicited approximately threefold lower titers of anti-gp120 antibodies. Priming with DNA encoding a membrane-bound form of the SOS gp140 protein, followed by several immunizations with soluble SOSIP gp140 trimers, resulted in antibodies capable of neutralizing sensitive strains at high titers. A subset of these sera also neutralized, at lower titers, HIV-1JR-FL and some other primary isolates in pseudovirus and/or whole-virus assays. Neutralization of these viruses was immunoglobulin mediated and was predominantly caused by antibodies to gp120 epitopes, but not the V3 region. PMID:15994775

  11. Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells

    SciTech Connect

    Rusche, J.R.; Lynn, D.L.; Robert-Guroff, M.; Langlois, A.J.; Lyerly, H.K.; Carson, H.; Krohn, K.; Ranki, A.; Gallo, R.C.; Bolognesi, D.P.; Putney, S.D.

    1987-10-01

    The human immunodeficiency virus envelope gene was expressed in insect cells by using a Baculovirus expression vector. The protein has an apparent molecular mass of 160 kDa, appears on the surface of infected insect cells, and does not appear to be cleaved to glycoproteins gp120 and gp41. Goats immunized with the 160-kDa protein have high titers of antibody that neutralizes virus infection as measured by viral gene expression or cell cytolysis. In addition, immune sera can block fusion of human immunodeficiency virus-infected cells in culture. Both neutralization and fusion-blocking activities are bound to and eluted from immobilized gp120.

  12. ADS-J1 Inhibits HIV-1 Entry by Interacting with gp120 and Does Not Block Fusion-Active gp41 Core Formation▿

    PubMed Central

    González-Ortega, Emmanuel; Mena, Maria-Pau; Permanyer, Marc; Ballana, Ester; Clotet, Bonaventura; Esté, José A.

    2010-01-01

    We had shown that virus resistance to ADS-J1 was associated with amino acid changes in the envelope glycoprotein, mostly located in the gp120 coding region. Time-of-addition and endocytic virus transfer assays clearly demonstrated that ADS-J1 behaved as a gp120 inhibitor. ADS-J1-resistant virus was cross-resistant to the polyanion dextran sulfate, and recombination of gp120 recovered only the ADS-J1-resistant phenotype. In summary, ADS-J1 blocks an early step of virus entry that appears to be driven by gp120 alone. PMID:20643898

  13. ADS-J1 inhibits HIV-1 entry by interacting with gp120 and does not block fusion-active gp41 core formation.

    PubMed

    González-Ortega, Emmanuel; Mena, Maria-Pau; Permanyer, Marc; Ballana, Ester; Clotet, Bonaventura; Esté, José A

    2010-10-01

    We had shown that virus resistance to ADS-J1 was associated with amino acid changes in the envelope glycoprotein, mostly located in the gp120 coding region. Time-of-addition and endocytic virus transfer assays clearly demonstrated that ADS-J1 behaved as a gp120 inhibitor. ADS-J1-resistant virus was cross-resistant to the polyanion dextran sulfate, and recombination of gp120 recovered only the ADS-J1-resistant phenotype. In summary, ADS-J1 blocks an early step of virus entry that appears to be driven by gp120 alone.

  14. Comparative functional role of PC7 and furin in the processing of the HIV envelope glycoprotein gp160.

    PubMed

    Decroly, E; Benjannet, S; Savaria, D; Seidah, N G

    1997-03-17

    The intracellular proteolytic processing of HIV envelope glycoprotein gp160 into gp120/gp41 is an essential step for virus infectivity. Several convertases, belonging to the pro-protein convertase family, have been proposed as candidate gp160 processing enzymes. Here we demonstrate using RT-PCR that resting human T4 lymphocytes weakly express PC7, furin, and PC5 mRNA whereas lymphocytes activated under conditions favoring HIV replication express 5-10-fold higher levels of furin and PC7. In this report, we examined the capability of the newly cloned convertase PC7 to cleave gp160 into gp120/gp41 and compared it to furin. This was carried out in a cell-based assay whereby both gp160 and the cognate convertase were co-expressed in the constitutively secreting BSC40 cells and in the regulated AtT20 cells, as well as using two in vitro assays which examined the cleavage of gp160 or of a synthetic peptide spanning the cleavage site. The data demonstrate that PC7 can cleave specifically and in a cell-type specific manner gp160 into gp120gp41, suggesting that both furin and PC7 are so far the major PC-like candidate gp160 convertase in T4 lymphocytes.

  15. Release of gp120 Restraints Leads to an Entry-Competent Intermediate State of the HIV-1 Envelope Glycoproteins.

    PubMed

    Herschhorn, Alon; Ma, Xiaochu; Gu, Christopher; Ventura, John D; Castillo-Menendez, Luis; Melillo, Bruno; Terry, Daniel S; Smith, Amos B; Blanchard, Scott C; Munro, James B; Mothes, Walther; Finzi, Andrés; Sodroski, Joseph

    2016-10-25

    Primary human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimers [(gp120/gp41)3] typically exist in a metastable closed conformation (state 1). Binding the CD4 receptor triggers Env to undergo extensive conformational changes to mediate virus entry. We identified specific gp120 residues that restrain Env in state 1. Alteration of these restraining residues destabilized state 1, allowing Env to populate a functional conformation (state 2) intermediate between state 1 and the full CD4-bound state (state 3). Increased state 2 occupancy was associated with lower energy barriers between the states. State 2 was an obligate intermediate for all transitions between state 1 and state 3. State 2-enriched Envs required lower CD4 concentrations to trigger virus entry and more efficiently infected cells expressing low levels of CD4. These Envs were resistant to several broadly neutralizing antibodies and small-molecule inhibitors. Thus, state 2 is an Env conformation on the virus entry pathway; sampling state 2 increases the adaptability of HIV-1 to different host cell receptor levels and immune environments. Our results provide new insights into the conformational regulation of HIV-1 entry. The envelope glycoproteins (Env) of HIV-1 mediate virus entry and are the sole targets of neutralizing antibodies. Understanding the way that Env promotes HIV-1 entry can expedite drug and vaccine development. By destabilizing Env, we found that it assumes an intermediate state that is functional and obligate for transitions to entry-competent conformations. Increased sampling of this state enhances the ability of HIV-1 to infect cells that express low levels of the CD4 receptor and allows the virus to evade neutralizing antibodies and small-molecule inhibitors. These findings provide new mechanistic insights into the function and inhibition of HIV-1 Env and will contribute to ongoing therapeutic and prevention efforts to combat HIV-1. Copyright © 2016 Herschhorn et al.

  16. Contribution of mdr1b-type P-glycoprotein to okadaic acid resistance in rat pituitary GH3 cells.

    PubMed

    Ritz, V; Marwitz, J; Sieder, S; Ziemann, C; Hirsch-Ernst, K I; Quentin, I; Steinfelder, H J

    1999-08-01

    Okadaic acid as well as other, structurally different, inhibitors of serine/threonine phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells. Incubation with stepwise raised concentrations of okadaic acid resulted in the isolation of cells that were increasingly less sensitive to the cytotoxic effect of this agent. After about 18 months cells were selected that survived at 300 nM okadaic acid, which is about 30 times the initially lethal concentration. This study revealed that a major pharmacokinetic mechanism underlying cell survival was the development of a P-glycoprotein-mediated multidrug resistance (MDR) phenotype. The increase in mRNA levels of the mdr1b P-glycoprotein isoform correlated with the extent of drug resistance. Functional assays revealed that increasing drug resistance was paralleled by a decreased accumulation of rhodamine 123, a fluorescent dye which is a substrate of mdr1-mediated efflux activity. Resistance could be abolished by structurally different chemosensitizers of P-glycoprotein function like verapamil and reserpine but not by the leukotriene receptor antagonist MK571 which is a modulator of the multidrug resistance-associated protein (MRP). Okadaic acid resistance included cross-resistance to other cytotoxic agents that are substrates of mdr1-type P-glycoproteins, like doxorubicin and actinomycin D, but not to non-substrates of mdr1, e.g. cytosine arabinoside. Thus, functional as well as biochemical features support the conclusion that okadaic acid is a substrate of the mdr1-mediated efflux activity in rat pituitary GH3 cells. Maintenance of resistance after withdrawal of okadaic acid as well as metaphase spreads of 100 nM okadaic acid-resistant cells suggested a stable MDR genotype without indications for the occurrence of extrachromosomal amplifications, e.g. double minute chromosomes.

  17. Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer.

    PubMed

    Ciczora, Yann; Callens, Nathalie; Montpellier, Claire; Bartosch, Birke; Cosset, François-Loïc; Op de Beeck, Anne; Dubuisson, Jean

    2005-10-01

    The envelope glycoproteins of Hepatitis C virus (HCV), E1 and E2, form a heterodimer that is retained in the endoplasmic reticulum (ER). The transmembrane (TM) domains play a major role in E1E2 heterodimerization and in ER retention. Two fully conserved charged residues in the middle of the TM domain of E2 (Asp and Arg) are crucial for these functions. Replacement of the Asp residue by a Leu impaired E1E2 heterodimerization, whereas the Arg-to-Leu mutation had a milder effect. Both Asp and Arg residues were shown to contribute to the ER retention function of E2. In addition, the entry function of HCV envelope glycoproteins was affected by these mutations. Together, these data indicate that the charged residues present in the TM domain of E2 play a major role in the biogenesis and the entry function of the E1E2 heterodimer. However, the Asp and Arg residues do not contribute equally to these functions.

  18. Improbability of harmful autoimmune responses resulting from immunization with HIV-1 envelope glycoproteins.

    PubMed

    Neurath, A R; Strick, N; Li, Y Y; Jiang, S

    1993-12-01

    Autoimmunity mediated by cross-reactive antibodies, elicited by HIV-1 envelope glycoproteins gp120/gp160, has been postulated to contribute to the pathogenesis of AIDS. Partial amino acid sequence homology between gp120/gp160 and several human host proteins, including MHC antigens and immunoglobulins, has been perceived as the basis for immunological cross-reactivity. Binding of antibodies from sera of HIV-1-infected individuals to selected host proteins and/or to synthetic peptides derived from them and the inhibitory activity of such sera in assays measuring the functional activity of T cells provided apparent support for the autoimmunity hypothesis, which is also relevant to the issue of safety of anti-HIV-1 vaccines. Considering the possibility that the detected autoantibodies may arise for reasons other than antibody responses to gp120/gp160, the immunological cross-reactivity between gp120/gp160 and the relevant host proteins was investigated using hyperimmune rabbit anti-gp120/gp160 and monoclonal antibodies. As determined from dilution end-point comparisons for polyclonal anti-gp120, the cross-reactivity of anti-gp120 with CD4 was undetectable (< 10(-5)%). The cross-reactivity of anti-gp120/gp160 with HLA-I and HLA-II antigens was also undetectable (< 4 x 10(-4)%) and that with other human proteins reported to have partial sequence homology with gp120/gp41 was < or = 0.013%. Anti-gp120/gp160 did not have detectable inhibitory effects in functional assays measuring proliferative T cell responses. Therefore, immunization with gp120/gp160 is unlikely to elicit harmful autoimmune responses.

  19. Basic residues in hypervariable region 1 of hepatitis C virus envelope glycoprotein e2 contribute to virus entry.

    PubMed

    Callens, Nathalie; Ciczora, Yann; Bartosch, Birke; Vu-Dac, Ngoc; Cosset, François-Loïc; Pawlotsky, Jean-Michel; Penin, François; Dubuisson, Jean

    2005-12-01

    The N terminus of hepatitis C virus (HCV) envelope glycoprotein E2 contains a hypervariable region (HVR1) which has been proposed to play a role in viral entry. Despite strong amino acid variability, HVR1 is globally basic, with basic residues located at specific sequence positions. Here we show by analyzing a large number of HVR1 sequences that the frequency of basic residues at each position is genotype dependent. We also used retroviral pseudotyped particles (HCVpp) harboring genotype 1a envelope glycoproteins to study the role of HVR1 basic residues in entry. Interestingly, HCVpp infectivity globally increased with the number of basic residues in HVR1. However, a shift in position of some charged residues also modulated HCVpp infectivity. In the absence of basic residues, infectivity was reduced to the same level as that of a mutant deleted of HVR1. We also analyzed the effect of these mutations on interactions with some potential HCV receptors. Recognition of CD81 was not affected by changes in the number of charged residues, and we did not find a role for heparan sulfates in HCVpp entry. The involvement of the scavenger receptor class B type I (SR-BI) was indirectly analyzed by measuring the enhancement of infectivity of the mutants in the presence of the natural ligand of SR-BI, high-density lipoproteins (HDL). However, no correlation between the number of basic residues within HVR1 and HDL enhancement effect was observed. Despite the lack of evidence of the involvement of known potential receptors, our results demonstrate that the presence of basic residues in HVR1 facilitates virus entry.

  20. Basic Residues in Hypervariable Region 1 of Hepatitis C Virus Envelope Glycoprotein E2 Contribute to Virus Entry

    PubMed Central

    Callens, Nathalie; Ciczora, Yann; Bartosch, Birke; Vu-Dac, Ngoc; Cosset, François-Loïc; Pawlotsky, Jean-Michel; Penin, François; Dubuisson, Jean

    2005-01-01

    The N terminus of hepatitis C virus (HCV) envelope glycoprotein E2 contains a hypervariable region (HVR1) which has been proposed to play a role in viral entry. Despite strong amino acid variability, HVR1 is globally basic, with basic residues located at specific sequence positions. Here we show by analyzing a large number of HVR1 sequences that the frequency of basic residues at each position is genotype dependent. We also used retroviral pseudotyped particles (HCVpp) harboring genotype 1a envelope glycoproteins to study the role of HVR1 basic residues in entry. Interestingly, HCVpp infectivity globally increased with the number of basic residues in HVR1. However, a shift in position of some charged residues also modulated HCVpp infectivity. In the absence of basic residues, infectivity was reduced to the same level as that of a mutant deleted of HVR1. We also analyzed the effect of these mutations on interactions with some potential HCV receptors. Recognition of CD81 was not affected by changes in the number of charged residues, and we did not find a role for heparan sulfates in HCVpp entry. The involvement of the scavenger receptor class B type I (SR-BI) was indirectly analyzed by measuring the enhancement of infectivity of the mutants in the presence of the natural ligand of SR-BI, high-density lipoproteins (HDL). However, no correlation between the number of basic residues within HVR1 and HDL enhancement effect was observed. Despite the lack of evidence of the involvement of known potential receptors, our results demonstrate that the presence of basic residues in HVR1 facilitates virus entry. PMID:16306604

  1. Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

    PubMed

    Vogel, Erica P; Curtis-Fisk, Jaime; Young, Kaitlin M; Weliky, David P

    2011-11-22

    Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41. © 2011 American Chemical Society

  2. Individual contributions of the human metapneumovirus F, G, and SH surface glycoproteins to the induction of neutralizing antibodies and protective immunity

    SciTech Connect

    Skiadopoulos, Mario H. . E-mail: mskiadopoulos@niaid.nih.gov; Biacchesi, Stephane; Buchholz, Ursula J.; Amaro-Carambot, Emerito; Surman, Sonja R.; Collins, Peter L.; Murphy, Brian R.

    2006-02-20

    We evaluated the individual contributions of the three surface glycoproteins of human metapneumovirus (HMPV), namely the fusion F, attachment G, and small hydrophobic SH proteins, to the induction of serum HMPV-binding antibodies, serum HMPV-neutralizing antibodies, and protective immunity. Using reverse genetics, each HMPV protein was expressed individually from an added gene in recombinant human parainfluenza virus type 1 (rHPIV1) and used to infect hamsters once or twice by the intranasal route. The F protein was highly immunogenic and protective, whereas G and SH were only weakly or negligibly immunogenic and protective, respectively. Thus, in contrast to other paramyxoviruses, the HMPV attachment G protein is not a major neutralization or protective antigen. Also, although the SH protein of HMPV is a virion protein that is much larger than its counterparts in previously studied paramyxoviruses, it does not appear to be a significant neutralization or protective antigen.

  3. A broad HIV-1 inhibitor blocks envelope glycoprotein transitions critical for entry

    PubMed Central

    Herschhorn, Alon; Gu, Christopher; Espy, Nicole; Richard, Jonathan; Finzi, Andrés; Sodroski, Joseph G.

    2014-01-01

    Binding to the primary receptor, CD4, triggers conformational changes in the metastable envelope glycoprotein (Env) trimer (gp1203/gp413) of human immunodeficiency virus (HIV-1) that are important for virus entry into host cells. These changes include an “opening” of the trimer, creation of a binding site for the CCR5 coreceptor, and formation/exposure of a gp41 coiled coil. Here we identify a new compound, 18A (1), that specifically inhibits the entry of a wide range of HIV-1 isolates. 18A does not interfere with CD4 or CCR5 binding, but inhibits the CD4-induced disruption of quaternary structures at the trimer apex and the formation/exposure of the gp41 HR1 coiled coil. Analysis of HIV-1 variants exhibiting increased or reduced sensitivity to 18A suggests that the inhibitor can distinguish distinct conformational states of gp120 in the unliganded Env trimer. The broad-range activity and observed hypersensitivity of resistant mutants to antibody neutralization support further investigation of 18A. PMID:25174000

  4. Glycoprotein synthesis

    SciTech Connect

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

    2009-07-14

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  5. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-08-28

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  6. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-05-15

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  7. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-07-03

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  8. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2010-11-02

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  9. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2006-10-31

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  10. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2010-11-16

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  11. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2005-08-09

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  12. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-02-27

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  13. Glycoprotein synthesis

    DOEpatents

    Shultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-04-03

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  14. Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody.

    PubMed

    Trott, Maria; Weiβ, Svenja; Antoni, Sascha; Koch, Joachim; von Briesen, Hagen; Hust, Michael; Dietrich, Ursula

    2014-01-01

    HIV neutralizing antibodies (nAbs) represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP) and elite controllers (EC), represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env) proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb) A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR) in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.

  15. Functional Characterization of Two scFv-Fc Antibodies from an HIV Controller Selected on Soluble HIV-1 Env Complexes: A Neutralizing V3- and a Trimer-Specific gp41 Antibody

    PubMed Central

    Trott, Maria; Weiß, Svenja; Antoni, Sascha; Koch, Joachim; von Briesen, Hagen; Hust, Michael; Dietrich, Ursula

    2014-01-01

    HIV neutralizing antibodies (nAbs) represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP) and elite controllers (EC), represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env) proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb) A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR) in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike. PMID:24828352

  16. Mechanism of pluronic effect on P-glycoprotein efflux system in blood-brain barrier: contributions of energy depletion and membrane fluidization.

    PubMed

    Batrakova, E V; Li, S; Vinogradov, S V; Alakhov, V Y; Miller, D W; Kabanov, A V

    2001-11-01

    Pluronic block copolymer, P85, inhibits the P-glycoprotein (Pgp) drug efflux system and increases the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). This study examines the mechanisms by which P85 inhibits Pgp using bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB. The hypothesis was that simultaneous alterations in intracellular ATP levels and membrane fluidization in BBMEC monolayers by P85 results in inhibition of the drug efflux system. The methods included the use of 1) standard Pgp substrate rhodamine 123 to assay the Pgp efflux system in BBMEC, 2) luciferin/luciferase assay for ATP intracellular levels, and 3) 1,6-diphenyl-1,3,5-hexatriene for membrane microviscosity. Using 3H-labeled P85 and fluorescein-labeled P85 for confocal microscopy, this study suggests that P85 accumulates in the cells and intracellular organelles such as the mitochondria where it can interfere with metabolic processes. Following exposure of BBMEC to P85, the ATP levels were depleted, and microviscosity of the cell membranes was decreased. Furthermore, P85 treatment decreased Pgp ATPase activity in membranes expressing human Pgp. A combination of experiments examining the kinetics, concentration dependence, and directionality of P85 effects on Pgp-mediated efflux in BBMEC monolayers suggests that both energy depletion (decreasing ATP pool available for Pgp) and membrane fluidization (inhibiting Pgp ATPase activity) are critical factors contributing to the activity of the block copolymer in the BBB.

  17. An update on post-translational modifications of hydroxyproline-rich glycoproteins: toward a model highlighting their contribution to plant cell wall architecture.

    PubMed

    Hijazi, May; Velasquez, Silvia M; Jamet, Elisabeth; Estevez, José M; Albenne, Cécile

    2014-01-01

    Plant cell walls are composite structures mainly composed of polysaccharides, also containing a large set of proteins involved in diverse functions such as growth, environmental sensing, signaling, and defense. Research on cell wall proteins (CWPs) is a challenging field since present knowledge of their role into the structure and function of cell walls is very incomplete. Among CWPs, hydroxyproline (Hyp)-rich O-glycoproteins (HRGPs) were classified into three categories: (i) moderately glycosylated extensins (EXTs) able to form covalent scaffolds; (ii) hyperglycosylated arabinogalactan proteins (AGPs); and (iii) Hyp/proline (Pro)-Rich proteins (H/PRPs) that may be non-, weakly- or highly-glycosylated. In this review, we provide a description of the main features of their post-translational modifications (PTMs), biosynthesis, structure, and function. We propose a new model integrating HRGPs and their partners in cell walls. Altogether, they could form a continuous glyco-network with non-cellulosic polysaccharides via covalent bonds or non-covalent interactions, thus strongly contributing to cell wall architecture.

  18. An update on post-translational modifications of hydroxyproline-rich glycoproteins: toward a model highlighting their contribution to plant cell wall architecture

    PubMed Central

    Hijazi, May; Velasquez, Silvia M.; Jamet, Elisabeth; Estevez, José M.; Albenne, Cécile

    2014-01-01

    Plant cell walls are composite structures mainly composed of polysaccharides, also containing a large set of proteins involved in diverse functions such as growth, environmental sensing, signaling, and defense. Research on cell wall proteins (CWPs) is a challenging field since present knowledge of their role into the structure and function of cell walls is very incomplete. Among CWPs, hydroxyproline (Hyp)-rich O-glycoproteins (HRGPs) were classified into three categories: (i) moderately glycosylated extensins (EXTs) able to form covalent scaffolds; (ii) hyperglycosylated arabinogalactan proteins (AGPs); and (iii) Hyp/proline (Pro)-Rich proteins (H/PRPs) that may be non-, weakly- or highly-glycosylated. In this review, we provide a description of the main features of their post-translational modifications (PTMs), biosynthesis, structure, and function. We propose a new model integrating HRGPs and their partners in cell walls. Altogether, they could form a continuous glyco-network with non-cellulosic polysaccharides via covalent bonds or non-covalent interactions, thus strongly contributing to cell wall architecture. PMID:25177325

  19. Small molecule fusion inhibitors: design, synthesis and biological evaluation of (Z)-3-(5-(3-benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrroles and related derivatives targeting HIV-1 gp41.

    PubMed

    He, Xiao-Yang; Lu, Lu; Qiu, Jiayin; Zou, Peng; Yu, Fei; Jiang, Xing-Kai; Li, Lin; Jiang, Shibo; Liu, Shuwen; Xie, Lan

    2013-12-01

    By a scaffold elongation strategy, a series of (Z)-3-(5-(3-benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrroles and related derivatives with a linear multi-aromatic-ring skeleton were designed, synthesized, and evaluated in HIV-1 gp41 and cellular assays. Among them, the most active compounds, 12e, 12g, and 12k with a one-carbon linker (n=1) between the rhodanine (C) and phenyl (D) rings, exhibited very promising inhibitory potency with IC50 values of 1.8-2.6 μM and EC50 values of 0.3-1.5 μM against gp41 6-HB formation and HIV-1 replication in MT-2 cells, respectively. Additionally, they were almost equally effective against both T20-sensitive and resistant strains. The related SAR studies and molecular modeling results provided potential for further developing a new class of non-peptide small molecular fusion inhibitors targeting the HIV-1 gp41.

  20. Glycoprotein Nonmetastatic Melanoma B (Gpnmb)-Positive Macrophages Contribute to the Balance between Fibrosis and Fibrolysis during the Repair of Acute Liver Injury in Mice

    PubMed Central

    Kumagai, Kotaro; Tabu, Kazuaki; Sasaki, Fumisato; Takami, Yoichiro; Morinaga, Yuko; Mawatari, Seiichi; Hashimoto, Shinichi; Tanoue, Shiroh; Kanmura, Shuji; Tamai, Tsutomu; Moriuchi, Akihiro; Uto, Hirofumi; Tsubouchi, Hirohito; Ido, Akio

    2015-01-01

    Background and aims Glycoprotein nonmetastatic melanoma B (Gpnmb), a transmembrane glycoprotein that is expressed in macrophages, negatively regulates inflammation. We have reported that Gpnmb is strongly expressed in the livers of rats fed a choline-deficient, L-amino acid-defined (CDAA) diet. However, the role of macrophage-expressed Gpnmb in liver injury is still unknown. This study aimed to clarify the characteristics of infiltrating macrophages that express Gpnmb, and the involvement of Gpnmb in the repair process in response to liver injury. Methods C57BL/6J, DBA/2J [DBA] and DBA/2J-Gpnmb+ [DBA-g+] mice were treated with a single intraperitoneal injection of carbon tetrachloride (CCl4) at a dose of 1.0 mL/kg body weight. Mice were sacrificed at predetermined time points, followed by measurement of serum alanine aminotransferase (ALT) levels and histological examination. Expression of Gpnmb, pro-/anti-inflammatory cytokines, and profibrotic/antifibrotic factors were examined by quantitative RT-PCR and/or Western blotting. Immunohistochemistry, fluorescent immunostaining and flow cytometry were used to determine the expression of Gpnmb, CD68, CD11b and α-SMA, phagocytic activity, and the presence of apoptotic bodies. We used quantitative RT-PCR and ELISA to examine TGF-β and MMP-13 expression and the concentrations and supernatants of isolated infiltrating hepatic macrophages transfected with siGpnmb. Results In C57BL/6J mice, serum ALT levels increased at two days after CCl4 injection and decreased at four days. Gpnmb expression in the liver was stimulated four days after CCl4 injection. Histological examination and flow cytometry showed that Gpnmb-positive cells were almost positive for CD68-positive macrophages, contained engulfed apoptotic bodies and exhibited enhanced phagocytic activity. Isolated infiltrating hepatic macrophages transfected with siGpnmb showed high MMP-13 secretion. There was no significant difference in the magnitude of CCl4-induced

  1. Binding of soluble CD4 proteins to human immunodeficiency virus type 1 and infected cells induces release of envelope glycoprotein gp120.

    PubMed Central

    Hart, T K; Kirsh, R; Ellens, H; Sweet, R W; Lambert, D M; Petteway, S R; Leary, J; Bugelski, P J

    1991-01-01

    Human immunodeficiency virus (HIV) infects cells after binding of the viral envelope glycoprotein gp120 to the cell surface recognition marker CD4. gp120 is noncovalently associated with the HIV transmembrane envelope glycoprotein gp41, and this complex is believed responsible for the initial stages of HIV infection and cytopathic events in infected cells. Soluble constructs of CD4 that contain the gp120 binding site inhibit HIV infection in vitro. This is believed to occur by competitive inhibition of viral binding to cellular CD4. Here we suggest an alternative mechanism of viral inhibition by soluble CD4 proteins. We demonstrate biochemically and morphologically that following binding, the soluble CD4 proteins sT4, V1V2,DT, and V1[106] (amino acids 1-369, 1-183, and -2 to 106 of mature CD4) induced the release of gp120 from HIV-1 and HIV-1-infected cells. gp120 release was concentration-, time-, and temperature-dependent. The reaction was biphasic at 37 degrees C and did not take place at 4 degrees C, indicating that binding of soluble CD4 was not sufficient to release gp120. The appearance of free gp120 in the medium after incubation with sT4 correlated with a decrease in envelope glycoprotein spikes on virions and exposure of a previously cryptic epitope near the amino terminus of gp41 on virions and infected cells. The concentration of soluble CD4 proteins needed to induce the release of gp120 from virally infected cells also correlated with those required to inhibit HIV-mediated syncytium formation. These results suggest that soluble CD4 constructs may inactivate HIV by inducing the release of gp120. We propose that HIV envelope-mediated fusion is initiated following rearrangement and/or dissociation of gp120 from the gp120-gp41 complex upon binding to cellular CD4, thus exposing the fusion domain of gp41. Images PMID:2006155

  2. The V1-V3 region of a brain-derived HIV-1 envelope glycoprotein determines macrophage tropism, low CD4 dependence, increased fusogenicity and altered sensitivity to entry inhibitors

    PubMed Central

    Rossi, Fiorella; Querido, Bianca; Nimmagadda, Manideepthi; Cocklin, Simon; Navas-Martín, Sonia; Martín-García, Julio

    2008-01-01

    Background HIV-1 infects macrophages and microglia in the brain and can cause neurological disorders in infected patients. We and others have shown that brain-derived envelope glycoproteins (Env) have lower CD4 dependence and higher avidity for CD4 than those from peripheral isolates, and we have also observed increased fusogenicity and reduced sensitivity to the fusion inhibitor T-1249. Due to the genetic differences between brain and spleen env from one individual throughout gp120 and in gp41's heptad repeat 2 (HR2), we investigated the viral determinants for the phenotypic differences by performing functional studies with chimeric and mutant Env. Results Chimeric Env showed that the V1/V2-C2-V3 region in brain's gp120 determines the low CD4 dependence and high avidity for CD4, as well as macrophage tropism and reduced sensitivity to the small molecule BMS-378806. Changes in brain gp41's HR2 region did not contribute to the increased fusogenicity or to the reduced sensitivity to T-1249, since a T-1249-based peptide containing residues found in brain's but not in spleen's HR2 had similar potency than T-1249 and interacted similarly with an immobilized heptad repeat 1-derived peptide in surface plasmon resonance analysis. However, the increased fusogenicity and reduced T-1249 sensitivity of brain and certain chimeric Env mostly correlated with the low CD4 dependence and high avidity for CD4 determined by brain's V1-V3 region. Remarkably, most but not all of these low CD4-dependent, macrophage tropic envelopes glycoproteins also had increased sensitivity to the novel allosteric entry inhibitor HNG-105. The gp120's C2 region asparagine 283 (N283) has been previously associated with macrophage tropism, brain infection, lower CD4 dependence and higher CD4 affinity. Therefore, we introduced the N283T mutation into an env clone from a brain-derived isolate and into a brain tissue-derived env clone, and the T283N change into a spleen-derived env from the same individual

  3. Maturation of HIV envelope glycoprotein precursors by cellular endoproteases.

    PubMed

    Moulard, M; Decroly, E

    2000-11-10

    . Furthermore, relationships between differential pathogenicity of influenza strains and their susceptibility to cleavage are molecularly funded. Here we review the most recent data and recent insights demonstrating the crucial role played by this activation step in virus infectivity. We discuss the cellular endoproteases that are implicated in HIV gp160 endoproteolytical maturation into gp120 and gp41.

  4. cis Expression of the F12 Human Immunodeficiency Virus (HIV) Nef Allele Transforms the Highly Productive NL4-3 HIV Type 1 to a Replication-Defective Strain: Involvement of both Env gp41 and CD4 Intracytoplasmic Tails

    PubMed Central

    Olivetta, Eleonora; Pugliese, Katherina; Bona, Roberta; D'Aloja, Paola; Ferrantelli, Flavia; Santarcangelo, Anna Claudia; Mattia, Gianfranco; Verani, Paola; Federico, Maurizio

    2000-01-01

    F12 human immunodeficiency virus type 1 (HIV-1) nef is a naturally occurring nef mutant cloned from the provirus of a nonproductive, nondefective, and interfering HIV-1 variant (F12-HIV). We have already shown that cells stably transfected with a vector expressing the F12-HIV nef allele do not downregulate CD4 receptors and, more peculiarly, become resistant to the replication of wild type (wt) HIV. In order to investigate the mechanism of action of such an HIV inhibition, the F12-HIV nef gene was expressed in the context of the NL4-3 HIV-1 infectious molecular clone by replacing the wt nef gene (NL4-3/chi). Through this experimental approach we established the following. First, NL4-3/chi and nef-defective (Δnef) NL4-3 viral particles behave very similarly in terms of viral entry and HIV protein production during the first replicative cycle. Second, no viral particles were produced from cells infected with NL4-3/chi virions, whatever the multiplicity of infection used. The viral inhibition apparently occurs at level of viral assembling and/or release. Third, this block could not be relieved by in-trans expression of wt nef. Finally, NL4-3/chi reverts to a producer HIV strain when F12-HIV Nef is deprived of its myristoyl residue. Through a CD4 downregulation competition assay, we demonstrated that F12-HIV Nef protein potently inhibits the CD4 downregulation induced by wt Nef. Moreover, we observed a redistribution of CD4 receptors at the cell margin induced by F12-HIV Nef. These observations strongly suggest that F12-HIV Nef maintains the ability to interact with the intracytoplasmic tail of the CD4 receptor molecule. Remarkably, we distinguished the intracytoplasmic tails of Env gp41 and CD4 as, respectively, viral and cellular targets of the F12-HIV Nef-induced viral retention. For the first time, the inhibition of the viral life cycle by means of in-cis expression of a Nef mutant is here reported. Delineation of the F12-HIV Nef mechanism of action may offer

  5. Effect of trimerization motifs on quaternary structure, antigenicity, and immunogenicity of a noncleavable HIV-1 gp140 envelope glycoprotein

    SciTech Connect

    Du, Sean X.; Idiart, Rebecca J.; Mariano, Ellaine B.; Chen, Helen; Jiang Peifeng; Xu Li; Ostrow, Kristin M.; Wrin, Terri; Phung, Pham; Binley, James M.; Petropoulos, Christos J.; Ballantyne, John A.; Whalen, Robert G.

    2009-12-05

    The external domains of the HIV-1 envelope glycoprotein (gp120 and the gp41 ectodomain, collectively known as gp140) contain all known viral neutralization epitopes. Various strategies have been used to create soluble trimers of the envelope to mimic the structure of the native viral protein, including mutation of the gp120-gp41 cleavage site, introduction of disulfide bonds, and fusion to heterologous trimerization motifs. We compared the effects on quaternary structure, antigenicity, and immunogenicity of three such motifs: T4 fibritin, a GCN4 variant, and the Escherichia coli aspartate transcarbamoylase catalytic subunit. Fusion of each motif to the C-terminus of a noncleavable JRCSF gp140(-) envelope protein led to enhanced trimerization but had limited effects on the antigenic profile and CD4-binding ability of the trimers. Immunization of rabbits provided no evidence that the trimerized gp140(-) constructs induced significantly improved neutralizing antibodies to several HIV-1 pseudoviruses, compared to gp140 lacking a trimerization motif. However, modest differences in both binding specificity and neutralizing antibody responses were observed among the various immunogens.

  6. Design and characterization of swapped-domain constructs of HIV-1 glycoprotein-41 as receptors for drug discovery

    PubMed Central

    Walsh, Joseph D.; Chu, Shidong; Zhang, Shao-Qing; Gochin, Miriam

    2015-01-01

    Four new swapped-domain constructs of the ectodomain of human immunodeficiency virus type 1 glycoprotein-41 (gp41) were prepared. The gp41 ectodomain consists of 50-residue N-heptad repeat (NHR), 36-residue disulfide-bonded loop and 39-residue C-heptad repeat (CHR). It folds into a hairpin structure that forms a trimer along the NHR axis. The swapped-domain proteins feature CHR domains of length 39, 28 or 21 residues preceding a 4-residue loop and a 49- or 50-residue NHR domain. The effect of CHR truncation was to expose increasing lengths of the NHR groove, including the conserved hydrophobic pocket, an important drug target. A novel method for preparing proteins with extended exposed hydrophobic surfaces was demonstrated. Biophysical measurements, including analytical ultracentrifugation and ligand-detected Water-Ligand Observed via Gradient Spectroscopy and 1H–15N-HSQC NMR experiments, were used to confirm that the proteins formed stable trimers in solution with exposed binding surfaces. These proteins could play an important role as receptors in structure-based drug discovery. PMID:25792539

  7. NMR-assisted computational studies of peptidomimetic inhibitors bound in the hydrophobic pocket of HIV-1 glycoprotein 41

    NASA Astrophysics Data System (ADS)

    Gochin, Miriam; Whitby, Landon R.; Phillips, Aaron H.; Boger, Dale L.

    2013-07-01

    Due to the inherently flexible nature of a protein-protein interaction surface, it is difficult both to inhibit the association with a small molecule, and to predict how it might bind to the surface. In this study, we have examined small molecules that mediate the interaction between a WWI motif on the C-helix of HIV-1 glycoprotein-41 (gp41) and a deep hydrophobic pocket contained in the interior N-helical trimer. Association between these two components of gp41 leads to virus-cell and cell-cell fusion, which could be abrogated in the presence of an inhibitor that binds tightly in the pocket. We have studied a comprehensive combinatorial library of α-helical peptidomimetics, and found that compounds with strongly hydrophobic side chains had the highest affinity. Computational docking studies produced multiple possible binding modes due to the flexibility of both the binding site and the peptidomimetic compounds. We applied a transferred paramagnetic relaxation enhancement experiment to two selected members of the library, and showed that addition of a few experimental constraints enabled definitive identification of unique binding poses. Computational docking results were extremely sensitive to side chain conformations, and slight variations could preclude observation of the experimentally validated poses. Different receptor structures were required for docking simulations to sample the correct pose for the two compounds. The study demonstrated the sensitivity of predicted poses to receptor structure and indicated the importance of experimental verification when docking to a malleable protein-protein interaction surface.

  8. Identification of two distinct intracellular sites that contribute to the modulation of multidrug resistance in P388/ADR cells expressing P-glycoprotein.

    PubMed

    Mayer, Lawrence D; Lim, Kye-Taek; Hartley, Daria

    2002-01-01

    Although the ability of chemosensitizers to modulate P-glycoprotein (PGP)-based multidrug resistance (MDR) has been extensively studied, relatively little is known about the cellular pharmacology of the PGP inhibitors themselves in MDR cells. The studies described here have correlated the in vitro accumulation and retention properties of verapamil (VRP) in murine P388 (sensitive) and P388/ADR (MDR) cells with doxorubicin (DOX) uptake and cytotoxicity modulation characteristics in order to better understand VRP-tumor cell interactions that give rise to MDR modulation. VRP is rapidly taken up by DOX-sensitive and -resistant P388 cells where greater than 50% maximal VRP uptake occurs within 10 min of initial exposure at 37 degrees C. Whereas chemosensitization and DOX uptake in P388/ADR cells increase with increasing VRP concentration until a plateau is achieved at approximately 5 microM VRP, cellular modulator levels increase proportionally with increasing VPR concentrations beyond 20 microM. Subsequent to removal of noncell-associated modulator, VRP levels in both sensitive and resistant cells rapidly fall below 10% of those obtained at uptake equilibrium. However, a residual amount of VRP remains associated with the cells for extended time periods after the cells are washed. Pulse exposures of P388/ADR cells to high concentrations of VRP (50-100 microM) are capable of providing extended cell-associated VRP levels comparable to those obtained with continuous exposure at biologically active VRP concentrations (1-3 microM) and this leads to chemosensitization. These results are consistent with the existence of high- and low-affinity intracellular VRP pools in P388 MDR cells, both of which can contribute to the reversal of drug resistance. It is suggested that these properties should be taken into consideration during the design and evaluation of preclinical in vivo MDR models where pulsed exposure to high concentrations of resistance modulators often occurs. Special

  9. The Envelope Cytoplasmic Tail of HIV-1 Subtype C Contributes to Poor Replication Capacity through Low Viral Infectivity and Cell-to-Cell Transmission

    PubMed Central

    Lemaire, Morgane; Masquelier, Cécile; Beraud, Cyprien; Rybicki, Arkadiusz; Servais, Jean-Yves; Iserentant, Gilles; Schmit, Jean-Claude; Seguin-Devaux, Carole; Perez Bercoff, Danielle

    2016-01-01

    The cytoplasmic tail (gp41CT) of the HIV-1 envelope (Env) mediates Env incorporation into virions and regulates Env intracellular trafficking. Little is known about the functional impact of variability in this domain. To address this issue, we compared the replication of recombinant virus pairs carrying the full Env (Env viruses) or the Env ectodomain fused to the gp41CT of NL4.3 (EnvEC viruses) (12 subtype C and 10 subtype B pairs) in primary CD4+ T-cells and monocyte-derived-macrophages (MDMs). In CD4+ T-cells, replication was as follows: B-EnvEC = B-Env>C-EnvEC>C-Env, indicating that the gp41CT of subtype C contributes to the low replicative capacity of this subtype. In MDMs, in contrast, replication capacity was comparable for all viruses regardless of subtype and of gp41CT. In CD4+ T-cells, viral entry, viral release and viral gene expression were similar. However, infectivity of free virions and cell-to-cell transmission of C-Env viruses released by CD4+ T-cells was lower, suggestive of lower Env incorporation into virions. Subtype C matrix only minimally rescued viral replication and failed to restore infectivity of free viruses and cell-to-cell transmission. Taken together, these results show that polymorphisms in the gp41CT contribute to viral replication capacity and suggest that the number of Env spikes per virion may vary across subtypes. These findings should be taken into consideration in the design of vaccines. PMID:27598717

  10. An amphipathic peptide from the C-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes.

    PubMed Central

    Chernomordik, L; Chanturiya, A N; Suss-Toby, E; Nora, E; Zimmerberg, J

    1994-01-01

    The peptide fragment of the carboxy-terminal region of the human immunodeficiency virus (HIV) transmembrane protein (gp41) has been implicated in T-cell death. This positively charged, amphipathic helix (amino acids 828 to 848) of the envelope protein is located within virions or cytoplasm. We studied the interaction of the isolated, synthetic amphipathic helix of gp41 with planar phospholipid bilayer membranes and with Sf9 cells using voltage clamp, potentiodynamic, and single-cell recording techniques. We found that the peptide binds strongly to planar membranes, especially to the negatively charged phosphatidylserine bilayer. In the presence of micromolar concentrations of peptide sufficient to make its surface densities comparable with those of envelope glycoprotein molecules in HIV virions, an increase in bilayer conductance and a decrease in bilayer stability were observed, showing pore formation in the planar lipid bilayers. These pores were permeable to both monovalent and divalent cations, as well as to chloride. The exposure of the inner leaflet of cell membranes to even 25 nM peptide increased membrane conductance. We suggest that the carboxy-terminal fragment of the HIV type 1 envelope protein may interact with the cell membrane of infected T cells to create lipidic pores which increase membrane permeability, leading to sodium and calcium flux into cells, osmotic swelling, and T-cell necrosis or apoptosis. PMID:7933093

  11. Glycosylation Engineering of Glycoproteins

    NASA Astrophysics Data System (ADS)

    Sadamoto, Reiko; Nishimura, Shin-Ichiro

    Naturally occurring glycosylation of glycoproteins varies in glycosylation site and in the number and structure of glycans. The engineering of well-defined glycoproteins is an important technology for the preparation of pharmaceutically relevant glycoproteins and in the study of the relationship between glycans and proteins on a structure-function level. In pharmaceutical applications of glycoproteins, the presence of terminal sialic acids on glycans is particularly important for the in vivo circulatory half life, since sialic acid-terminated glycans are not recognized by asialoglycoprotein receptors. Therefore, there have been a number of attempts to control or modify cellular metabolism toward the expression of glycoproteins with glycosylation profiles similar to that of human glycoproteins. In this chapter, recent methods for glycoprotein engineering in various cell culture systems (mammalian cells, plant, yeast, and E. coli) and advances in the chemical approach to glycoprotein formation are described.

  12. Persistence of HIV-1 structural proteins and glycoproteins in lymph nodes of patients under highly active antiretroviral therapy.

    PubMed

    Popovic, Mikulas; Tenner-Racz, Klara; Pelser, Colleen; Stellbrink, Hans-Jurgen; van Lunzen, Jan; Lewis, George; Kalyanaraman, Vaniambadi S; Gallo, Robert C; Racz, Paul

    2005-10-11

    Here we report a long-term persistence of HIV-1 structural proteins and glycoproteins in germinal centers (GCs) of lymph nodes (LNs) in the absence of detectable virus replication in patients under highly active antiretroviral therapy (HAART). The persistence of viral structural proteins and glycoproteins in GCs was accompanied by specific antibody responses to HIV-1. Seven patients during the chronic phase of HIV-1 infection were analyzed for the presence of the capsid protein (HIV-1p24), matrix protein (HIV-1p17), and envelope glycoproteins (HIV-1gp120/gp41), as well as for viral RNA (vRNA) in biopsy specimens from LNs obtained before initiation of therapy and during HAART that lasted from 5 to 13 months. In parallel, these patients were also monitored for viremia and specific anti-HIV-1 antibody responses to structural proteins and glycoproteins both before and during treatment. Before-therapy viral levels, as determined by RT-PCR, ranged from 3 x 10(3) to 6.3 x 10(5) copies of vRNA per ml, whereas during treatment, vRNA was under detectable levels (<25 copies per ml). The pattern of vRNA detection in peripheral blood was concordant with in situ hybridization results of LN specimens. Before treatment, vRNA associated with follicular dendritic cells (FDCs) was readily detected in GCs of LNs of the patients, whereas during therapy, vRNA was consistently absent in the GCs of LN biopsies of treated patients. In contrast to vRNA hybridization results, viral structural proteins and glycoproteins, evaluated by immunohistochemical staining, were present and persisted in the GC light zone of LNs in abundant amounts not only before initiation of therapy but also during HAART, when no vRNA was detected in GCs. Consistent with immunohistochemical findings, specific antibody responses to HIV-1p17, -p24, and -gp120/gp41, as evaluated by ELISA and virus neutralization, persisted in patients under therapy for up to 13 months of follow-up. The implications of these findings are

  13. Antigenic analysis of classical swine fever virus E2 glycoprotein using pig antibodies identifies residues contributing to antigenic variation of the vaccine C-strain and group 2 strains circulating in China.

    PubMed

    Chen, Ning; Tong, Chao; Li, Dejiang; Wan, Jing; Yuan, Xuemei; Li, Xiaoliang; Peng, Jinrong; Fang, Weihuan

    2010-12-31

    Glycoprotein E2, the immunodominant protein of classical swine fever virus (CSFV), can induce neutralizing antibodies and confer protective immunity in pigs. Our previous phylogenetic analysis showed that subgroup 2.1 viruses branched away from subgroup 1.1, the vaccine C-strain lineage, and became dominant in China. The E2 glycoproteins of CSFV C-strain and recent subgroup 2.1 field isolates are genetically different. However, it has not been clearly demonstrated how this diversity affects antigenicity of the protein. Antigenic variation of glycoprotein E2 was observed not only between CSFV vaccine C-strain and subgroup 2.1 strains, but also among strains of the same subgroup 2.1 as determined by ELISA-based binding assay using pig antisera to the C-strain and a representative subgroup 2.1 strain QZ-07 currently circulating in China. Antigenic incompatibility of E2 proteins markedly reduced neutralization efficiency against heterologous strains. Single amino acid substitutions of D705N, L709P, G713E, N723S, and S779A on C-strain recombinant E2 (rE2) proteins significantly increased heterologous binding to anti-QZ-07 serum, suggesting that these residues may be responsible for the antigenic variation between the C-strain and subgroup 2.1 strains. Notably, a G713E substitution caused the most dramatic enhancement of binding of the variant C-strain rE2 protein to anti-QZ-07 serum. Multiple sequence alignment revealed that the glutamic acid residue at this position is conserved within group 2 strains, while the glycine residue is invariant among the vaccine strains, highlighting the role of the residue at this position as a major determinant of antigenic variation of E2. A variant Simpson's index analysis showed that both codons and amino acids of the residues contributing to antigenic variation have undergone similar diversification. These results demonstrate that CSFV vaccine C-strain and group 2 strains circulating in China differ in the antigenicity of their E2

  14. Determining the Structure of an Unliganded and Fully Glycosylated SIV gp120 Envelope Glycoprotein

    SciTech Connect

    Chen, Bing; Vogan, Erik M.; Gong, Haiyun; Skehel, John J.; Wiley, Don C.; Harrison, Stephen C.

    2010-07-13

    HIV/SIV envelope glycoproteins mediate the first steps in viral infection. They are trimers of a membrane-anchored polypeptide chain, cleaved into two fragments known as gp120 and gp41. The structure of HIV gp120 bound with receptor (CD4) has been known for some time. We have now determined the structure of a fully glycosylated SIV gp120 envelope glycoprotein in an unliganded conformation by X-ray crystallography at 4.0 {angstrom} resolution. We describe here our experimental and computational approaches, which may be relevant to other resolution-limited crystallographic problems. Key issues were attention to details of beam geometry mandated by small, weakly diffracting crystals, and choice of strategies for phase improvement, starting with two isomorphous derivatives and including multicrystal averaging. We validated the structure by analyzing composite omit maps, averaged among three distinct crystal lattices, and by calculating model-based, SeMet anomalous difference maps. There are at least four ordered sugars on many of the thirteen oligosaccharides.

  15. Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

    PubMed Central

    Ringe, Rajesh P.; Yasmeen, Anila; Ozorowski, Gabriel; Go, Eden P.; Pritchard, Laura K.; Guttman, Miklos; Ketas, Thomas A.; Cottrell, Christopher A.; Wilson, Ian A.; Sanders, Rogier W.; Cupo, Albert; Crispin, Max; Lee, Kelly K.; Desaire, Heather; Ward, Andrew B.; Klasse, P. J.

    2015-01-01

    ABSTRACT We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140UNC-Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41ECTO) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ∼20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140UNC-Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components

  16. Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers.

    PubMed

    Ringe, Rajesh P; Yasmeen, Anila; Ozorowski, Gabriel; Go, Eden P; Pritchard, Laura K; Guttman, Miklos; Ketas, Thomas A; Cottrell, Christopher A; Wilson, Ian A; Sanders, Rogier W; Cupo, Albert; Crispin, Max; Lee, Kelly K; Desaire, Heather; Ward, Andrew B; Klasse, P J; Moore, John P

    2015-12-01

    We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140UNC-Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41ECTO) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ∼20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140UNC-Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components. Soluble

  17. Elite Control, Gut CD4 T Cell Sparing, and Enhanced Mucosal T Cell Responses in Macaca nemestrina Infected by a Simian Immunodeficiency Virus Lacking a gp41 Trafficking Motif

    PubMed Central

    Breed, Matthew W.; Elser, Samra E.; Torben, Workineh; Jordan, Andrea P. O.; Aye, Pyone P.; Midkiff, Cecily; Schiro, Faith; Sugimoto, Chie; Alvarez-Hernandez, Xavier; Blair, Robert V.; Somasunderam, Anoma; Utay, Netanya S.; Kuroda, Marcelo J.; Pahar, Bapi; Wiseman, Roger W.; O'Connor, David H.; LaBranche, Celia C.; Montefiori, David C.; Marsh, Mark; Li, Yuan; Piatak, Michael; Lifson, Jeffrey D.; Keele, Brandon F.; Fultz, Patricia N.; Lackner, Andrew A.

    2015-01-01

    ABSTRACT Deletion of Gly-720 and Tyr-721 from a highly conserved GYxxØ trafficking signal in the SIVmac239 envelope glycoprotein cytoplasmic domain, producing a virus termed ΔGY, leads to a striking perturbation in pathogenesis in rhesus macaques (Macaca mulatta). Infected macaques develop immune activation and progress to AIDS, but with only limited and transient infection of intestinal CD4+ T cells and an absence of microbial translocation. Here we evaluated ΔGY in pig-tailed macaques (Macaca nemestrina), a species in which SIVmac239 infection typically leads to increased immune activation and more rapid progression to AIDS than in rhesus macaques. In pig-tailed macaques, ΔGY also replicated acutely to high peak plasma RNA levels identical to those for SIVmac239 and caused only transient infection of CD4+ T cells in the gut lamina propria and no microbial translocation. However, in marked contrast to rhesus macaques, 19 of 21 pig-tailed macaques controlled ΔGY replication with plasma viral loads of <15 to 50 RNA copies/ml. CD4+ T cells were preserved in blood and gut for up to 100 weeks with no immune activation or disease progression. Robust antiviral CD4+ T cell responses were seen, particularly in the gut. Anti-CD8 antibody depletion demonstrated CD8+ cellular control of viral replication. Two pig-tailed macaques progressed to disease with persisting viremia and possible compensatory mutations in the cytoplasmic tail. These studies demonstrate a marked perturbation in pathogenesis caused by ΔGY's ablation of the GYxxØ trafficking motif and reveal, paradoxically, that viral control is enhanced in a macaque species typically predisposed to more pathogenic manifestations of simian immunodeficiency virus (SIV) infection. IMPORTANCE The pathogenesis of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) reflects a balance between viral replication, host innate and adaptive antiviral immune responses, and sustained immune activation

  18. Comprehensive Analysis of Contributions from Protein Conformational Stability and Major Histocompatibility Complex Class II-Peptide Binding Affinity to CD4+ Epitope Immunogenicity in HIV-1 Envelope Glycoprotein

    PubMed Central

    Li, Tingfeng; Steede, N. Kalaya; Nguyen, Hong-Nam P.; Freytag, Lucy C.; McLachlan, James B.; Mettu, Ramgopal R.; Robinson, James E.

    2014-01-01

    ABSTRACT Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-terminal flanks of flexible segments that are likely to be proteolytic cleavage sites. In this study, the influence of gp120 conformation on the dominance pattern in gp120 from HIV strain 89.6 was examined in CBA mice, whose MHC class II protein has one of the most well defined peptide-binding preferences. Only one of six dominant epitopes contained the most conserved element of the I-Ak binding motif, an aspartic acid. Destabilization of the gp120 conformation by deletion of single disulfide bonds preferentially enhanced responses to the cryptic I-Ak motif-containing sequences, as reported by T-cell proliferation or cytokine secretion. Conversely, inclusion of CpG in the adjuvant with gp120 enhanced responses to the dominant CD4+ T-cell epitopes. The gp120 destabilization affected secretion of some cytokines more than others, suggesting that antigen conformation could modulate T-cell functions through mechanisms of antigen processing. IMPORTANCE CD4+ helper T cells play an essential role in protection against HIV and other pathogens. Thus, the sites of helper T-cell recognition, the dominant epitopes, are targets for vaccine design; and the corresponding T cells may provide markers for monitoring infection and immunity. However, T-cell epitopes are difficult to identify and predict. It is also unclear whether CD4+ T cells specific for one epitope are more protective than T cells specific for other epitopes. This work shows that the three-dimensional (3D) structure of an

  19. Glycoproteins: Occurrence and Significance

    NASA Astrophysics Data System (ADS)

    Wittmann, Valentin

    Protein glycosylation is regarded as the most complex form of post-translational modification leading to a heterogeneous expression of glycoproteins as mixtures of glycoforms. This chapter describes the structure and occurrence of glycoproteins with respect to their glycan chains. Discussed are different carbohydrate-peptide linkages including GPI anchors, common structures of N- and O-glycans, and the structure of glycosaminoglycans contained in proteoglycans. Also covered are the bacterial cell wall polymer peptidoglycan and the glycopeptide antibiotics of the vancomycin group. Properties and functions of the glycans contained in glycoproteins are dealt with in the next chapter of this book.

  20. Are integrin alpha(2)beta(1), glycoprotein Ib and vWf levels correlated with their contributions to platelet adhesion on collagen under high-shear flow?

    PubMed

    Jung, Stephanie M; Sonoda, Mamiko; Tsuji, Kayoko; Jimi, Atsuo; Nomura, Shosaku; Kanaji, Taisuke; Moroi, Masaaki

    2010-01-01

    Platelets in flowing blood at high-shear stress are recruited to exposed subendothelial collagen of injured vessels by GPIb-von Willebrand factor (vWf) and integrin alpha(2)beta(1) (alpha(2)beta(1))-collagen interactions. Platelet adhesion to type I collagen depends mainly on the alpha(2)beta(1)-collagen interaction and that to type III collagen depends on the GPIb-vWf interaction due to vWf's weak affinity for type I collagen. Contributions of these two interactions would differ depending on expressions of alpha(2)beta(1), vWf, or GPIb. We quantitated platelet adhesion to low- and high-density collagen under high-shear flow conditions in the presence of anti-alpha(2)beta(1) (Gi9) and anti-GPIb (NNKY5-5) antibodies to determine if their inhibitory effects were correlated with the amounts of alpha(2)beta(1), GPIb and vWf. Gi9 inhibition of adhesion to type I collagen was decreased in platelets with more integrin alpha(2)beta(1). Gi9 and NNKY5-5 are more inhibitory against adhesion to low-density type III and I, respectively. Higher alpha(2)beta(1) expression decreases adhesion to low-density type III and increases Gi9 inhibition of adhesion to high-density type III, suggesting crosstalk between the alpha(2)beta(1)-collagen and GPIb-vWf interactions in adhesion to type III. Integrin alpha(2)beta(1)-collagen and GPIb-vWf interactions both contribute to platelet adhesion to collagen under high-shear flow. In adhesion under high-shear stress, the two interactions would compensate for each other, when there is a deficiency in one or the other. The alpha(2)beta(1)-collagen interaction was also suggested to have an inhibitory effect on platelet adhesion to type III collagen, through a yet undefined mechanism.

  1. Bloch spin waves and emergent structure in protein folding with HIV envelope glycoprotein as an example

    NASA Astrophysics Data System (ADS)

    Dai, Jin; Niemi, Antti J.; He, Jianfeng; Sieradzan, Adam; Ilieva, Nevena

    2015-11-01

    We inquire how structure emerges during the process of protein folding. For this we scrutinise col- lective many-atom motions during all-atom molecular dynamics simulations. We introduce, develop and employ various topological techniques, in combination with analytic tools that we deduce from the concept of integrable models and structure of discrete nonlinear Schroedinger equation. The example we consider is an alpha-helical subunit of the HIV envelope glycoprotein gp41. The helical structure is stable when the subunit is part of the biological oligomer. But in isolation the helix becomes unstable, and the monomer starts deforming. We follow the process computationally. We interpret the evolving structure both in terms of a backbone based Heisenberg spin chain and in terms of a side chain based XY spin chain. We find that in both cases the formation of protein super-secondary structure is akin the formation of a topological Bloch domain wall along a spin chain. During the process we identify three individual Bloch walls and we show that each of them can be modelled with a very high precision in terms of a soliton solution to a discrete nonlinear Schroedinger equation.

  2. Bloch spin waves and emergent structure in protein folding with HIV envelope glycoprotein as an example

    NASA Astrophysics Data System (ADS)

    Dai, Jin; Niemi, Antti J.; He, Jianfeng; Sieradzan, Adam; Ilieva, Nevena

    2016-03-01

    We inquire how structure emerges during the process of protein folding. For this we scrutinize collective many-atom motions during all-atom molecular dynamics simulations. We introduce, develop, and employ various topological techniques, in combination with analytic tools that we deduce from the concept of integrable models and structure of discrete nonlinear Schrödinger equation. The example we consider is an α -helical subunit of the HIV envelope glycoprotein gp41. The helical structure is stable when the subunit is part of the biological oligomer. But in isolation, the helix becomes unstable, and the monomer starts deforming. We follow the process computationally. We interpret the evolving structure both in terms of a backbone based Heisenberg spin chain and in terms of a side chain based XY spin chain. We find that in both cases the formation of protein supersecondary structure is akin the formation of a topological Bloch domain wall along a spin chain. During the process we identify three individual Bloch walls and we show that each of them can be modelled with a precision of tenths to several angstroms in terms of a soliton solution to a discrete nonlinear Schrödinger equation.

  3. What Do Chaotrope-Based Avidity Assays for Antibodies to HIV-1 Envelope Glycoproteins Measure?

    PubMed Central

    Alexander, Marina R.; Ringe, Rajesh; Sanders, Rogier W.; Voss, James E.; Moore, John P.

    2015-01-01

    ABSTRACT When HIV-1 vaccine candidates that include soluble envelope glycoproteins (Env) are tested in humans and other species, the resulting antibody responses to Env are sifted for correlates of protection or risk. One frequently used assay measures the reduction in antibody binding to Env antigens by an added chaotrope (such as thiocyanate). Based on that assay, an avidity index was devised for assessing the affinity maturation of antibodies of unknown concentration in polyclonal sera. Since a high avidity index was linked to protection in animal models of HIV-1 infection, it has become a criterion for evaluating antibody responses to vaccine candidates. But what does the assay measure and what does an avidity index mean? Here, we have used a panel of monoclonal antibodies to well-defined epitopes on Env (gp120, gp41, and SOSIP.664 trimers) to explore how the chaotrope acts. We conclude that the chaotrope sensitivity of antibody binding to Env depends on several properties of the epitopes (continuity versus tertiary- and quaternary-structural dependence) and that the avidity index has no simple relationship to antibody affinity for functional Env spikes on virions. We show that the binding of broadly neutralizing antibodies against quaternary-structural epitopes is particularly sensitive to chaotrope treatment, whereas antibody binding to epitopes in variable loops and to nonneutralization epitopes in gp41 is generally resistant. As a result of such biases, the avidity index may at best be a mere surrogate for undefined antibody or other immune responses that correlate weakly with protection. IMPORTANCE An effective HIV-1 vaccine is an important goal. Such a vaccine will probably need to induce antibodies that neutralize typically transmitted variants of HIV-1, preventing them from infecting target cells. Vaccine candidates have so far failed to induce such antibody responses, although some do protect weakly against infection in animals and, possibly, humans

  4. The V4 and V5 Variable Loops of HIV-1 Envelope Glycoprotein Are Tolerant to Insertion of Green Fluorescent Protein and Are Useful Targets for Labeling.

    PubMed

    Nakane, Shuhei; Iwamoto, Aikichi; Matsuda, Zene

    2015-06-12

    The mature human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) comprises the non-covalently associated gp120 and gp41 subunits generated from the gp160 precursor. Recent structural analyses have provided quaternary structural models for gp120/gp41 trimers, including the variable loops (V1-V5) of gp120. In these models, the V3 loop is located under V1/V2 at the apical center of the Env trimer, and the V4 and V5 loops project outward from the trimeric protomers. In addition, the V4 and V5 loops are predicted to have less movement upon receptor binding during membrane fusion events. We performed insertional mutagenesis using a GFP variant, GFPOPT, placed into the variable loops of HXB2 gp120. This allowed us to evaluate the current structural models and to simultaneously generate a GFP-tagged HIV-1 Env, which was useful for image analyses. All GFP-inserted mutants showed similar levels of whole-cell expression, although certain mutants, particularly V3 mutants, showed lower levels of cell surface expression. Functional evaluation of their fusogenicities in cell-cell and virus-like particle-cell fusion assays revealed that V3 was the most sensitive to the insertion and that the V1/V2 loops were less sensitive than V3. The V4 and V5 loops were the most tolerant to insertion, and certain tag proteins other than GFPOPT could also be inserted without functional consequences. Our results support the current structural models and provide a GFPOPT-tagged Env construct for imaging studies.

  5. The V4 and V5 Variable Loops of HIV-1 Envelope Glycoprotein Are Tolerant to Insertion of Green Fluorescent Protein and Are Useful Targets for Labeling*

    PubMed Central

    Nakane, Shuhei; Iwamoto, Aikichi; Matsuda, Zene

    2015-01-01

    The mature human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) comprises the non-covalently associated gp120 and gp41 subunits generated from the gp160 precursor. Recent structural analyses have provided quaternary structural models for gp120/gp41 trimers, including the variable loops (V1–V5) of gp120. In these models, the V3 loop is located under V1/V2 at the apical center of the Env trimer, and the V4 and V5 loops project outward from the trimeric protomers. In addition, the V4 and V5 loops are predicted to have less movement upon receptor binding during membrane fusion events. We performed insertional mutagenesis using a GFP variant, GFPOPT, placed into the variable loops of HXB2 gp120. This allowed us to evaluate the current structural models and to simultaneously generate a GFP-tagged HIV-1 Env, which was useful for image analyses. All GFP-inserted mutants showed similar levels of whole-cell expression, although certain mutants, particularly V3 mutants, showed lower levels of cell surface expression. Functional evaluation of their fusogenicities in cell-cell and virus-like particle-cell fusion assays revealed that V3 was the most sensitive to the insertion and that the V1/V2 loops were less sensitive than V3. The V4 and V5 loops were the most tolerant to insertion, and certain tag proteins other than GFPOPT could also be inserted without functional consequences. Our results support the current structural models and provide a GFPOPT-tagged Env construct for imaging studies. PMID:25911103

  6. Glycoprotein biosynthesis in calf kidney. Glycoprotein sialyltransferase activities towards serum glycoproteins and calf Tamm-Horsfall glycoprotein.

    PubMed

    van Dijk, W; Lasthuis, A M; van den Eijnden, D H

    1979-04-18

    CMP-AcNeu:glycoprotein sialyltransltransltransltransltransferase of calf kidney cortex was characterized using serum glycoproteins and Tamm-Horsfall glycoprotein, obtained from calf urine, as acceptors. Native calf Tamm-Horsfall glycoprotein showed the best acceptor properties, followed by desialylated calf fetuin and desialylated human alpha 1-acid glycoprotein exhibiting V values of, respectively, 114, 63 and 41 nmol/h per g wet wt. of kidney cortex and Km values of 0.12, 0.16 and 0.26 mM glycoprotein acceptor. Desialylated ovine submaxillary mucine appeared to be a very poor acceptor. Tamm-Horsfall glycoprotein sialyltransferase could be distinguished from serum glycoprotein sialyltransferase by competition studies. In addition the two glycoprotein sialyltransferase activities showed different distributions over the three regions of the calf kidney: the ratios of the Tamm-Horsfall to serum glycoprotein sialyltransferase activities decreased from 3.3 in the cortex to 0.8 and 0.4 in the medulla and the papilla, respectively. It was concluded that in calf kidney at least two different sialyltransferases exist. The high cortical Tamm-Horsfall glycoprotein sialyltransferases activity corresponds markedly to the origin of the urinary Tamm-Horsfall glycoprotein, namely the distal part of the kidney tubule. Inactivation of glycoprotein sialyltransferase activity by preincubation at various temperatures and during storage at 0 degree C, could be reduced by the addition of CMP-AcNeu. The possible relevance towards the in vivo sialylation of this finding is discussed.

  7. Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140

    PubMed Central

    Go, Eden P.; Herschhorn, Alon; Gu, Christopher; Castillo-Menendez, Luis; Zhang, Shijian; Mao, Youdong; Chen, Haiyan; Ding, Haitao; Wakefield, John K.; Hua, David; Liao, Hua-Xin; Kappes, John C.; Sodroski, Joseph

    2015-01-01

    ABSTRACT The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, which consists of the gp120 and gp41 subunits, is the focus of multiple strategies for vaccine development. Extensive Env glycosylation provides HIV-1 with protection from the immune system, yet the glycans are also essential components of binding epitopes for numerous broadly neutralizing antibodies. Recent studies have shown that when Env is isolated from virions, its glycosylation profile differs significantly from that of soluble forms of Env (gp120 or gp140) predominantly used in vaccine discovery research. Here we show that exogenous membrane-anchored Envs, which can be produced in large quantities in mammalian cells, also display a virion-like glycan profile, where the glycoprotein is extensively decorated with high-mannose glycans. Additionally, because we characterized the glycosylation with a high-fidelity profiling method, glycopeptide analysis, an unprecedented level of molecular detail regarding membrane Env glycosylation and its heterogeneity is presented. Each glycosylation site was characterized individually, with about 500 glycoforms characterized per Env protein. While many of the sites contain exclusively high-mannose glycans, others retain complex glycans, resulting in a glycan profile that cannot currently be mimicked on soluble gp120 or gp140 preparations. These site-level studies are important for understanding antibody-glycan interactions on native Env trimers. Additionally, we report a newly observed O-linked glycosylation site, T606, and we show that the full O-linked glycosylation profile of membrane-associated Env is similar to that of soluble gp140. These findings provide new insight into Env glycosylation and clarify key molecular-level differences between membrane-anchored Env and soluble gp140. IMPORTANCE A vaccine that protects against human immunodeficiency virus type 1 (HIV-1) infection should elicit antibodies that bind to the surface

  8. HIV-1 envelope glycoprotein

    DOEpatents

    Caulfield, Michael; Cupo, Albert; Dean, Hansi; Hoffenberg, Simon; King, C. Richter; Klasse, P. J.; Marozsan, Andre; Moore, John P.; Sanders, Rogier W.; Ward, Andrew; Wilson, Ian; Julien, Jean-Philippe

    2017-08-22

    The present application relates to novel HIV-1 envelope glycoproteins, which may be utilized as HIV-1 vaccine immunogens, and antigens for crystallization, electron microscopy and other biophysical, biochemical and immunological studies for the identification of broad neutralizing antibodies. The present invention encompasses the preparation and purification of immunogenic compositions, which are formulated into the vaccines of the present invention.

  9. Residues in the membrane-spanning domain core modulate conformation and fusogenicity of the HIV-1 envelope glycoprotein

    SciTech Connect

    Shang Liang; Hunter, Eric

    2010-09-01

    The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant 'core' structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD 'core' do not influence specific virus-cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD 'core' delayed the kinetics and reduced the efficiency of cell-cell fusion mediated by Env. Although we observed no evidence that membrane fusion mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process.

  10. Release of gp120 Restraints Leads to an Entry-Competent Intermediate State of the HIV-1 Envelope Glycoproteins

    PubMed Central

    Ma, Xiaochu; Gu, Christopher; Ventura, John D.; Castillo-Menendez, Luis; Melillo, Bruno; Terry, Daniel S.; Smith, Amos B.; Blanchard, Scott C.; Munro, James B.; Mothes, Walther; Finzi, Andrés

    2016-01-01

    ABSTRACT Primary human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimers [(gp120/gp41)3] typically exist in a metastable closed conformation (state 1). Binding the CD4 receptor triggers Env to undergo extensive conformational changes to mediate virus entry. We identified specific gp120 residues that restrain Env in state 1. Alteration of these restraining residues destabilized state 1, allowing Env to populate a functional conformation (state 2) intermediate between state 1 and the full CD4-bound state (state 3). Increased state 2 occupancy was associated with lower energy barriers between the states. State 2 was an obligate intermediate for all transitions between state 1 and state 3. State 2-enriched Envs required lower CD4 concentrations to trigger virus entry and more efficiently infected cells expressing low levels of CD4. These Envs were resistant to several broadly neutralizing antibodies and small-molecule inhibitors. Thus, state 2 is an Env conformation on the virus entry pathway; sampling state 2 increases the adaptability of HIV-1 to different host cell receptor levels and immune environments. Our results provide new insights into the conformational regulation of HIV-1 entry. PMID:27795397

  11. Structure–Activity Relationship Studies of Indole-Based Compounds as Small Molecule HIV-1 Fusion Inhibitors Targeting Glycoprotein 41

    PubMed Central

    2015-01-01

    We previously described indole-containing compounds with the potential to inhibit HIV-1 fusion by targeting the hydrophobic pocket of transmembrane glycoprotein gp41. Here we report optimization and structure–activity relationship studies on the basic scaffold, defining the role of shape, contact surface area, and molecular properties. Thirty new compounds were evaluated in binding, cell–cell fusion, and viral replication assays. Below a 1 μM threshold, correlation between binding and biological activity was diminished, indicating an amphipathic requirement for activity in cells. The most active inhibitor 6j exhibited 0.6 μM binding affinity and 0.2 μM EC50 against cell–cell fusion and live virus replication and was active against T20 resistant strains. Twenty-two compounds with the same connectivity displayed a consensus pose in docking calculations, with rank order matching the biological activity. The work provides insight into requirements for small molecule inhibition of HIV-1 fusion and demonstrates a potent low molecular weight fusion inhibitor. PMID:24856833

  12. NMR-assisted computational studies of peptidomimetic inhibitors bound in the hydrophobic pocket of HIV-1 glycoprotein 41

    PubMed Central

    Gochin, Miriam; Whitby, Landon R.; Phillips, Aaron H.; Boger, Dale L.

    2013-01-01

    Due to the inherently flexible nature of a protein – protein interaction surface, it is difficult both to inhibit the association with a small molecule, and to predict how it might bind to the surface. In this study, we have examined small molecules that mediate the interaction between a WWI motif on the C-helix of HIV-1 glycoprotein-41 and a deep hydrophobic pocket contained in the interior N-helical trimer. Association between these two components of gp41 leads to virus–cell and cell–cell fusion, which could be abrogated in the presence of an inhibitor that binds tightly in the pocket. We have studied a comprehensive combinatorial library of α-helical peptidomimetics, and found that compounds with strongly hydrophobic side chains had the highest affinity. Computational docking studies produced multiple possible binding modes due to the flexibility of both the binding site and the peptidomimetic compounds. We applied a transferred paramagnetic relaxation enhancement (PRE) experiment to two selected members of the library, and showed that addition of a few experimental constraints enabled definitive identification of unique binding poses. Computational docking results were extremely sensitive to side chain conformations, and slight variations could preclude observation of the experimentally validated poses. Different receptor structures were required for docking simulations to sample the correct pose for the two compounds. The study demonstrated the sensitivity of predicted poses to receptor structure and indicated the importance of experimental verification when docking to a malleable protein – protein interaction surface. PMID:23893342

  13. Egg envelope glycoprotein gp37 as a Xenopus homolog of mammalian ZP1, based on cDNA cloning.

    PubMed

    Kubo, H; Kawano, T; Tsubuki, S; Kotani, M; Kawasaki, H; Kawashima, S

    2000-08-01

    The egg envelope is a kind of extracellular matrix, which surrounds growing oocytes, ovulated eggs and early embryos. Among the glycoprotein components of the Xenopus laevis egg envelope, gp43/gp41 and gp69/64 have already been shown to be frog homologs of the mammalian zona pellucida components ZP3 and ZP2, respectively. To determine the structure of another major component of egg envelope, gp37, the peptides isolated from the lysyl endopeptidase digests of gp37 were sequenced for amino acids to design degenerate primers for polymerase chain reaction. By reverse transcription-polymerase chain reaction with a poly(A)+ RNA from the ovary of a postovulated female Xenopus, a specifically amplified band was obtained and sequenced. The upstream and downstream sequences of the sequenced region were completed by 5'- and 3'-rapid amplification of cDNA ends, respectively. The gp37 cDNA comprises 1674 bp and contains one open reading frame encoding a polypeptide with 543 amino acids. The predicted amino acid sequence of the gp37 cDNA has a close similarity to that of mammalian ZP1. Northern blot and in situ hybridization studies indicated that the transcript (1.8 kb) is exclusively expressed in the oocytes, particularly in the previtellogenic young oocytes, just like the expression pattern of gp43 mRNA, suggesting a coordinate transcription of the gp43 and gp37 genes in Xenopus.

  14. Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies.

    PubMed

    Gnanakaran, S; Daniels, Marcus G; Bhattacharya, Tanmoy; Lapedes, Alan S; Sethi, Anurag; Li, Ming; Tang, Haili; Greene, Kelli; Gao, Hongmei; Haynes, Barton F; Cohen, Myron S; Shaw, George M; Seaman, Michael S; Kumar, Amit; Gao, Feng; Montefiori, David C; Korber, Bette

    2010-10-07

    A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for

  15. Genetic Signatures in the Envelope Glycoproteins of HIV-1 that Associate with Broadly Neutralizing Antibodies

    PubMed Central

    Gnanakaran, S.; Daniels, Marcus G.; Bhattacharya, Tanmoy; Lapedes, Alan S.; Sethi, Anurag; Li, Ming; Tang, Haili; Greene, Kelli; Gao, Hongmei; Haynes, Barton F.; Cohen, Myron S.; Shaw, George M.; Seaman, Michael S.; Kumar, Amit; Gao, Feng; Montefiori, David C.; Korber, Bette

    2010-01-01

    A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for

  16. A Mechanistic Understanding of Allosteric Immune Escape Pathways in the HIV-1 Envelope Glycoprotein

    PubMed Central

    Sethi, Anurag; Tian, Jianhui; Derdeyn, Cynthia A.; Korber, Bette; Gnanakaran, S.

    2013-01-01

    The HIV-1 envelope (Env) spike, which consists of a compact, heterodimeric trimer of the glycoproteins gp120 and gp41, is the target of neutralizing antibodies. However, the high mutation rate of HIV-1 and plasticity of Env facilitates viral evasion from neutralizing antibodies through various mechanisms. Mutations that are distant from the antibody binding site can lead to escape, probably by changing the conformation or dynamics of Env; however, these changes are difficult to identify and define mechanistically. Here we describe a network analysis-based approach to identify potential allosteric immune evasion mechanisms using three known HIV-1 Env gp120 protein structures from two different clades, B and C. First, correlation and principal component analyses of molecular dynamics (MD) simulations identified a high degree of long-distance coupled motions that exist between functionally distant regions within the intrinsic dynamics of the gp120 core, supporting the presence of long-distance communication in the protein. Then, by integrating MD simulations with network theory, we identified the optimal and suboptimal communication pathways and modules within the gp120 core. The results unveil both strain-dependent and -independent characteristics of the communication pathways in gp120. We show that within the context of three structurally homologous gp120 cores, the optimal pathway for communication is sequence sensitive, i.e. a suboptimal pathway in one strain becomes the optimal pathway in another strain. Yet the identification of conserved elements within these communication pathways, termed inter-modular hotspots, could present a new opportunity for immunogen design, as this could be an additional mechanism that HIV-1 uses to shield vulnerable antibody targets in Env that induce neutralizing antibody breadth. PMID:23696718

  17. Dicalcin Inhibits Fertilization through Its Binding to a Glycoprotein in the Egg Envelope in Xenopus laevis*

    PubMed Central

    Miwa, Naofumi; Ogawa, Motoyuki; Shinmyo, Yukiko; Hiraoka, Yoshiki; Takamatsu, Ken; Kawamura, Satoru

    2010-01-01

    Fertilization comprises oligosaccharide-mediated sperm-egg interactions, including sperm binding to an extracellular egg envelope, sperm penetration through the envelope, and fusion with an egg plasma membrane. We show that Xenopus dicalcin, an S100-like Ca2+-binding protein, present in the extracellular egg envelope (vitelline envelope (VE)), is a suppressive mediator of sperm-egg interaction. Preincubation with specific antibody greatly increased the efficiency of in vitro fertilization, whereas prior application of exogenous dicalcin substantially inhibited fertilization as well as sperm binding to an egg and in vitro sperm penetration through the VE protein layer. Dicalcin showed binding to protein cores of gp41 and gp37, constituents of VE, in a Ca2+-dependent manner and increased in vivo reactivity of VE with a lectin, Ricinus communis agglutinin I, which was accounted for by increased binding ability of gp41 to the lectin and greater exposure of gp41 to an external environment. Our findings strongly suggest that dicalcin regulates the distribution of oligosaccharides within the VE through its binding to the protein core of gp41, probably by modulating configuration of oligosaccharides on gp41 and the three-dimensional structure of VE framework, and thereby plays a pivotal role in sperm-egg interactions during fertilization. PMID:20299459

  18. Envelope glycoprotein of arenaviruses.

    PubMed

    Burri, Dominique J; da Palma, Joel Ramos; Kunz, Stefan; Pasquato, Antonella

    2012-10-17

    Arenaviruses include lethal human pathogens which pose serious public health threats. So far, no FDA approved vaccines are available against arenavirus infections, and therapeutic options are limited, making the identification of novel drug targets for the development of efficacious therapeutics an urgent need. Arenaviruses are comprised of two RNA genome segments and four proteins, the polymerase L, the envelope glycoprotein GP, the matrix protein Z, and the nucleoprotein NP. A crucial step in the arenavirus life-cycle is the biosynthesis and maturation of the GP precursor (GPC) by cellular signal peptidases and the cellular enzyme Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P) yielding a tripartite mature GP complex formed by GP1/GP2 and a stable signal peptide (SSP). GPC cleavage by SKI-1/S1P is crucial for fusion competence and incorporation of mature GP into nascent budding virion particles. In a first part of our review, we cover basic aspects and newer developments in the biosynthesis of arenavirus GP and its molecular interaction with SKI-1/S1P. A second part will then highlight the potential of SKI-1/S1P-mediated processing of arenavirus GPC as a novel target for therapeutic intervention to combat human pathogenic arenaviruses.

  19. Folding of synthetic homogeneous glycoproteins in the presence of a glycoprotein folding sensor enzyme.

    PubMed

    Dedola, Simone; Izumi, Masayuki; Makimura, Yutaka; Seko, Akira; Kanamori, Akiko; Sakono, Masafumi; Ito, Yukishige; Kajihara, Yasuhiro

    2014-03-10

    UDP-glucose:glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded glycoproteins in the glycoprotein quality control system of the endoplasmic reticulum. UGGT detects misfolded glycoproteins and re-glucosylates them as a tag for misfolded glycoproteins. A flexible model to reproduce in vitro folding of a glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded glycoproteins is described. The data demonstrates that UGGT can re-glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the glycoprotein folding intermediates.

  20. Salivary Mucin 19 Glycoproteins

    PubMed Central

    Culp, David J.; Robinson, Bently; Cash, Melanie N.; Bhattacharyya, Indraneel; Stewart, Carol; Cuadra-Saenz, Giancarlo

    2015-01-01

    Saliva functions in innate immunity of the oral cavity, protecting against demineralization of teeth (i.e. dental caries), a highly prevalent infectious disease associated with Streptococcus mutans, a pathogen also linked to endocarditis and atheromatous plaques. Gel-forming mucins are a major constituent of saliva. Because Muc19 is the dominant salivary gel-forming mucin in mice, we studied Muc19−/− mice for changes in innate immune functions of saliva in interactions with S. mutans. When challenged with S. mutans and a cariogenic diet, total smooth and sulcal surface lesions are more than 2- and 1.6-fold higher in Muc19−/− mice compared with wild type, whereas the severity of lesions are up to 6- and 10-fold higher, respectively. Furthermore, the oral microbiota of Muc19−/− mice display higher levels of indigenous streptococci. Results emphasize the importance of a single salivary constituent in the innate immune functions of saliva. In vitro studies of S. mutans and Muc19 interactions (i.e. adherence, aggregation, and biofilm formation) demonstrate Muc19 poorly aggregates S. mutans. Nonetheless, aggregation is enhanced upon adding Muc19 to saliva from Muc19−/− mice, indicating Muc19 assists in bacterial clearance through formation of heterotypic complexes with salivary constituents that bind S. mutans, thus representing a novel innate immune function for salivary gel-forming mucins. In humans, expression of salivary MUC19 is unclear. We find MUC19 transcripts in salivary glands of seven subjects and demonstrate MUC19 glycoproteins in glandular mucous cells and saliva. Similarities and differences between mice and humans in the expression and functions of salivary gel-forming mucins are discussed. PMID:25512380

  1. CD4-Induced Antibodies Promote Association of the HIV-1 Envelope Glycoprotein with CD4-Binding Site Antibodies

    PubMed Central

    Fellinger, Christoph H.; Prasad, Neha R.; Zhou, Amber S.; Kondur, Hema R.; Joshi, Vinita R.; Quinlan, Brian D.; Farzan, Michael

    2016-01-01

    ABSTRACT The HIV-1 envelope glycoprotein (Env) is a trimer of gp120/gp41 heterodimers that mediates viral entry. Env binds cellular CD4, an association which stabilizes a conformation favorable to its subsequent association with a coreceptor, typically CCR5 or CXCR4. The CD4- and coreceptor-binding sites serve as epitopes for two classes of HIV-1-neutralizing antibodies: CD4-binding site (CD4bs) and CD4-induced (CD4i) antibodies, respectively. Here we observed that, at a fixed total concentration, mixtures of the CD4i antibodies (E51 or 412d) and the CD4bs antibody VRC01 neutralized the HIV-1 isolates 89.6, ADA, SG3, and SA32 more efficiently than either antibody alone. We found that E51, and to a lesser extent 412d and 17b, promoted association of four CD4bs antibodies to the Env trimer but not to monomeric gp120. We further demonstrated that the binding of the sulfotyrosine-binding pocket by CCR5mim2-Ig was sufficient for promoting CD4bs antibody binding to Env. Interestingly, the relationship is not reciprocal: CD4bs antibodies were not as efficient as CD4-Ig at promoting E51 or 412d binding to Env trimer. Consistent with these observations, CD4-Ig, but none of the CD4bs antibodies tested, substantially increased HIV-1 infection of a CD4-negative, CCR5-positive cell line. We conclude that the ability of CD4i antibodies to promote VRC01 association with Env trimers accounts for the increase potency of VRC01 and CD4i antibody mixtures. Our data further suggest that potent CD4bs antibodies avoid inducing Env conformations that bind CD4i antibodies or CCR5. IMPORTANCE Potent HIV-1-neutralizing antibodies can prevent viral transmission and suppress an ongoing infection. Here we show that CD4-induced (CD4i) antibodies, which recognize the conserved coreceptor-binding site of the HIV-1 envelope glycoprotein (Env), can increase the association of Env with potent broadly neutralizing antibodies that recognize the CD4-binding site (CD4bs antibodies). We further show that

  2. Variable Constraints on the Principal Immunodominant Domain of the Transmembrane Glycoprotein of Human Immunodeficiency Virus Type 1

    PubMed Central

    Merat, Rastine; Raoul, Herve; Leste-Lasserre, Thierry; Sonigo, Pierre; Pancino, Gianfranco

    1999-01-01

    Lentiviruses have in their transmembrane glycoprotein (TM) a highly immunogenic structure referred to as the principal immunodominant domain (PID). The PID forms a loop of 5 to 7 amino acids between two conserved cysteines. Previous studies showed that envelope (Env) glycoprotein functions of feline immunodeficiency virus (FIV) could be retained after extensive mutation of the PID loop sequence, in spite of its high conservation. In order to compare Env function in different lentiviruses, either random mutations were introduced in the PID loop sequence of human immunodeficiency virus type 1 (HIV-1) or the entire HIV-1 PID loop was replaced by the corresponding PID loop of FIV or simian immunodeficiency virus (SIV). In the macrophage-tropic HIV-1 ADA Env, mutations impaired the processing of the gp160 Env precursor, thereby abolishing viral infectivity. However, 6 of the 108 random Env mutants that were screened retained the capacity to induce cell membrane fusion. The SIV and FIV sequences and five random mutations were then introduced in the context of T-cell-line-adapted HIV-1 LAI which, although phenotypically distant from HIV-1 ADA, has an identical PID loop sequence. In contrast to the situation for HIV-1 ADA mutants, the cleavage of the Env precursor was unaffected in most HIV-1 LAI mutants. Such mutations, however, resulted in increased shedding of the gp120 surface glycoprotein (SU) from the gp41 TM. The HIV-1 LAI Env mutants showed high fusogenic efficiency. Three Env mutants retained the capacity to mediate virus entry in target cells, although less efficiently than the wild-type Env, and allowed the reconstitution of infectious molecular clones. These results indicated that in HIV-1, like FIV, the conserved PID sequence can be changed without impairing Env function. However, functional constraints on the PID of HIV-1 vary depending on the structural context of Env, presumably in relation to the role of the PID in the interaction of the SU and TM subunits

  3. Anti-glycoprotein g antibodies of herpes simplex virus 2 contribute to complete protection after vaccination in mice and induce antibody-dependent cellular cytotoxicity and complement-mediated cytolysis.

    PubMed

    Görander, Staffan; Ekblad, Maria; Bergström, Tomas; Liljeqvist, Jan-Åke

    2014-11-12

    We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2) of herpes simplex virus 2 (HSV-2) in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1) and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (ACMC) activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials.

  4. Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

    SciTech Connect

    Guan, Yongjun; Pazgier, Marzena; Sajadi, Mohammad M.; Kamin-Lewis, Roberta; Al-Darmarki, Salma; Flinko, Robin; Lovo, Elena; Wu, Xueji; Robinson, James E.; Seaman, Michael S.; Fouts, Timothy R.; Gallo, Robert C.; DeVico, Anthony L.; Lewis, George K.

    2012-12-13

    The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. In conclusion, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.

  5. Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

    DOE PAGES

    Guan, Yongjun; Pazgier, Marzena; Sajadi, Mohammad M.; ...

    2012-12-13

    The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain;more » and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. In conclusion, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.« less

  6. Functional stability of unliganded envelope glycoprotein spikes among isolates of human immunodeficiency virus type 1 (HIV-1).

    PubMed

    Agrawal, Nitish; Leaman, Daniel P; Rowcliffe, Eric; Kinkead, Heather; Nohria, Raman; Akagi, Junya; Bauer, Katherine; Du, Sean X; Whalen, Robert G; Burton, Dennis R; Zwick, Michael B

    2011-01-01

    The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T(90) values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34). For select Envs (n = 10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T(90) (p = 0.029), though two 'outliers' were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1(ADA) was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1(JR-CSF). Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines.

  7. Functional Stability of Unliganded Envelope Glycoprotein Spikes among Isolates of Human Immunodeficiency Virus Type 1 (HIV-1)

    PubMed Central

    Kinkead, Heather; Nohria, Raman; Akagi, Junya; Bauer, Katherine; Du, Sean X.; Whalen, Robert G.; Burton, Dennis R.; Zwick, Michael B.

    2011-01-01

    The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T90 values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34). For select Envs (n = 10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T90 (p = 0.029), though two ‘outliers’ were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1ADA was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1JR-CSF. Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines. PMID:21738637

  8. Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

    PubMed Central

    Sarkar, Pampi; Labranche, Celia; Go, Eden P.; Clark, Daniel F.; Sun, Yide; Nandi, Avishek; Hartog, Karin; Desaire, Heather; Montefiori, David; Carfi, Andrea; Srivastava, Indresh K.; Barnett, Susan W.

    2013-01-01

    Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules. PMID:24146829

  9. Glycan analysis of therapeutic glycoproteins

    PubMed Central

    Zhang, Lei; Luo, Shen; Zhang, Baolin

    2016-01-01

    ABSTRACT Therapeutic monoclonal antibodies (mAbs) are glycoproteins produced by living cell systems. The glycan moieties attached to the proteins can directly affect protein stability, bioactivity, and immunogenicity. Therefore, glycan variants of a glycoprotein product must be adequately analyzed and controlled to ensure product quality. However, the inherent complexity of protein glycosylation poses a daunting analytical challenge. This review provides an update of recent advances in glycan analysis, including the potential utility of lectin-based microarray for high throughput glycan profiling. Emphasis is placed on comparison of the major types of analytics for use in determining unique glycan features such as glycosylation site, glycan structure, and content. PMID:26599345

  10. Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins.

    PubMed

    Lentz, T L; Wilson, P T; Hawrot, E; Speicher, D W

    1984-11-16

    Evidence was presented earlier that a host-cell receptor for the highly neurotropic rabies virus might be the acetylcholine receptor. The amino acid sequence of the glycoprotein of rabies virus was compared by computer analysis with that of snake venom curaremimetic neurotoxins, potent ligands of the acetylcholine receptor. A statistically significant sequence relation was found between a segment of the rabies glycoprotein and the entire sequence of long neurotoxins. The greatest identity occurs with residues considered most important in neurotoxicity, including those interacting with the acetylcholine binding site of the acetylcholine receptor. Because of the similarity between the glycoprotein and the receptor-binding region of the neurotoxins, this region of the viral glycoprotein may function as a recognition site for the acetylcholine receptor. Direct binding of the rabies virus glycoprotein to the acetylcholine receptor could contribute to the neurotropism of this virus.

  11. Inflammatory glycoproteins in cardiometabolic disorders, autoimmune diseases and cancer.

    PubMed

    Connelly, Margery A; Gruppen, Eke G; Otvos, James D; Dullaart, Robin P F

    2016-08-01

    The physiological function initially attributed to the oligosaccharide moieties or glycans on inflammatory glycoproteins was to improve protein stability. However, it is now clear that glycans play a prominent role in glycoprotein structure and function and in some cases contribute to disease states. In fact, glycan processing contributes to pathogenicity not only in autoimmune disorders but also in atherosclerotic cardiovascular disease, diabetes and malignancy. While most clinical laboratory tests measure circulating levels of inflammatory proteins, newly developed diagnostic and prognostic tests are harvesting the information that can be gleaned by measuring the amount or structure of the attached glycans, which may be unique to individuals as well as various diseases. As such, these newer glycan-based tests may provide future means for more personalized approaches to patient stratification and improved patient care. Here we will discuss recent progress in high-throughput laboratory methods for glycomics (i.e. the study of glycan structures) and glycoprotein quantification by methods such as mass spectrometry and nuclear magnetic resonance spectroscopy. We will also review the clinical utility of glycoprotein and glycan measurements in the prediction of common low-grade inflammatory disorders including cardiovascular disease, diabetes and cancer, as well as for monitoring autoimmune disease activity.

  12. Recent Progress in Electrochemical Biosensors for Glycoproteins

    PubMed Central

    Akiba, Uichi; Anzai, Jun-ichi

    2016-01-01

    This review provides an overview of recent progress in the development of electrochemical biosensors for glycoproteins. Electrochemical glycoprotein sensors are constructed by combining metal and carbon electrodes with glycoprotein-selective binding elements including antibodies, lectin, phenylboronic acid and molecularly imprinted polymers. A recent trend in the preparation of glycoprotein sensors is the successful use of nanomaterials such as graphene, carbon nanotube, and metal nanoparticles. These nanomaterials are extremely useful for improving the sensitivity of glycoprotein sensors. This review focuses mainly on the protocols for the preparation of glycoprotein sensors and the materials used. Recent improvements in glycoprotein sensors are discussed by grouping the sensors into several categories based on the materials used as recognition elements. PMID:27916961

  13. Effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins

    SciTech Connect

    Schwarz, P.M.; Elbein, A.D.

    1985-11-25

    Influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. To determine what structural features of the oligosaccharide were required for fucosylation influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, (5,6-TH)fucose and (1- UC)mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the ( UC)mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the ( UC)mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the (TH)fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the ( UC)mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure.

  14. A sweet code for glycoprotein folding.

    PubMed

    Caramelo, Julio J; Parodi, Armando J

    2015-11-14

    Glycoprotein synthesis is initiated in the endoplasmic reticulum (ER) lumen upon transfer of a glycan (Glc3Man9GlcNAc2) from a lipid derivative to Asn residues (N-glycosylation). N-Glycan-dependent quality control of glycoprotein folding in the ER prevents exit to Golgi of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones (calnexin and calreticulin) that recognize monoglucosylated polymannose protein-linked glycans, lectin-associated oxidoreductase acting on monoglucosylated glycoproteins (ERp57), a glucosyltransferase that creates monoglucosylated epitopes in protein-linked glycans (UGGT) and a glucosidase (GII) that removes the glucose units added by UGGT. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded glycoproteins or in not completely assembled multimeric glycoprotein complexes. Glycoproteins that fail to properly fold are eventually driven to proteasomal degradation in the cytosol following the ER-associated degradation pathway, in which the extent of N-glycan demannosylation by ER mannosidases play a relevant role in the identification of irreparably misfolded glycoproteins.

  15. Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins.

    PubMed

    Sheppard, Neil C; Davies, Sarah L; Jeffs, Simon A; Vieira, Sueli M; Sattentau, Quentin J

    2007-02-01

    Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.

  16. Dominant-negative effect of hetero-oligomerization on the function of the human immunodeficiency virus type 1 envelope glycoprotein complex

    SciTech Connect

    Herrera, Carolina; Klasse, Per Johan; Kibler, Christopher W.; Michael, Elizabeth; Moore, John P. . E-mail: jpm2003@med.cornell.edu; Beddows, Simon

    2006-07-20

    The human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein forms trimers that mediate interactions with the CD4 receptor and a co-receptor on the target cell surface, thereby triggering viral fusion with the cell membrane. Cleavage of Env into its surface, gp120, and transmembrane, gp41, moieties is necessary for activation of its fusogenicity. Here, we produced pseudoviruses with phenotypically mixed wild-type (Wt) and mutant, cleavage-incompetent Env in order to quantify the effects of incorporating uncleaved Env on virion infectivity, antigenicity and neutralization sensitivity. We modeled the relative infectivity of three such phenotypically mixed viral strains, JR-FL, HXBc2 and a derivative of the latter, 3.2P, as a function of the relative amount of Wt Env. The data were fit very closely (R {sup 2} > 0.99) by models which assumed that only Wt homotrimers were functional, with different approximate thresholds of critical numbers of functional trimers per virion for the three strains. We also produced 3.2P pseudoviruses containing both a cleavage-competent Env that is defective for binding the neutralizing monoclonal antibody (NAb) 2G12, and a cleavage-incompetent Env that binds 2G12. The 2G12 NAb was not able to reduce the infectivity of these pseudoviruses detectably. Their neutralization by the CD4-binding site-directed agents CD4-IgG2 and NAb b12 was also unaffected by 2G12 binding to uncleaved Env. These results further strengthen the conclusion that only homotrimers consisting of cleaved Env are functional. They also imply that the function of a trimer is unaffected sterically by the binding of an antibody to an adjacent trimer.

  17. Circular dichroism of erythrocyte membrane glycoproteins.

    PubMed

    Decker, R V; Carraway, K L

    1975-03-28

    The circular dichroism spectra were obtained for purified equine, human and bovine membrane glycoproteins, which have 40, 55 and 70% carbohydrate, respectively. The spectra in aqueous buffer show similar shapes, maxima and minima but somewhat different peak amplitudes. Analysis of the spectra indicated that the glycoproteins can be pictured as existing primarily in an unordered form in dilute aqueous buffer with small amounts of alpha-helix (13-23%) present. In 2-chloroethanol, a helix-promoting solvent, the amount of alpha-helix is increased to 60-70%. The glycoproteins underwent denaturation in guanidine hydrochloride, although evidence of some residual structure did remain. The spectra of the glycoproteins change relatively little on going from aqueous buffer to dodecylsulfate solutions. Removal of 60% of the sialic acid does not induce significant conformational alterations. The anomalous behavior of the glycoproteins during molecular weight determinations does not appear to be related primarily to conformational restrictions on the polypeptide chain.

  18. Role of the Rhesus glycoprotein, Rh B glycoprotein, in renal ammonia excretion

    PubMed Central

    Bishop, Jesse M.; Verlander, Jill W.; Lee, Hyun-Wook; Nelson, Raoul D.; Weiner, Arthur J.; Handlogten, Mary E.

    2010-01-01

    Rh B glycoprotein (Rhbg) is a member of the Rh glycoprotein family of ammonia transporters. In the current study, we examine Rhbg's role in basal and acidosis-stimulated acid-base homeostasis. Metabolic acidosis induced by HCl administration increased Rhbg expression in both the cortex and outer medulla. To test the functional significance of increased Rhbg expression, we used a Cre-loxP approach to generate mice with intercalated cell-specific Rhbg knockout (IC-Rhbg-KO). On normal diet, intercalated cell-specific Rhbg deletion did not alter urine ammonia excretion, pH, or titratable acid excretion significantly, but it did decrease glutamine synthetase expression in the outer medulla significantly. After metabolic acidosis was induced, urinary ammonia excretion was significantly less in IC-Rhbg-KO than in control (C) mice on days 2–4 of acid loading, but not on day 5. Urine pH and titratable acid excretion and dietary acid intake did not differ significantly between acid-loaded IC-Rhcg-KO and C mice. In IC-Rhbg-KO mice, acid loading increased connecting segment (CNT) cell and outer medullary collecting duct principal cell Rhbg expression. In both C and IC-Rhbg-KO mice, acid loading decreased glutamine synthetase in both the cortex and outer medulla; the decrease on day 3 was similar in IC-Rhbg-KO and C mice, but on day 5 it was significantly greater in IC-Rhbg-KO than in C mice. We conclude 1) intercalated cell Rhbg contributes to acidosis-stimulated renal ammonia excretion, 2) Rhbg in CNT and principal cells may contribute to renal ammonia excretion, and 3) decreased glutamine synthetase expression may enable normal rates of ammonia excretion under both basal conditions and on day 5 of acid loading in IC-Rhbg-KO mice. PMID:20719974

  19. The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein

    SciTech Connect

    Vogler, Meike; Dickens, David; Dyer, Martin J.S.; Owen, Andrew; Pirmohamed, Munir; Cohen, Gerald M.

    2011-05-06

    Highlights: {yields} The BCL2-inhibitor ABT-263 is a substrate for P-glycoprotein. {yields} Apoptosis is inhibited by P-glycoprotein expression. {yields} Overexpression of P-glycoprotein may contribute to resistance to ABT-263 or ABT-737. -- Abstract: Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using {sup 3}H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

  20. Sweating the small stuff: Glycoproteins in human sweat and their unexplored potential for microbial adhesion.

    PubMed

    Peterson, Robyn A; Gueniche, Audrey; Adam de Beaumais, Ségolène; Breton, Lionel; Dalko-Csiba, Maria; Packer, Nicolle H

    2016-03-01

    There is increasing evidence that secretory fluids such as tears, saliva and milk play an important role in protecting the human body from infection via a washing mechanism involving glycan-mediated adhesion of potential pathogens to secretory glycoproteins. Interaction of sweat with bacteria is well established as the cause of sweat-associated malodor. However, the role of sweat glycoproteins in microbial attachment has received little, if any, research interest in the past. In this review, we demonstrate how recent published studies involving high-throughput proteomic analysis have inadvertently, and fortuitously, exposed an abundance of glycoproteins in sweat, many of which have also been identified in other secretory fluids. We bring together research demonstrating microbial adhesion to these secretory glycoproteins in tears, saliva and milk and suggest a similar role of the sweat glycoproteins in mediating microbial attachment to sweat and/or skin. The contribution of glycan-mediated microbial adhesion to sweat glycoproteins, and the associated impact on sweat derived malodor and pathogenic skin infections are unchartered new research areas that we are beginning to explore.

  1. Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants

    PubMed Central

    Wilbers, Ruud H. P.; Westerhof, Lotte B.; van Noort, Kim; Obieglo, Katja; Driessen, Nicole N.; Everts, Bart; Gringhuis, Sonja I.; Schramm, Gabriele; Goverse, Aska; Smant, Geert; Bakker, Jaap; Smits, Hermelijn H.; Yazdanbakhsh, Maria; Schots, Arjen; Hokke, Cornelis H.

    2017-01-01

    Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein’s Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens. PMID:28393916

  2. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum

    PubMed Central

    Ferris, Sean P.; Jaber, Nikita S.; Molinari, Maurizio; Arvan, Peter; Kaufman, Randal J.

    2013-01-01

    Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: null Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER. PMID:23864712

  3. Due to interleukin-6 type cytokine redundancy only glycoprotein 130 receptor blockade efficiently inhibits myeloma growth

    PubMed Central

    Burger, Renate; Günther, Andreas; Klausz, Katja; Staudinger, Matthias; Peipp, Matthias; Penas, Eva Maria Murga; Rose-John, Stefan; Wijdenes, John; Gramatzki, Martin

    2017-01-01

    Interleukin-6 has an important role in the pathophysiology of multiple myeloma where it supports the growth and survival of the malignant plasma cells in the bone marrow. It belongs to a family of cytokines which use the glycoprotein 130 chain for signal transduction, such as oncostatin M or leukemia inhibitory factor. Targeting interleukin-6 in plasma cell diseases is currently evaluated in clinical trials with monoclonal antibodies. Here, efforts were made to elucidate the contribution of interleukin-6 and glycoprotein 130 signaling in malignant plasma cell growth in vivo. In the xenograft severe combined immune deficiency model employing our interleukin-6-dependent plasma cell line INA-6, the lack of human interleukin-6 induced autocrine interleukin-6 production and a proliferative response to other cytokines of the glycoprotein 130 family. Herein, mice were treated with monoclonal antibodies against human interleukin-6 (elsilimomab/B-E8), the interleukin-6 receptor (B-R6), and with an antibody blocking glycoprotein 130 (B-R3). While treatment of mice with interleukin-6 and interleukin-6 receptor antibodies resulted in a modest delay in tumor growth, the development of plasmacytomas was completely prevented with the anti-glycoprotein 130 antibody. Importantly, complete inhibition was also achieved using F(ab’)2-fragments of monoclonal antibody B-R3. Tumors harbor activated signal transducer and activator of transcription 3, and in vitro, the antibody inhibited leukemia inhibitory factor stimulated signal transducer and activator of transcription 3 phosphorylation and cell growth, while being less effective against interleukin-6. In conclusion, the growth of INA-6 plasmacytomas in vivo under interleukin-6 withdrawal remains strictly dependent on glycoprotein 130, and other glycoprotein 130 cytokines may substitute for interleukin-6. Antibodies against glycoprotein 130 are able to overcome this redundancy and should be explored for a possible therapeutic window

  4. Calnexin, calreticulin and the folding of glycoproteins.

    PubMed

    1997-05-01

    Calnexin and calreticulin are molecular chaperones in the endoplasmic reticulum (ERJ. They are lectins that interact with newly synthesized glycoproteins that have undergone partial trimming of their core N-linked oligosaccharides. Together with the enzymes responsible for glucose removal and a glucosyltransferase that re-glucosylates already-trimmed glycoproteins, they provide a novel mechanism for promoting folding, oligomeric assembly and quality control in the ER.

  5. [Biological role of heterogeneous glycoprotein structures].

    PubMed

    Jakab, Lajos

    2016-07-01

    Carbohydrate molecules connected mostly with covalent junctions to protein chains are called glycoproteins. These carbohydrate molecules are attached to the protein core in different qualities and order. When the protein core is connected with acidic components such as uronic acid or SO4 radicals, they are called proteoglycans. The currently used name "glycosaminoglycan" in this case is not entirely correct. In the living world polymannane structures occur, too. Glycoproteins do not only exceptionally hold acidic groups but they have neuraminic acid derivatives. Tissue, cellular and matrix structures, and mostly all serum "proteins" are mainly glycoproteins. In the everyday clinical practice glycoproteins are mentioned as proteins. Nevertheless, the inadequate use of the concept may cause errors in the attitudes, too. This paper aims to correct this notion, because the term of "glycobiology" has already been expanded to be an independent scientific field. The practical clinical consequences of recent knowledge in this field are also summarized including novel findings on glycoprotein structures and functions. The importance of the quantity of carbohydrates, and their structural arrangements are also presented. In short, significance of glycoprotein-carbohydrate structures, as well as their physiological and pathological roles are reviewed in order to introduce the field of "glycobiology". Orosomucoid and immunoglobulins are discussed separately. Orv. Hetil., 2016, 157(30), 1185-1192.

  6. Secondary structure of gp160 and gp120 envelope glycoproteins of human immunodeficiency virus type 1: a Fourier transform infrared spectroscopic study.

    PubMed Central

    Decroly, E; Cornet, B; Martin, I; Ruysschaert, J M; Vandenbranden, M

    1993-01-01

    The secondary structure of the precursor (gp160) of the envelope protein of human immunodeficiency virus type 1 (BH10) and its receptor-binding subunit (gp120) was studied by Fourier-transformed attenuated total reflection spectroscopy. A higher alpha-helix/beta-sheet ratio in the gp120 subunit than in the precursor indicates a structural heterogeneity between the two subunits (gp120 and gp41), in agreement with classical secondary-structure predictions. The secondary structure of gp41 was estimated and compared with existing models. The high alpha-helical content in gp41 and the dominant beta-sheet content in gp120 resemble the distribution in influenza virus hemagglutinin subunits. PMID:8497064

  7. Secondary structure of gp160 and gp120 envelope glycoproteins of human immunodeficiency virus type 1: a Fourier transform infrared spectroscopic study.

    PubMed

    Decroly, E; Cornet, B; Martin, I; Ruysschaert, J M; Vandenbranden, M

    1993-06-01

    The secondary structure of the precursor (gp160) of the envelope protein of human immunodeficiency virus type 1 (BH10) and its receptor-binding subunit (gp120) was studied by Fourier-transformed attenuated total reflection spectroscopy. A higher alpha-helix/beta-sheet ratio in the gp120 subunit than in the precursor indicates a structural heterogeneity between the two subunits (gp120 and gp41), in agreement with classical secondary-structure predictions. The secondary structure of gp41 was estimated and compared with existing models. The high alpha-helical content in gp41 and the dominant beta-sheet content in gp120 resemble the distribution in influenza virus hemagglutinin subunits.

  8. Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway.

    PubMed

    Ninagawa, Satoshi; Okada, Tetsuya; Sumitomo, Yoshiki; Horimoto, Satoshi; Sugimoto, Takehiro; Ishikawa, Tokiro; Takeda, Shunichi; Yamamoto, Takashi; Suzuki, Tadashi; Kamiya, Yukiko; Kato, Koichi; Mori, Kazutoshi

    2015-11-23

    Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ-ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ-ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER.

  9. Bioanalytical tools for the discovery of eukaryotic glycoproteins applied to the analysis of bacterial glycoproteins.

    PubMed

    Balonova, Lucie; Hernychova, Lenka; Bilkova, Zuzana

    2009-02-01

    The commonly accepted theory that prokaryotes lack the ability to glycosylate their proteins has been disproved recently. The field of bacterial glycoprotein research is no longer considered novel owing to the rapid progress in analytical technologies and genome sequencing that has been made in the last few years. Enhanced interest in glycoprotein discovery in bacteria can be explained by a proven correlation between the presence of glycosylation and bacterial pathogenicity. Eukaryotic and prokaryotic organisms' features share certain similarities. However, with respect to inherent differences between these two distinct domains of life, the use of bioanalytical tools for glycoprotein analysis in eukaryotic systems often needs modification to be applied successfully to bacteria. In this article, we draw attention to the differences between eukaryotic and prokaryotic glycoproteins. We also focus on the main bottlenecks that may be encountered in the search for glycosylation in concrete bacterium and outline a possible work-flow for the exploration of glycoproteins in bacteria.

  10. Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment.

    PubMed

    Song, Ehwang; Mechref, Yehia

    2015-01-01

    Protein glycosylation is an important and common post-translational modification. More than 50% of human proteins are believed to be glycosylated to modulate the functionality of proteins. Aberrant glycosylation has been correlated to several diseases, such as inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer's and prion diseases, and cancer. Many approved cancer biomarkers are glycoproteins which are not highly abundant proteins. Therefore, effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. This chapter summarizes glycoprotein enrichment methods, including lectin affinity, immunoaffinity, hydrazide chemistry, hydrophilic interaction liquid chromatography, and click chemistry. The use of these enrichment approaches in assessing the qualitative and quantitative changes of glycoproteins in different types of cancers are presented and discussed. This chapter highlights the importance of glycoprotein enrichment techniques for the identification and characterization of new reliable cancer biomarkers.

  11. Requirements for cell rounding and surface protein down-regulation by Ebola virus glycoprotein.

    PubMed

    Francica, Joseph R; Matukonis, Meghan K; Bates, Paul

    2009-01-20

    Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of beta1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects.

  12. Pharmacokinetic role of P-glycoprotein in oral bioavailability and intestinal secretion of grepafloxacin in vivo.

    PubMed

    Yamaguchi, Hiroaki; Yano, Ikuko; Saito, Hideyuki; Inui, Ken-ichi

    2002-03-01

    The purpose of this study was to clarify the contribution of P-glycoprotein to the bioavailability and intestinal secretion of grepafloxacin and levofloxacin in vivo. Plasma concentrations of grepafloxacin and levofloxacin after intravenous and intraintestinal administration were increased by cyclosporin A, a P-glycoprotein inhibitor, in rats. The total body clearance and volume of distribution at steady state of grepafloxacin were significantly decreased to 60 and 63% of the corresponding control values by cyclosporin A. The apparent oral clearance of grepafloxacin was decreased to 33% of the control, and the bioavailability of grepafloxacin was increased to 95% by cyclosporin A from 53% in the controls. Intestinal clearance of grepafloxacin and levofloxacin were decreased to one-half and one-third of the control, respectively, and biliary clearance of grepafloxacin was also decreased to one-third with cyclosporin A in rats. Intestinal secretion of grepafloxacin in mdr1a/1b (-/-) mice, which lack mdr1-type P-glycoproteins, was significantly decreased compared with wild-type mice, although the biliary secretion was similar. Intestinal secretion of grepafloxacin in wild-type mice treated with cyclosporin A was comparable to those in mdr1a/1b (-/-) mice with or without cyclosporin A, indicating that cyclosporin A completely inhibited P-glycoprotein-mediated intestinal transport of grepafloxacin. In conclusion, our results indicated that P-glycoprotein mediated the intestinal secretion of grepafloxacin and limited the bioavailability of this drug in vivo.

  13. Traceless labeling of glycoproteins and its application to the study of glycoprotein-protein interactions.

    PubMed

    Yang, Yung-Lin; Lee, Yen-Pin; Yang, Yen-Ling; Lin, Po-Chiao

    2014-02-21

    A new chemical method for the traceless labeling of glycoproteins with synthetic boronic acid (BA)-tosyl probes was successfully developed. The BA moiety acts as an affinity head to direct the formation of a cyclic boronate diester with the diol groups of glycans. Following this step, the electrophilic tosyl group is displaced by an SN2 reaction with a nucleophilic residue of the boronated glycoprotein, and finally, a reporter group is tagged onto the glycoprotein via an ether linkage. In the presence of polyols, a competition reaction recovers the native glycan of the tagged glycoprotein, conserving its biological significance. The BA-tosyl probes were used successfully for the specific labeling of glycosylated fetuins in a mixed protein pool and from crude Escherichia coli (E. coli) lysate. Further, a BA-tosyl-functionalized glass slide was used to fabricate glycoprotein microarrays with highly conserved glycans. By interacting with various lectins (carbohydrate-binding proteins), such as Concanavalin A (Con A) and wheat germ agglutinin (WGA), the types of carbohydrates and specific linkages of glycoproteins (α or β) could be systematically monitored. It is believed that the newly developed method will greatly accelerate the understanding of glycoproteins.

  14. Structural details of HIV-1 recognition by the broadly neutralizing monoclonal antibody 2F5: epitope conformation, antigen-recognition loop mobility, and anion-binding site.

    PubMed

    Julien, Jean-Philippe; Bryson, Steve; Nieva, Jose L; Pai, Emil F

    2008-12-12

    2F5 is a monoclonal antibody with potent and broadly neutralizing activity against HIV-1. It targets the membrane-proximal external region (MPER) of the gp41 subunit of the envelope glycoprotein and interferes with the process of fusion between viral and host cell membranes. This study presents eight 2F5 F(ab)' crystal structures in complex with various gp41 peptide epitopes. These structures reveal several key features of this antibody-antigen interaction. (1) Whenever free of contacts caused by crystal artifacts, the extended complementarity-determining region H3 loop is mobile; this is true for ligand-free and epitope-bound forms. (2) The interaction between the antibody and the gp41 ELDKWA epitope core is absolutely critical, and there are also close and specific contacts with residues located N-terminal to the epitope core. (3) Residues located at the C-terminus of the gp41 ELDKWA core do not interact as tightly with the antibody. However, in the presence of a larger peptide containing the gp41 fusion peptide segment, these residues adopt a conformation consistent with the start of an alpha-helix. (4) At high sulfate concentrations, the electron density maps of 2F5 F(ab)'-peptide complexes contain a peak that may mark a binding site for phosphate groups of negatively charged lipid headgroups. The refined atomic-level details of 2F5 paratope-epitope interactions revealed here should contribute to a better understanding of the mechanism of 2F5-based virus neutralization, in general, and prove important for the design of potential vaccine candidates intended to elicit 2F5-like antibody production.

  15. Identification of a Protein for Prostate-Specific Infection

    DTIC Science & Technology

    2007-12-01

    tissue-specific manner. In the third year, we worked on using gp41 HIV glycoprotein to fuse with these two peptides in a lentiviral vector. The gp41 ...HIV gp41 protein, we could increase gene delivery by 30% to 70% into LNCaP prostate cancer cells (Fig. 3C). Although the the fusion of the...in Fig. 4. We use influenza HA protein instead of Sindbis E1 gp41 Vector genome C. C-terminus N-terminus C-terminus N-terminus Modified with our

  16. Roundtable for the Development of Drugs and Vaccines Against Acquired Immunodeficiency Syndrome (AIDS).

    DTIC Science & Technology

    1992-11-01

    to mediating HIV binding and entry into target cells, the gpl20/ gp41 envelope glycoprotein complex also plays a major role in the cytopathic...consequences of HIV infection. Expression of the gpl2O/ gp41 complex in a CD4+ T cell can result in a process of membrane fusion between neighboring cells, which

  17. Characterization of disease-associated N-linked glycoproteins.

    PubMed

    Tian, Yuan; Zhang, Hui

    2013-02-01

    N-linked glycoproteins play important roles in biological processes, including cell-to-cell recognition, growth, differentiation, and programmed cell death. Specific N-linked glycoprotein changes are associated with disease progression and identification of these N-linked glycoproteins has potential for use in disease diagnosis, prognosis, and prediction of treatments. In this review, we summarize common strategies for N-linked glycoprotein characterization and applications of these strategies to identification of glycoprotein changes associated with disease states. We also review the N-linked glycoproteins altered in diseases such as breast cancer, lung cancer, and prostate cancer. Although assays for these glycoproteins have potential clinical utility, research is needed to translate these glycoproteins to clinical biomarkers.

  18. Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability

    NASA Astrophysics Data System (ADS)

    Kong, Leopold; He, Linling; de Val, Natalia; Vora, Nemil; Morris, Charles D.; Azadnia, Parisa; Sok, Devin; Zhou, Bin; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Zhu, Jiang

    2016-06-01

    The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains.

  19. Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability

    PubMed Central

    Kong, Leopold; He, Linling; de Val, Natalia; Vora, Nemil; Morris, Charles D.; Azadnia, Parisa; Sok, Devin; Zhou, Bin; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Zhu, Jiang

    2016-01-01

    The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains. PMID:27349805

  20. Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability.

    PubMed

    Kong, Leopold; He, Linling; de Val, Natalia; Vora, Nemil; Morris, Charles D; Azadnia, Parisa; Sok, Devin; Zhou, Bin; Burton, Dennis R; Ward, Andrew B; Wilson, Ian A; Zhu, Jiang

    2016-06-28

    The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains.

  1. Glycoprotein enrichment through lectin affinity techniques.

    PubMed

    Mechref, Yehia; Madera, Milan; Novotny, Milos V

    2008-01-01

    Posttranslational modifications (PTM) of proteins are among the key biological regulators of function, activity, localization, and interaction. The fact that no more than 30,000-50,000 proteins are encoded by the human genome underlines the importance of posttranslational modifications in modulating the activities and functions of proteins in health and disease. With approximately 50% of all proteins now considered to be glycosylated, its physiological importance in mammalian systems is imperative. Aberrant glycosylation has now been recognized as an attribute of many mammalian diseases, including hereditary disorders, immune deficiencies, neurodegenerative diseases, cardiovascular conditions, and cancer. As many potential disease biomarkers may be glycoproteins present in only minute quantities in tissue extracts and physiological fluids, glycoprotein isolation and enrichment may be critical in a search for such biomarkers. For decades, efforts have been focused on the development of glycoprotein enrichment from complex biological samples. Logically, the great majority of these enrichment methodologies rely on the use of immobilized lectins, which permit selective enrichment of the pools of glycoproteins for proteomic/glycomic studies. In this chapter, lectin affinity chromatography in different formats are described, including tubes; packed columns, and microfluidic channels.

  2. Glycoprotein NMB: an Emerging Role in Neurodegenerative Disease.

    PubMed

    Budge, Kevin M; Neal, Matthew L; Richardson, Jason R; Safadi, Fayez F

    2017-08-30

    Neurodegeneration is characterized by severe neuronal loss leading to the cognitive and physical impairments that define various neurodegenerative diseases. Neuroinflammation is one hallmark of neurodegenerative diseases and can ultimately contribute to disease progression. Increased inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1β (IL-1 β), and tumor necrosis factor-α (TNF-α) are associated with Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Unfortunately, current therapeutic options lack ability to stop or effectively slow progression of these diseases and are primarily aimed at alleviating symptoms. Thus, it is crucial to discover novel treatment candidates for neurodegenerative diseases. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type-I transmembrane glycoprotein first identified in a melanoma cell line. GPNMB augments bone mineral deposition by stimulating osteoblast differentiation. Aside from its anabolic function in the bone, emerging evidence suggests that GPNMB has anti-inflammatory and reparative functions. GPNMB has also been demonstrated to be neuroprotective in an animal model of ALS, cerebral ischemia, and other disease models. Given these discoveries, GPNMB should be investigated as a potential therapeutic option for multiple neurodegenerative diseases.

  3. Using proximity biotinylation to detect herpesvirus entry glycoprotein interactions: Limitations for integral membrane glycoproteins.

    PubMed

    Lajko, Michelle; Haddad, Alexander F; Robinson, Carolyn A; Connolly, Sarah A

    2015-09-01

    Herpesvirus entry into cells requires coordinated interactions among several viral transmembrane glycoproteins. Viral glycoproteins bind to receptors and interact with other glycoproteins to trigger virus-cell membrane fusion. Details of these glycoprotein interactions are not well understood because they are likely transient and/or low affinity. Proximity biotinylation is a promising protein-protein interaction assay that can capture transient interactions in live cells. One protein is linked to a biotin ligase and a second protein is linked to a short specific acceptor peptide (AP). If the two proteins interact, the ligase will biotinylate the AP, without requiring a sustained interaction. To examine herpesvirus glycoprotein interactions, the ligase and AP were linked to herpes simplex virus 1 (HSV1) gD and Epstein Barr virus (EBV) gB. Interactions between monomers of these oligomeric proteins (homotypic interactions) served as positive controls to demonstrate assay sensitivity. Heterotypic combinations served as negative controls to determine assay specificity, since HSV1 gD and EBV gB do not interact functionally. Positive controls showed strong biotinylation, indicating that viral glycoprotein proximity can be detected. Unexpectedly, the negative controls also showed biotinylation. These results demonstrate the special circumstances that must be considered when examining interactions among glycosylated proteins that are constrained within a membrane.

  4. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    PubMed

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism.

  5. Effect of reduced renal mass on renal ammonia transporter family, Rh C glycoprotein and Rh B glycoprotein, expression.

    PubMed

    Kim, Hye-Young; Baylis, Chris; Verlander, Jill W; Han, Ki-Hwan; Reungjui, Sirirat; Handlogten, Mary E; Weiner, I David

    2007-10-01

    Kidneys can maintain acid-base homeostasis, despite reduced renal mass, through adaptive changes in net acid excretion, of which ammonia excretion is the predominant component. The present study examines whether these adaptations are associated with changes in the ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). We used normal Sprague-Dawley rats and a 5/6 ablation-infarction model of reduced renal mass; control rats underwent sham operation. After 1 wk, glomerular filtration rate, assessed as creatinine clearance, was decreased, serum bicarbonate was slightly increased, and Na(+) and K(+) were unchanged. Total urinary ammonia excretion was unchanged, but urinary ammonia adjusted for creatinine clearance, an index of per nephron ammonia metabolism, increased significantly. Although reduced renal mass did not alter total Rhcg protein expression, both light microscopy and immunohistochemistry with quantitative morphometric analysis demonstrated hypertrophy of both intercalated cells and principal cells in the cortical and outer medullary collecting duct that was associated with increased apical and basolateral Rhcg polarization. Rhbg expression, analyzed using immunoblot analysis, immunohistochemistry, and measurement of cell-specific expression, was unchanged. We conclude that altered subcellular localization of Rhcg contributes to adaptive changes in single-nephron ammonia metabolism and maintenance of acid-base homeostasis in response to reduced renal mass.

  6. Intracellular lectins are involved in quality control of glycoproteins

    PubMed Central

    YAMAMOTO, Kazuo

    2014-01-01

    Glycoprotein quality control is categorized into three kinds of reactions; the folding of nascent glycoproteins, ER-associated degradation of misfolded or unassembled glycoproteins, and transport and sorting of correctly folded glycoproteins. In all three processes, N-glycans on the glycoproteins are used as tags that are recognized by intracellular lectins. We analyzed the functions of these intracellular lectins and their sugar-binding specificities. The results clearly showed that the A, B, and C-arms of high mannose-type glycans participate in the folding, transport and sorting, and degradation, respectively, of newly synthesized peptides. After correctly folded glycoproteins are transported to the Golgi apparatus, N-glycans are trimmed into Man3GlcNAc2 and then rebuilt into various complex-type glycans in the Golgi, resulting in the addition of diverse sugar structures that allow glycoproteins to play various roles outside of the cells. PMID:24522156

  7. Glycoprotein patterns in normal and malignant cervical tissue.

    PubMed

    O'Brien, M E; Souberbielle, B E; Cowan, M E; Allen, C A; Luesley, D M; Mould, J J; Blackledge, G R; Skinner, G R

    1991-07-04

    Glycoproteins from normal and malignant human cervix were studied using an organ culture system and compared by gel electrophoresis and autoradiography. Five glycoproteins of 178 kDa, 95 kDa, 93 kDa, 82 kDa and 38 kDa and 1 glycolipid (46 kDa) were detected more frequently in squamous carcinomas. Certain glycoproteins were shown to be oncofoetal and some had affinity for Concanavalin A (Con A). The 82 kDa glycoprotein was present in 16/17 squamous carcinomas but in only 1/13 normal cervices. This band represented a glycoprotein containing glucosamine, mannose, small quantities of methionine and no fucose. These preliminary results suggest that these glycoproteins and in particular the 82-kDa glycoprotein are worthy of further investigation and characterisation.

  8. Human Milk Glycoproteins Protect Infants Against Human Pathogens

    PubMed Central

    Liu, Bo

    2013-01-01

    Abstract Breastfeeding protects the neonate against pathogen infection. Major mechanisms of protection include human milk glycoconjugates functioning as soluble receptor mimetics that inhibit pathogen binding to the mucosal cell surface, prebiotic stimulation of gut colonization by favorable microbiota, immunomodulation, and as a substrate for bacterial fermentation products in the gut. Human milk proteins are predominantly glycosylated, and some biological functions of these human milk glycoproteins (HMGPs) have been reported. HMGPs range in size from 14 kDa to 2,000 kDa and include mucins, secretory immunoglobulin A, bile salt-stimulated lipase, lactoferrin, butyrophilin, lactadherin, leptin, and adiponectin. This review summarizes known biological roles of HMGPs that may contribute to the ability of human milk to protect neonates from disease. PMID:23697737

  9. Human milk glycoproteins protect infants against human pathogens.

    PubMed

    Liu, Bo; Newburg, David S

    2013-08-01

    Breastfeeding protects the neonate against pathogen infection. Major mechanisms of protection include human milk glycoconjugates functioning as soluble receptor mimetics that inhibit pathogen binding to the mucosal cell surface, prebiotic stimulation of gut colonization by favorable microbiota, immunomodulation, and as a substrate for bacterial fermentation products in the gut. Human milk proteins are predominantly glycosylated, and some biological functions of these human milk glycoproteins (HMGPs) have been reported. HMGPs range in size from 14 kDa to 2,000 kDa and include mucins, secretory immunoglobulin A, bile salt-stimulated lipase, lactoferrin, butyrophilin, lactadherin, leptin, and adiponectin. This review summarizes known biological roles of HMGPs that may contribute to the ability of human milk to protect neonates from disease.

  10. Small-angle scattering study of Aspergillus awamori glycoprotein glucoamylase

    NASA Astrophysics Data System (ADS)

    Schmidt, A. E.; Shvetsov, A. V.; Kuklin, A. I.; Lebedev, D. V.; Surzhik, M. A.; Sergeev, V. R.; Isaev-Ivanov, V. V.

    2016-01-01

    Glucoamylase from fungus Aspergillus awamori is glycoside hydrolase that catalyzes the hydrolysis of α-1,4- and α-1,6-glucosidic bonds in glucose polymers and oligomers. This glycoprotein consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated polypeptide chain. The conformation of the linker, the relative arrangement of the domains, and the structure of the full-length enzyme are unknown. The structure of the recombinant glucoamylase GA1 was studied by molecular modelling and small-angle neutron scattering (SANS) methods. The experimental SANS data provide evidence that glucoamylase exists as a monomer in solution and contains a glycoside component, which makes a substantial contribution to the scattering. The model of full-length glucoamylase, which was calculated without taking into account the effect of glycosylation, is consistent with the experimental data and has a radius of gyration of 33.4 ± 0.6 Å.

  11. An analysis of amino acid sequences surrounding archaeal glycoprotein sequons.

    PubMed

    Abu-Qarn, Mehtap; Eichler, Jerry

    2007-05-01

    Despite having provided the first example of a prokaryal glycoprotein, little is known of the rules governing the N-glycosylation process in Archaea. As in Eukarya and Bacteria, archaeal N-glycosylation takes place at the Asn residues of Asn-X-Ser/Thr sequons. Since not all sequons are utilized, it is clear that other factors, including the context in which a sequon exists, affect glycosylation efficiency. As yet, the contribution to N-glycosylation made by sequon-bordering residues and other related factors in Archaea remains unaddressed. In the following, the surroundings of Asn residues confirmed by experiment as modified were analyzed in an attempt to define sequence rules and requirements for archaeal N-glycosylation.

  12. Small-angle scattering study of Aspergillus awamori glycoprotein glucoamylase

    SciTech Connect

    Schmidt, A. E. Shvetsov, A. V.; Kuklin, A. I.; Lebedev, D. V.; Surzhik, M. A.; Sergeev, V. R.; Isaev-Ivanov, V. V.

    2016-01-15

    Glucoamylase from fungus Aspergillus awamori is glycoside hydrolase that catalyzes the hydrolysis of α-1,4- and α-1,6-glucosidic bonds in glucose polymers and oligomers. This glycoprotein consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated polypeptide chain. The conformation of the linker, the relative arrangement of the domains, and the structure of the full-length enzyme are unknown. The structure of the recombinant glucoamylase GA1 was studied by molecular modelling and small-angle neutron scattering (SANS) methods. The experimental SANS data provide evidence that glucoamylase exists as a monomer in solution and contains a glycoside component, which makes a substantial contribution to the scattering. The model of full-length glucoamylase, which was calculated without taking into account the effect of glycosylation, is consistent with the experimental data and has a radius of gyration of 33.4 ± 0.6 Å.

  13. Platelet glycoprotein Ibα supports experimental lung metastasis

    PubMed Central

    Jain, Shashank; Zuka, Masahiko; Liu, Jungling; Russell, Susan; Dent, Judith; Guerrero, José A.; Forsyth, Jane; Maruszak, Brigid; Gartner, T. Kent; Felding-Habermann, Brunhilde; Ware, Jerry

    2007-01-01

    The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the α-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy. PMID:17494758

  14. Characterization of human immunodeficiency virus type 2 envelope glycoproteins: Dimerization of the glycoprotein precursor during processing

    SciTech Connect

    Rey, M.A.; Krust, B.; Laurent, A.G.; Montagnier, L.; Hovanessian, A.G.

    1989-02-01

    For glycoproteins with apparent molecular weights of 300,000, 140,000, 125,000, and 36,000 (gp300, gp140, gp125, and gp36) were detectable in human immunodeficiency virus type 2 (HIV-2)-infected cells. They have identical isoelectric points, suggesting that gp300 might be a dimeric form of the immature precursor, gp140. The purified gp300 can be dissociated in a slightly acidic buffer to give rise to monomers of 140,000 molecular weight. Such dissociated monomers and the purified gp140 showed identical patterns of polypeptides after partial proteolysis with Staphylococcus aureus V8 protease. Pulse-chase experiments indicated that gp300 is formed after synthesis of gp140 and before the detection of the mature external envelope glycoprotein, gp125. These results were confirmed by using various inhibitors of glycosylation and inhibitors of trimming enzymes. Dimer formation of the envelope glycoprotein precursor was also observed in cells infected with simian immunodeficiency virus (SIV), a virus closely related to HIV-2. On the other hand, the envelope glycoprotein precursor of HIV-1 did not form a dimer during its processing. Therefore, dimer formation seems to be a specific property of HIV-2 and SIV envelope gene expression. Such transient dimerization of the glycoprotein precursor might be required for its efficient transport to the Golgi apparatus and for its processing.

  15. Paramyxovirus Glycoprotein Incorporation, Assembly and Budding: A Three Way Dance for Infectious Particle Production

    PubMed Central

    El Najjar, Farah; Schmitt, Anthony P.; Dutch, Rebecca Ellis

    2014-01-01

    Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout. PMID:25105277

  16. Glycoprotein enrichment method using a selective magnetic nano-probe platform (MNP) functionalized with lectins.

    PubMed

    Cova, Marta; Oliveira-Silva, Rui; Ferreira, José Alexandre; Ferreira, Rita; Amado, Francisco; Daniel-da-Silva, Ana Luísa; Vitorino, Rui

    2015-01-01

    Protein post-translational modifications (PTMs) have increasingly become a research field of incredible importance to fully understand the regulation of biological processes in health and disease. Among PTMs, glycosylation is one of the most studied for which contributed the development and improvement of enrichment techniques. Nowadays, glycoprotein enrichment methods are based on lectin affinity, covalent interactions, and hydrophilic interaction liquid chromatography (HILIC). Nonetheless, the nanotechnology era has fetched new methods to enrich glycoproteins from complex samples as human biological fluids. For instance, magnetic nanoparticles (MNPs) are being used as an interesting enrichment approach allowing a better characterization of glycoproteins and glycopeptides.In this chapter, we describe an enrichment method based on MNPs functionalized with lectins (Concavalin A, wheat germ agglutinin, and Maackia amurensis lectin) to enrich specific sets of glycoproteins from biological fluids. Moreover, it is proposed a bioinformatic strategy to deal with data retrieved from mass spectrometry analysis of enriched samples aiming the identification of relevant biological processes modulated by a given stimuli and, ultimately, of new biomarkers for disease screening/management.

  17. Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function

    PubMed Central

    Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

    2014-01-01

    Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review. PMID:25324850

  18. Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function.

    PubMed

    Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

    2014-01-01

    Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewis(a) substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review.

  19. Chemical and Chemoenzymatic Synthesis of Glycoproteins for Deciphering Functions

    PubMed Central

    Wang, Lai-Xi; Amin, Mohammed N.

    2014-01-01

    Summary Glycoproteins are an important class of biomolecules involved in a number of biological recognition processes. However, natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ in the structures of the pendent glycans, which are difficult to separate in pure glycoforms. As a result, synthetic homogeneous glycopeptides and glycoproteins have become indispensable probes for detailed structural and functional studies. A number of elegant chemical and biological strategies have been developed for synthetic construction of tailor-made, full-size glycoproteins to address specific biological problems. In this review, we highlight recent advances in chemical and chemoenzymatic synthesis of homogeneous glycoproteins. Selected examples are given to demonstrate the applications of tailor-made, glycan-defined glycoproteins for deciphering glycosylation functions. PMID:24439206

  20. Glycoproteins as biological antifreeze agents in antarctic fishes.

    PubMed

    DeVries, A L

    1971-06-11

    The blood serums of Antarctic fishes freeze at -2 degrees C, which is approximately 1 degrees C below the melting points of their serums. This thermal hysteresis is due to the influence of serum glycoproteins. The temperatures of freezing and melting of aqueous solutions of the purified glycoproteins suggest that this thermal hysteresis results from the adsorption of the glycoprotein molecule onto the surface of ice crystals.

  1. Immunochemical analysis of the H and M glycoproteins from Histoplasma capsulatum.

    PubMed Central

    Zancopé-Oliveira, R M; Bragg, S L; Reiss, E; Peralta, J M

    1994-01-01

    The H and M antigens of Histoplasma capsulatum are glycoproteins, and both possess epitopes found on the C antigen, a cross-reactive galactomannan shared by the major genera of systemic dimorphic fungi. We modified the H and M glycoproteins by chemical and enzymatic digestion to determine the relative contributions of the carbohydrate and protein moieties to the immunological reactivities and the apparent molecular weights of these antigens. Endoglycosidases with known action patterns were used to determine the nature of the glycopeptide bonds in the H and M antigens. The effects of these treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lectin binding, and enzyme-linked immunoelectrotransfer blots probed with polyclonal and monoclonal antibodies (MAbs). Oxidation with 100 mM periodate destroyed the common fungal epitope recognized by MAb CA1-CB4 and nearly all of the concanavalin A-binding sites on both the H and M antigens; it also caused the molecular mass of the M antigen to shift from 94 to 88 kDa. Treatment of samples with O-glycanase had little, if any, effect on the H and M glycoproteins. On the other hand, treatments with endo-beta-N-acetylglucosaminidase H, and particularly peptide N-glycoproteins F (PNGase F), produced pronounced shifts in the M(r) but did not completely eliminate concanavalin A- or MAb CA1-CB4-binding sites. PNGase F treatment caused the molecular mass of the H antigen to shift from 116 to 94 kDa and that of the M antigen to shift from 94 to 74 kDa. The susceptibilities of the H and M glycoproteins to endo-N-acetyl-beta-D-glucosaminidases suggest that their glycosidic moieties are N linked.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8556502

  2. Dimerization of the P-glycoprotein in membranes.

    PubMed

    Boscoboinik, D; Debanne, M T; Stafford, A R; Jung, C Y; Gupta, R S; Epand, R M

    1990-09-07

    Plasma membranes from a CHO cell line, CHRC5, which exhibits multidrug resistance was studied using radiation inactivation analysis. The P-glycoprotein content of the membrane was determined by Western blots. Irradiation resulted in the loss of P-glycoprotein. The dependence of this loss on radiation dose corresponded to a target size of 250 kDa which is the molecular mass of a dimer of the P-glycoprotein. This is strong evidence to indicate that the P-glycoprotein self associates in the membrane.

  3. Progesterone regulates the expression and activity of two mouse isoforms of the glycoprotein folding sensor UDP-Glc: glycoprotein glucosyltransferase (UGGT).

    PubMed

    Prados, María B; Caramelo, Julio J; Miranda, Silvia E

    2013-12-01

    UDP-Glucose:glycoprotein glucosyltransferase (UGGT) is a central component of the endoplasmic reticulum (ER) glycoprotein-folding quality control system, which prevents the exit of partially folded species. UGGT activity can be regulated by the accumulation of misfolded proteins in the ER, a stimulus that triggers a complex signaling pathway known as unfolded protein response (UPR) which is closely associated with inflammation and disease. In this work, we investigated the effect of progesterone (P4) on the expression and activity of UGGT in a mouse hybridoma. We detected the expression of two UGGT isoforms, UGGT1 and UGGT2, and demonstrated that both isoforms are active in these cells. Interestingly, the expression of each isoform is regulated by high physiological P4 concentrations. This work provides the first evidence of a hormonal regulation of UGGT isoform expression and activity, which might influence the glycoprotein quality control mechanism. These findings could contribute to the study of pathologies triggered by the accumulation of misfolded proteins.

  4. Glycoprotein Degradation in the Blind Loop Syndrome

    PubMed Central

    Prizont, Roberto

    1981-01-01

    Contents obtained from jejunum of normal controls, self-emptying and self-filling blind loop rats were analyzed for the presence of glycoprotein-degrading glycosidases. The blind loop syndrome was documented by the increased fat excretion and slower growth rate of self-filling blind loop rats 6 wk after surgery. With p-nitrophenylglycosides as substrate, the specific activity of α-N-acetylgalactosaminidase, a potential blood group A destroying glycosidase, was 0.90±0.40 mU/mg of protein. This level was 23-fold higher than the specific activity of normal controls. In partially purified self-filling blind loop contents, the activity of α-N-acetylgalactosaminidase was 9- to 70-fold higher than activities of self-emptying and normal controls. Antibiotic treatment with chloromycetin and polymyxin decreased 24-fold the glycosidase levels in self-filling blind loops. In experiments with natural substrate, the blood group A titer of a20,000g supernate from normal jejunal homogenates decreased 128-fold after 24-h incubation with blind loop contents. Normal contents failed to diminish the blood group reactivity of the natural substrate. Furthermore, blind loop contents markedly decreased the blood group A titer of isolated brush borders. Incubation between blind loop bacteria and mucosal homogenates or isolated brush borders labeled with d-[U-14C]glucosamine revealed increased production of labeled ether extractable organic acids. Likewise, intraperitoneal injection of d-[U-14C]glucosamine into self-filling blind loop rats resulted in incorporation of the label into luminal short chain fatty acids. These results suggest that glycosidases may provide a mechanism by which blind loop bacteria obtain sugars from intestinal glycoproteins. The released sugars are used and converted by bacteria into energy and organic acids. This use of the host's glycoproteins would allow blind loop bacteria to grow and survive within the lumen independent of exogenous sources. PMID:6257760

  5. A cytotoxic ribonuclease reduces the expression level of P-glycoprotein in multidrug-resistant cell lines.

    PubMed

    Castro, Jessica; Ribó, Marc; Puig, Teresa; Colomer, Ramon; Vilanova, Maria; Benito, Antoni

    2012-06-01

    We have previously described a cytotoxic human pancreatic-ribonuclease variant, named PE5, which is able to cleave nuclear RNA, inducing the apoptosis of cancer cells. We have investigated whether PE5 could specifically inhibit the accumulation of P-glycoprotein in multidrug-resistant cells, since P-glycoprotein overexpression is one of the most important mechanisms contributing to the multiple drug resistance phenotype. We show that PE5 is able to reduce the amount of P-glycoprotein in two different multidrug-resistant cell lines, NCI/H460-R and NCI/ADR-RES, while glutathione S-transferase-л is not affected. We also show that onconase, an amphibian ribonuclease that is undergoing phase II/III clinical trials as an antitumor drug, does not affect the expression of these proteins. The reduction of P-glycoprotein accumulation, which has been functionally confirmed by flow cytometry analysis, may be caused by the previously reported underphosphorylation of JNK induced by PE5. We also show that PE5 has synergistic cytotoxicity with doxorubicin on the NCI/ADR-RES multidrug-resistant cell line. In conclusion, PE5 is a cytotoxic ribonuclease that cleaves nuclear RNA and decreases the expression of P-glycoprotein, showing anticancer activity in multidrug-resistant cell lines.

  6. Characterization of Murine Gammaherpesvirus 68 Glycoprotein B

    PubMed Central

    Lopes, Filipa B.; Colaco, Susanna; May, Janet S.; Stevenson, Philip G.

    2004-01-01

    Murine gammaherpesvirus 68 (MHV-68) glycoprotein B (gB) was identified in purified virions by immunoblotting, immunoprecipitation, and immunoelectron microscopy. It was synthesized as a 120-kDa precursor in infected cells and cleaved into 65-kDa and 55-kDa disulfide-linked subunits close to the time of virion release. The N-linked glycans on the cleaved, virion gB remained partially endoglycosidase H sensitive. The processing of MHV-68 gB therefore appears similar to that of Kaposi's sarcoma-associated herpesvirus gB and human cytomegalovirus gB. PMID:15542690

  7. [Lactoferrin - a glycoprotein of great therapeutic potentials].

    PubMed

    Lauterbach, Ryszard; Kamińska, Ewa; Michalski, Piotr; Lauterbach, Jan Paweł

    2016-01-01

    Lactoferrin is an iron-binding glycoprotein, which is present in most biological fluids with particularly high levels in colostrum and in mammalian milk. Bovine lactoferrin is more than 70% homologous with human lactoferrin. Most of the clinical trials have used bovine lactoferrin for supplementation. This review summarizes the recent advances in explaining the mechanisms, which are responsible for the multifunctional roles of lactoferrin, and presents its potential prophylactic and therapeutic applications. On the ground of the results of preliminary clinical observations, authors suggest beneficial effect of lactoferrin supplementation on the prevalence of necrotizing enterocolitis in infants with birth weight below 1250 grams.

  8. Expression of ammonia transporter family members, Rh B glycoprotein and Rh C glycoprotein, in the developing rat kidney

    PubMed Central

    Han, Ki-Hwan; Lee, Su-Youn; Kim, Wan-Young; Shin, Jung-A; Kim, Jin

    2010-01-01

    Ammonia metabolism is a primary component of acid-base homeostasis but is incompletely developed at time of birth. Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg) are recently recognized ammonia transporter family members expressed in the mammalian kidney. This study's purpose was to establish the expression and localization of Rhbg and Rhcg during kidney development. We examined kidneys from fetal days 16 (E16), 18 (E18), and 20 (E20), and from the first 21 days of postnatal development. Rhbg was expressed initially at E18, with expression only in the connecting tubule (CNT); at E20, Rhbg was expressed in both the CNT and the medullary collecting duct (MCD). In contrast, Rhcg was first expressed at E16 with basal expression in the ureteric bud; at E18, it was expressed in a subset of CNT cells with an apical pattern, followed by apical and basolateral expression in the MCD at E20. In the cortex, Rhbg and Rhcg expression increased in the CNT before expression in the cortical collecting duct during fetal development. In the MCD, both Rhbg and Rhcg expression was initially in cells in the papillary tip, with gradual removal from the tip during the late fetal period and transition during the early neonatal period to an adult pattern with predominant expression in the outer MCD and only rare expression in cells in the initial inner MCD. Double-labeling with intercalated cell-specific markers identified that Rhbg and Rhcg were expressed initially in CNT cells, CNT A-type intercalated cells and non-A, non-B intercalated cells, and in MCD A-type intercalated cells. We conclude that expression of Rhbg and Rhcg parallels intercalated cell development and that immature Rhbg and Rhcg expression at birth contributes to incomplete ammonia excretion capacity. PMID:20392801

  9. Expression of ammonia transporter family members, Rh B glycoprotein and Rh C glycoprotein, in the developing rat kidney.

    PubMed

    Han, Ki-Hwan; Lee, Su-Youn; Kim, Wan-Young; Shin, Jung-A; Kim, Jin; Weiner, I David

    2010-07-01

    Ammonia metabolism is a primary component of acid-base homeostasis but is incompletely developed at time of birth. Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg) are recently recognized ammonia transporter family members expressed in the mammalian kidney. This study's purpose was to establish the expression and localization of Rhbg and Rhcg during kidney development. We examined kidneys from fetal days 16 (E16), 18 (E18), and 20 (E20), and from the first 21 days of postnatal development. Rhbg was expressed initially at E18, with expression only in the connecting tubule (CNT); at E20, Rhbg was expressed in both the CNT and the medullary collecting duct (MCD). In contrast, Rhcg was first expressed at E16 with basal expression in the ureteric bud; at E18, it was expressed in a subset of CNT cells with an apical pattern, followed by apical and basolateral expression in the MCD at E20. In the cortex, Rhbg and Rhcg expression increased in the CNT before expression in the cortical collecting duct during fetal development. In the MCD, both Rhbg and Rhcg expression was initially in cells in the papillary tip, with gradual removal from the tip during the late fetal period and transition during the early neonatal period to an adult pattern with predominant expression in the outer MCD and only rare expression in cells in the initial inner MCD. Double-labeling with intercalated cell-specific markers identified that Rhbg and Rhcg were expressed initially in CNT cells, CNT A-type intercalated cells and non-A, non-B intercalated cells, and in MCD A-type intercalated cells. We conclude that expression of Rhbg and Rhcg parallels intercalated cell development and that immature Rhbg and Rhcg expression at birth contributes to incomplete ammonia excretion capacity.

  10. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  11. THE EFFECTS OF HIV INFECTION ON THE EXPRESSION OF THE DRUG EFFLUX PROTEINS P-GLYCOPROTEIN AND BREAST CANCER RESISTANCE PROTEIN IN A HUMAN INTESTINE MODEL

    PubMed Central

    Ellis, Kelstan; Marlin, Jerry; Taylor, Tracey AH; Fitting, Sylvia; Hauser, Kurt F.; Rice, Greg

    2015-01-01

    Objectives In HIV infection, decreased penetration of antiretroviral drugs is postulated to contribute to HIV persistence within lymphoid rich regions of the gastrointestinal (GI) tract. However, mechanistic explanations for this phenomenon remain unclear. Specifically, investigations of HIV effects on drug efflux proteins within intestinal models are minimal. Methods Using an in vitro co-culture model of the GI tract, effects of HIV infection on drug efflux proteins, P-glycoprotein and Breast Cancer Resistance Protein (BCRP) were evaluated. The influence of the HIV-1 protein, Tat, and oxidative stress on P-glycoprotein and BCRP also was evaluated. Key Findings P-glycoprotein expression demonstrated an HIV-induced upregulation in Caco-2 cells over time for cells grown in co-culture with resting lymphocytes. BCRP overall expression increased with HIV exposure in activated primary human lymphocytes co-cultured with Caco-2 cells. Tat treatment resulted in no significant alterations in P-glycoprotein (43% increase), BCRP expression, or oxidative stress. Conclusions HIV exposure within an in vitro intestinal model resulted in increases in, P-glycoprotein and BCRP in a cell specific manner. Additionally, observed changes were not mediated by Tat. Collectively, these results suggest that alterations in BCRP and P-glycoprotein may contribute, in part, to decreased antiretroviral concentrations within the gastrointestinal tract in HIV infection. PMID:25557407

  12. Glycosylation modulates arenavirus glycoprotein expression and function

    SciTech Connect

    Bonhomme, Cyrille J. Capul, Althea A. Lauron, Elvin J. Bederka, Lydia H. Knopp, Kristeene A. Buchmeier, Michael J.

    2011-01-20

    The glycoprotein of lymphocytic choriomeningitis virus (LCMV) contains nine potential N-linked glycosylation sites. We investigated the function of these N-glycosylations by using alanine-scanning mutagenesis. All the available sites were occupied on GP1 and two of three on GP2. N-linked glycan mutations at positions 87 and 97 on GP1 resulted in reduction of expression and absence of cleavage and were necessary for downstream functions, as confirmed by the loss of GP-mediated fusion activity with T87A and S97A mutants. In contrast, T234A and E379N/A381T mutants impaired GP-mediated cell fusion without altered expression or processing. Infectivity via virus-like particles required glycans and a cleaved glycoprotein. Glycosylation at the first site within GP2, not normally utilized by LCMV, exhibited increased VLP infectivity. We also confirmed the role of the N-linked glycan at position 173 in the masking of the neutralizing epitope GP-1D. Taken together, our results indicated a strong relationship between fusion and infectivity.

  13. Tamm-Horsfall glycoprotein and calcium nephrolithiasis.

    PubMed

    Hess, B

    1994-01-01

    Available data on the effects of Tamm-Horsfall glycoprotein (THP) on calcium oxalate crystallization processes are apparently conflicting. With the main emphasis on calcium oxalate crystal aggregation, this review demonstrates that THP has a dual role as a modifier of crystal aggregation: in solutions with high pH, low ionic strength (IS) and low concentrations of calcium and THP itself, the glycoprotein acts as a powerful inhibitor of calcium oxalate crystal aggregation. Conversely, low pH, high IS and high concentrations of calcium and THP all favor self-aggregation of THP molecules which lowers their inhibitory activity against calcium oxalate crystal aggregation. Some patients with severely recurrent Ca stone disease excrete abnormal THPs which self-aggregate at levels of pH, IS and concentrations of Ca and THP at which normal THPs remain in monomeric form. With high Ca concentrations, such abnormal THPs become strong promoters of crystal aggregation, since conformational changes in crystal-bound THP molecules induce strong viscous binding forces which overcome repulsive electrostatic surface charges. By chelating free Ca ions, citrate reduces self-aggregation of THP molecules and turns promoting THPs into inhibitors of calcium oxalate crystal aggregation.

  14. Enhanced detection of glycoproteins in polyacrylamide gels.

    PubMed

    Muñoz, G; Marshall, S; Cabrera, M; Horvat, A

    1988-05-01

    A highly sensitive and simple method to enhance detection of glycoproteins resolved by either one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F. Kato, and G. Chappuis (1984) Arch. Virol. 80, 69-82) that uses concanavalin A conjugated with fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially transferred to 50% methanol in deionized water and to absolute methanol. The result is an abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement of fluorescence is obtained as detected by exposing the treated gel to an appropriate uv source. The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their carbohydrate content in the range of 0.2 microgram of total protein. The specificity of the detection is demonstrated by a comparison with the corresponding polypeptide profile obtained by silver nitrate staining of the gel.

  15. Human immune responses to major human cytomegalovirus glycoprotein complexes.

    PubMed Central

    Liu, Y N; Kari, B; Gehrz, R C

    1988-01-01

    Sera from both human cytomegalovirus (HCMV)-seropositive adults and infants with congenital HCMV infection recognized two major HCMV glycoprotein complexes. However, proliferative responses of peripheral blood mononuclear cells to these complexes varied among seropositive adults and were not detected in any of the infants. Thus, these glycoproteins alone may not be sufficient to develop a subviral HCMV vaccine. Images PMID:2828655

  16. Ammonia transport in the kidney by Rhesus glycoproteins.

    PubMed

    Weiner, I David; Verlander, Jill W

    2014-05-15

    Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4(+) with a new model in which specific and regulated transport of both NH3 and NH4(+) across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport.

  17. Ammonia transport in the kidney by Rhesus glycoproteins

    PubMed Central

    Verlander, Jill W.

    2014-01-01

    Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4+ with a new model in which specific and regulated transport of both NH3 and NH4+ across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport. PMID:24647713

  18. Analgesic effects of glycoproteins from Panax ginseng root in mice.

    PubMed

    Wang, Ying; Chen, Yinghong; Xu, Hong; Luo, Haoming; Jiang, Ruizhi

    2013-07-30

    The root of Panax ginseng C.A. Mey has various beneficial pharmacological effects. The present study aimed to evaluate the analgesic activities of glycoproteins from the root of Panax ginseng C.A. Mey in mice. Glycoproteins were isolated and purified from the root of Panax ginseng C.A. Mey. Physicochemical properties and molecular mass were determined by chemical assay and HPLC. Acetic acid-induced writhing and hot-plate tests were employed to study the analgesic effect of glycoproteins and compared with that of aspirin or morphine. The locomotor activity was tested in mice by using actophometer. Four glycoproteins were obtained. The glycoproteins which protein content was the highest (73.04%) displayed dose-dependent analgesic effect. In writhing test, the glycoproteins significantly inhibited writhes (P<0.001) at the dose of 20 mg/kg by intraperitoneal injection. In hot-plate test, only at the dose of 20 mg/kg prolong the hot-plate latency (P<0.05, at 30 min). In the locomotor activity test, the glycoproteins were significant decrease of motility counts at the dose of 20 and 40 mg/kg. These findings collectively indicate that the glycoproteins from the root of Panax ginseng C.A. Mey exhibited significant analgesic activities and the proteins were the active site, providing evidence for its pharmacal use. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Decoration of proteins with sugar chains: recent advances in glycoprotein synthesis.

    PubMed

    Okamoto, Ryo; Izumi, Masayuki; Kajihara, Yasuhiro

    2014-10-01

    Chemical or chemoenzymatic synthesis is an emerging approach to produce homogeneous glycoproteins, which are hard to obtain by conventional biotechnology methods. Recent advances in the synthetic methodologies for the decoration of protein molecules with oligosaccharides provide several remarkable syntheses of homogeneous glycoproteins. This short review highlights several of the latest syntheses of glycoproteins including therapeutically important glycoproteins, a highly glycosylated protein, and unnatural glycoproteins in order to illustrate the power of the modern glycoprotein synthesis. Structurally defined glycoproteins are a novel material for understanding the molecular basis of glycoprotein functions and for the development of the next generation of biopharmaceuticals.

  20. Glycoprotein synthesis in the Golgi apparatus of spermatids during spermiogenesis of the rat

    SciTech Connect

    Clermont, Y.; Tang, X.M.

    1985-09-01

    During steps 1-7 of spermiogenesis the Golgi apparatus contributes to the formation of the acrosomic system which develops at the surface of the nucleus. Later, in step 8, the Golgi apparatus detaches from the acrosome and remains suspended in the elongated cytoplasm until it degenerates during step 16. Using /sup 3/H-fucose as a tracer and the radioautographic technique, we observed that the Golgi apparatus incorporates the tracer and delivers the labeled glycoproteins to the developing acrosomic system during steps 1-7 of spermiogenesis, to multivesicular bodies during steps 1-9, and to the remaining cytoplasm and plasma membrane during steps 1-15. Throughout these steps of spermiogenesis the Golgi apparatus does not show major changes in structure; it is composed of a cortex made up of connected stacks of saccules and a medulla showing a loose aggregate of vesicular profiles. Glycoprotein synthesis in this Golgi apparatus, before and after it contributes lysosomal glycoproteins to the growing acrosomic system, was quantitatively assessed in electron microscope EM radioautographs of tissue sections from animals sacrificed at 1, 4, 8, and 24 h of 3H-fucose injection. The incorporation of the labeled sugar was found to remain quantitatively similar during steps 1-15 of spermiogenesis, and therefore, no shift in glycoprotein synthesis took place following separation of the Golgi apparatus from the acrosomic system. Throughout these steps, fucose molecules are first incorporated in the cortex of the organelle and subsequently transported to the medulla, where they temporarily accumulate before being delivered, depending on the step of spermiogenesis, to the acrosomic system, to the multivesicular bodies, and also, presumably, to the plasma membrane.

  1. Solubilization of glycoproteins of envelope viruses by detergents

    SciTech Connect

    Berezin, V.E.; Zaides, V.M.; Artamsnov, A.F.; Isaeva, E.S.; Zhdanov, V.M.

    1986-11-20

    The action of a number of known ionic and nonionic detergents, as well as the new nonionic detergent MESK, on envelope viruses was investigated. It was shown that the nonionic detergents MESK, Triton X-100, and octyl-..beta..-D-glucopyranoside selectively solubilize the outer glycoproteins of the virus particles. The nonionic detergent MESK has the mildest action. Using MESK, purified glycoproteins of influenza, parainfluenza, Venezuelan equine encephalomyelitis, vesicular stomatitis, rabies, and herpes viruses were obtained. The procedure for obtaining glycoproteins includes incubation of the virus suspension with the detergent MESK, removal of subvirus structures by centrifuging, and purification of glycoproteins from detergents by dialysis. Isolated glycoproteins retain a native structure and biological activity and possess high immunogenicity. The detergent MESK is promising for laboratory tests and with respect to the production of subunit vaccines.

  2. The turnover rate of rabbit urinary Tamm–Horsfall glycoprotein

    PubMed Central

    Grant, Anne M. S.; Neuberger, Albert

    1973-01-01

    1. The turnover rate of urinary Tamm–Horsfall glycoprotein in rabbits was determined by two different methods. The first involved measurement of the pool size of the glycoprotein in rabbit kidney and the daily urinary excretion rate by a radioimmunoassay from which the turnover rate was calculated. 2. The second method made use of the incorporation in vivo of Na214CO3 and sodium [14C]acetate. After a single intramuscular injection of one of these compounds, urine collections were made every 24h and the glycoprotein was isolated and its specific radioactivity was determined. 3. Incorporation of the label into urinary HCO3−, urea and plasma fibrinogen was also examined. The specific radio-activities of the O-acetyl, sialic acid, aspartic acid and glutamic acid residues isolated from the Tamm–Horsfall glycoprotein were compared and their half-lives were compared with that of the intact glycoprotein. The two methods gave results in quite close agreement and indicated a half-life for the glycoprotein of approx. 9h. 4. An attempt was made to localize the glycoprotein within the kidney and within the cell. It is present throughout the kidney, but was not detected in the brush-border fraction isolated from the proximal tubules. From differential cell-centrifugation studies, the glycoprotein seemed to be predominantly present in the soluble fraction (100000g supernatant). This suggests that it is either largely a soluble cytoplasmic component or is very loosely bound to a membrane, being readily released under the gentlest homogenization procedure. 5. The half-life of Tamm–Horsfall glycoprotein in human kidney was found by the radioimmunoassay method to be approx. 16h. The similarity between the composition of Tamm–Horsfall glycoprotein and human erythropoietin is discussed. PMID:4780692

  3. Array-based analysis of secreted glycoproteins for rapid selection of a single cell producing a glycoprotein with desired glycosylation.

    PubMed

    Park, Sunyoung; Kim, Wanjung; Kim, Yongtae; Son, Young Dok; Lee, Sang-Chul; Kim, Eunkyung; Kim, Sung Ho; Kim, Jung Hoe; Kim, Hak-Sung

    2010-07-01

    The therapeutic efficacy and in vivo biological function of a glycoprotein is significantly affected by its glycosylation profile. For the development of glycoproteins with therapeutic applications, selection of cell lines producing a glycoprotein with adequate glycoform is crucial. Here, we demonstrate an array-based analysis of secreted glycoproteins for rapid and efficient selection of a single cell producing a glycoprotein with desirable glycosylation. Our approach relies on microengraving and interrogation of glycoproteins produced by individual cells in a microwell array in terms of glycosylation profile as well as the produced amount. On the basis of statistical analysis of the interrogation, single cells which are predicted to produce a desired glycoprotein are selected, retrieved, and expanded. We applied the approach to human recombinant erythropoietin (rhEPO)-producing CHO cells and verified the selection of a single CHO cell that produces rhEPO with a high sialylation degree. Human erythropoietin (hEPO) bearing highly sialylated oligosaccharide was shown to display a much longer plasma half-life, resulting in high therapeutic efficacy. This method may find widespread use in the clonal selection for the production of other glycoproteins with specific glycosylation as well as analysis of the heterogeneity in cell populations in a high-throughput manner.

  4. The primary structure of a procaryotic glycoprotein. Cloning and sequencing of the cell surface glycoprotein gene of halobacteria.

    PubMed

    Lechner, J; Sumper, M

    1987-07-15

    The hexagonally patterned surface layer of halobacteria consists of a true glycoprotein. This procaryotic glycoprotein has recently been shown to exhibit novel features with respect to saccharide structure and saccharide biosynthesis. The primary structure and the location of glycosylation sites were determined by cloning and sequencing of the glycoprotein gene of Halobacterium halobium. According to the predicted amino acid sequence, the glycoprotein is synthesized with a N-terminal leader sequence of 34 amino acid residues reminiscent of eucaryotic and procaryotic signal peptides. A hydrophobic stretch of 21 amino acid residues at the C terminus probably serves as a transmembrane domain. 14 threonine residues are clustered adjacent to this membrane anchor and linked to these threonines are all the disaccharides of the cell surface glycoprotein. 12 N-glycosylation sites are distributed over the polypeptide chain.

  5. Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins

    PubMed Central

    1978-01-01

    Rat liver microsomal glycoproteins were purified by affinity chromatography on concanavalin A Sepharose columns from membrane and content fractions, separated from rough microsomes (RM) treated with low concentrations of deoxycholate (DOC). All periodic acid-Schiff (PAS)-positive glycoproteins of RM showed affinity for concanavalin A Sepharose; even after sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, most of the microsomal glycoproteins bound [125I]concanavalin A added to the gels, as detected by autoradiography. Two distinct sets of glycoproteins are present in the membrane and content fractions derived from RM. SDS acrylamide gel electrophoresis showed that RM membranes contain 15--20 glycoproteins (15--22% of the total microsomal protein) which range in apparent mol wt from 23,000 to 240,000 daltons. A smaller set of glycoproteins (five to seven polypeptides), with apparent mol wt between 60,000 and 200,000 daltons, was present in the microsomal content fraction. The disposition of the membrane glycoproteins with respect to the membrane plane was determined by selective iodination with the lactoperoxidase (LPO) technique. Intact RM were labeled on their outer face with 131I and, after opening of the vesicles with 0.05% DOC, in both faces with 125I. An analysis of iodination ratios for individual proteins separated electrophoretically showed that in most membrane glycoproteins, tyrosine residues are predominantly exposed on the luminal face of the vesicles, which is the same face on which the carbohydrate moieties are exposed. Several membrane glycoproteins are also exposed on the cytoplasmic surface and therefore have a transmembrane disposition. In this study, ribophorins I and II, two integral membrane proteins (mol wt 65,000 and 63,000) characteristic of RM, were found to be transmembrane glycoproteins. It is suggested that the transmembrane disposition of the ribophorins may be related to their possible role in ribosome binding and in the

  6. Glycoproteins and glycoproteomics in pancreatic cancer

    PubMed Central

    Pan, Sheng; Brentnall, Teresa A; Chen, Ru

    2016-01-01

    Aberrations in protein glycosylation and polysaccharides play a pivotal role in pancreatic tumorigenesis, influencing cancer progression, metastasis, immuno-response and chemoresistance. Abnormal expression in sugar moieties can impact the function of various glycoproteins, including mucins, surface receptors, adhesive proteins, proteoglycans, as well as their effectors and binding ligands, resulting in an increase in pancreatic cancer invasiveness and a cancer-favored microenvironment. Recent advance in glycoproteomics, glycomics and other chemical biology techniques have been employed to better understand the complex mechanism of glycosylation events and how they orchestrate molecular activities in genomics, proteomics and metabolomics implicated in pancreatic adenocarcinoma. A variety of strategies have been demonstrated targeting protein glycosylation and polysaccharides for diagnostic and therapeutic development. PMID:27895417

  7. Glycoprotein expression in human milk during lactation.

    PubMed

    Froehlich, John W; Dodds, Eric D; Barboza, Mariana; McJimpsey, Erica L; Seipert, Richard R; Francis, Jimi; An, Hyun Joo; Freeman, Samara; German, J Bruce; Lebrilla, Carlito B

    2010-05-26

    While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first 10 days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor.

  8. Conformational Changes of the Flavivirus E Glycoprotein

    PubMed Central

    Zhang, Ying; Zhang, Wei; Ogata, Steven; Clements, David; Strauss, James H.; Baker, Timothy S.; Kuhn, Richard J.; Rossmann, Michael G.

    2014-01-01

    Summary Dengue virus, a member of the Flaviviridae family, has a surface composed of 180 copies each of the envelope (E) glycoprotein and the membrane (M) protein. The crystal structure of an N-terminal fragment of E has been determined and compared with a previously described structure. The primary difference between these structures is a 10° rotation about a hinge relating the fusion domain DII to domains DI and DIII. These two rigid body components were used for independent fitting of E into the cryo-electron microscopy maps of both immature and mature dengue viruses. The fitted E structures in these two particles showed a difference of 27° between the two components. Comparison of the E structure in its postfusion state with that in the immature and mature virions shows a rotation approximately around the same hinge. Flexibility of E is apparently a functional requirement for assembly and infection of flaviviruses. PMID:15341726

  9. Immunological aspects of pregnancy-associated glycoproteins.

    PubMed

    Dosogne, H; Massart-Leën, A M; Burvenich, C

    2000-01-01

    The incidence of severe cases of acute E. coli mastitis in dairy cows is highest during early lactation. This phenomenon has been associated with a decreased function and decreased numbers of circulating polymorphonuclear neutrophil leukocytes (PMN). The cause of this impaired function and decreased number is poorly understood. Stress, hormonal and metabolic alterations around parturition and the onset of lactation may play a role in this phenomenon. Several molecules, such as cortisol and beta-hydroxybutyrate have been found to alter the oxidative burst activity of circulating PMN around parturition. Pregnancy-Associated Glycoprotein (bPAG) could also be involved. The theory of immunosuppression by bPAG was investigated because analogous glycoproteins produced by the placenta of other species exert local immunosuppression in order to maintain the histoincompatible feto-maternal unit. The production and subsequent release into the maternal circulation of bPAG is ensured by the binucleate cells from the trophoblast and starts already at implantation. However, peak levels are only reached 1 week before parturition. Due to the long half-life time of this molecule, high levels are found in plasma until 2 weeks after calving. The co-occurrence of the impairment of PMN oxidative burst activity in the early postpartum period and a peak in plasma bPAG concentrations might support the hypothesis of an immunosuppressive effect of PAG. Moreover, an inhibitory effect of bPAG on the proliferation of bovine bone marrow progenitor cells has been found recently in our laboratory. bPAG occurs in colostrum, but its effect on milk cells has not been clarified. It is concluded that interaction between the physiology of reproduction and lactation on the one side and immune function on the other side in dairy cattle requires further research.

  10. P-glycoprotein in autoimmune rheumatic diseases.

    PubMed

    García-Carrasco, M; Mendoza-Pinto, C; Macias Díaz, S; Vera-Recabarren, M; Vázquez de Lara, L; Méndez Martínez, S; Soto-Santillán, P; González-Ramírez, R; Ruiz-Arguelles, A

    2015-07-01

    P-glycoprotein (Pgp) is a transmembrane protein of 170 kD encoded by the multidrug resistance 1 (MDR-1) gene, localized on chromosome 7. More than 50 polymorphisms of the MDR-1 gene have been described; a subset of these has been shown to play a pathophysiological role in the development of inflammatory bowel disease, femoral head osteonecrosis induced by steroids, lung cancer and renal epithelial tumors. Polymorphisms that have a protective effect on the development of conditions such as Parkinson disease have also been identified. P-glycoprotein belongs to the adenosine triphosphate binding cassette transporter superfamily and its structure comprises a chain of approximately 1280 aminoacid residues with an N-C terminal structure, arranged as 2 homologous halves, each of which has 6 transmembrane segments, with a total of 12 segments with 2 cytoplasmic nucleotide binding domains. Many cytokines like interleukin 2 and tumor necrosis factor alpha increase Pgp expression and activity. Pgp functions as an efflux pump for a variety of toxins in order to protect particular organs and tissues as the central nervous system. Pgp transports a variety of substrates including glucocorticoids while other drugs such as tacrolimus and cyclosporine A act as modulators of this protein. The most widely used method to measure Pgp activity is flow cytometry using naturally fluorescent substrates such as anthracyclines or rhodamine 123. The study of drug resistance and its association to Pgp began with the study of resistance to chemotherapy in the treatment of cancer and antiretroviral therapy for human immunodeficiency virus; however, the role of Pgp in the treatment of systemic lupus erythematosus, rheumatoid arthritis and psoriatic arthritis has been a focus of study lately and has emerged as an important mechanism by which treatment failure occurs. The present review analyzes the role of Pgp in these autoimmune diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein

    SciTech Connect

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai . E-mail: chyang@emory.edu

    2006-08-15

    The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.

  12. Immunomodulatory Effects of Nontoxic Glycoprotein Fraction Isolated from Rice Bran.

    PubMed

    Park, Ho-Young; Yu, A-Reum; Hong, Hee-Do; Kim, Ha Hyung; Lee, Kwang-Won; Choi, Hee-Don

    2016-05-01

    Rice bran, a by-product of brown rice milling, is a rich source of dietary fiber and protein, and its usage as a functional food is expected to increase. In this study, immunomodulatory effects of glycoprotein obtained from rice bran were studied in normal mice and mouse models of cyclophosphamide-induced immunosuppression. We prepared glycoprotein from rice bran by using ammonium precipitation and anion chromatography techniques. Different doses of glycoprotein from rice bran (10, 25, and 50 mg/kg) were administered orally for 28 days. On day 21, cyclophosphamide at a dose of 100 mg/kg was administered intraperitoneally. Glycoprotein from rice bran showed a significant dose-dependent restoration of the spleen index and white blood cell count in the immunocompromised mice. Glycoprotein from rice bran affected the immunomodulatory function by inducing the proliferation of splenic lymphocytes, which produce potential T and B cells. Moreover, it prevented cyclophosphamide-induced damage of Th1-type immunomodulatory function through enhanced secretion of Th1-type cytokines (interferon-γ and interleukin-12). These results indicate that glycoprotein from rice bran significantly recovered cyclophosphamide-induced immunosuppression. Based on these data, it was concluded that glycoprotein from rice bran is a potent immunomodulator and can be developed to recover the immunity of immunocompromised individuals.