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Joseph Sodroski - One of the best experts on this subject based on the ideXlab platform.
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identification of a human immunodeficiency virus type 1 envelope glycoprotein variant resistant to cold inactivation
2009Co-Authors: Aemro Kassa, Andres Finzi, Marie Pancera, Joel R Courter, Amos B Smith, Joseph SodroskiAbstract: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.
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cytolysis by ccr5 using human immunodeficiency virus type 1 envelope glycoproteins is dependent on membrane fusion and can be inhibited by high levels of cd4 expression
2003Co-Authors: Jason A Labonte, Navid Madani, Joseph SodroskiAbstract:T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4(+) CXCR4(+) T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5(+) target cells. As is the case for CXCR4(+) target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4(+) CCR5(+) cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4(+) T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4(+) T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.
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highly stable trimers formed by human immunodeficiency virus type 1 envelope glycoproteins fused with the trimeric motif of t4 bacteriophage fibritin
2002Co-Authors: Xinzhen Yang, Juliette Lee, Erin Mahony, Peter D Kwong, Richard T Wyatt, Joseph SodroskiAbstract:The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane proteins. Soluble gp140 glycoproteins composed of the uncleaved ectodomains of gp120 and gp41 form unstable, heterogeneous oligomers, but soluble gp140 trimers can be stabilized by fusion with a C-terminal, trimeric GCN4 motif (X. Yang et al., J. Virol. 74:5716-5725, 2000). To understand the influence of the C-terminal trimerization domain on the properties of soluble HIV-1 envelope glycoprotein trimers, uncleaved, soluble gp140 glycoproteins were stabilized by fusion with another trimeric motif derived from T4 bacteriophage fibritin. The fibritin construct was more stable to heat and reducing conditions than the GCN4 construct. Both GCN4- and fibritin-stabilized soluble gp140 glycoproteins exhibited patterns of neutralizing and nonneutralizing antibody binding expected for the functional envelope glycoprotein spike. Of note, two potently neutralizing antibodies, immunoglobulin G1b12 and 2G12, exhibited the greatest recognition of the stabilized, soluble trimers, relative to recognition of the gp120 monomer. The observed similarities between the GCN4 and fibritin constructs indicate that the HIV-1 envelope glycoprotein ectodomains dictate many of the antigenic and structural features of these fusion proteins. The melting temperatures and ligand recognition properties of the GCN4- and fibritin-stabilized soluble gp140 glycoproteins suggest that these molecules assume conformations distinct from that of the fusion-active, six-helix bundle.
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modifications that stabilize human immunodeficiency virus envelope glycoprotein trimers in solution
2000Co-Authors: Xinzhen Yang, Joseph Sodroski, Peter D Kwong, Lauren Florin, Michael Farzan, Peter Kolchinsky, Richard T WyattAbstract:The functional unit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins is a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modifications that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: disruption of the proteolytic cleavage site between gp120 and gp41, introduction of cysteines that form intersubunit disulfide bonds, and addition of GCN4 trimeric helices. Characterization of these secreted glycoproteins by immunologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing the trimers, the formation of intersubunit disulfide bonds between appropriately placed cysteines, and the ability of the trimers to interact with a helical, C-terminal gp41 peptide (DP178) support a model in which the N-terminal gp41 coiled coil exists in the envelope glycoprotein precursor and contributes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progress in understanding the structure and function of the virion envelope glycoprotein spikes.
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replicative function and neutralization sensitivity of envelope glycoproteins from primary and t cell line passaged human immunodeficiency virus type 1 isolates
1995Co-Authors: Nancy Sullivan, J Li, Wolfgang Hofmann, Joseph SodroskiAbstract:The structure, replicative properties, and sensitivity to neutralization by soluble CD4 and monoclonal antibodies were examined for molecularly cloned envelope glycoproteins derived from human immunodeficiency virus type 1 (HIV-1) viruses either isolated directly from patients or passaged in T-cell lines. Complementation of virus entry into peripheral blood mononuclear cell targets by primary patient envelope glycoproteins exhibited efficiencies ranging from that observed for the HXBc2 envelope glycoproteins, which are derived from a T-cell line-passaged virus, to approximately fivefold-lower values. The ability of the envelope glycoproteins to complement virus entry roughly correlated with sensitivity to neutralization by soluble CD4. Laboratory-adapted viruses were sensitive to neutralization by monoclonal antibodies directed against the CD4-binding site and the third variable (V3) loop of the gp120 glycoprotein. By comparison, viruses with envelope glycoproteins from primary patient isolates exhibited decreased sensitivity to neutralization by these monoclonal antibodies; for these viruses, neutralization sensitivity correlated with replicative ability. Subinhibitory concentrations of soluble CD4 and a CD4-binding site-directed antibody significantly enhanced the entry of viruses containing envelope glycoproteins from some primary patient isolates. The sensitivity of viruses containing the different envelope glycoproteins to neutralization by soluble CD4 or monoclonal antibodies could be predicted by assays dependent on the binding of the inhibitory molecule to the oligomeric envelope glycoprotein complex but less well by assays measuring binding to the monomeric gp120 glycoprotein. These results indicate that the intrinsic structure of the oligomeric envelope glycoprotein complex of primary HIV-1 isolates, while often less than optimal with respect to the mediation of early events in virus replication, allows a relative degree of resistance to neutralizing antibodies. The interplay of selective forces for higher virus replication efficiency and resistance to neutralizing antibodies could explain the temporal course described for the in vivo emergence of HIV-1 isolates with differing phenotypes.
David M. Lubman - One of the best experts on this subject based on the ideXlab platform.
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comparative serum glycoproteomics using lectin selected sialic acid glycoproteins with mass spectrometric analysis application to pancreatic cancer serum
2006Co-Authors: Jia Zhao, Diane M Simeone, David G Heidt, Michelle A Anderson, David M. LubmanAbstract:A strategy is developed in this study for identifying sialylated glycoprotein markers in human cancer serum. This method consists of three steps: lectin affinity selection, a liquid separation and characterization of the glycoprotein markers using mass spectrometry. In this work, we use three different lectins (Wheat Germ Agglutinin, (WGA) Elderberry lectin,(SNA), Maackia amurensis lectin, (MAL)) to extract sialylated glycoproteins from normal and cancer serum. Twelve highly abundant proteins are depleted from the serum using an IgY-12 antibody column. The use of the different lectin columns allows one to monitor the distribution of α(2,3) and α(2,6) linkage type sialylation in cancer serum vs that in normal samples. Extracted glycoproteins are fractionated using NPS−RP−HPLC followed by SDS-PAGE. Target glycoproteins are characterized further using mass spectrometry to eludicate the carbohydrate structure and glycosylation site. We applied this approach to the analysis of sialylated glycoproteins in panc...
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comparative serum glycoproteomics using lectin selected sialic acid glycoproteins with mass spectrometric analysis application to pancreatic cancer serum
2006Co-Authors: Jia Zhao, Diane M Simeone, David G Heidt, Michelle A Anderson, David M. LubmanAbstract:A strategy is developed in this study for identifying sialylated glycoprotein markers in human cancer serum. This method consists of three steps: lectin affinity selection, a liquid separation and characterization of the glycoprotein markers using mass spectrometry. In this work, we use three different lectins (Wheat Germ Agglutinin, (WGA) Elderberry lectin,(SNA), Maackia amurensis lectin, (MAL)) to extract sialylated glycoproteins from normal and cancer serum. Twelve highly abundant proteins are depleted from the serum using an IgY-12 antibody column. The use of the different lectin columns allows one to monitor the distribution of alpha(2,3) and alpha(2,6) linkage type sialylation in cancer serum vs that in normal samples. Extracted glycoproteins are fractionated using NPS-RP-HPLC followed by SDS-PAGE. Target glycoproteins are characterized further using mass spectrometry to elucidate the carbohydrate structure and glycosylation site. We applied this approach to the analysis of sialylated glycoproteins in pancreatic cancer serum. Approximately 130 sialylated glycoproteins are identified using microLC-MS/MS. Sialylated plasma protease C1 inhibitor is identified to be down-regulated in cancer serum. Changes in glycosylation sites in cancer serum are also observed by glycopeptide mapping using microLC-ESI-TOF-MS where the N83 glycosylation of alpha1-antitrypsin is down regulated. In addition, the glycan structures of the altered proteins are assigned using MALDI-QIT-MS. This strategy offers the ability to quantitatively analyze changes in glycoprotein abundance and detect the extent of glycosylation alteration as well as the carbohydrate structure that correlate with cancer.
Richard T Wyatt - One of the best experts on this subject based on the ideXlab platform.
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rational modifications of hiv 1 envelope glycoproteins for immunogen design
2007Co-Authors: Sanjay Phogat, Richard T WyattAbstract:: An effective vaccine against the human immunodeficiency virus type 1 (HIV-1) will likely require the elicitation of broadly neutralizing antibodies as well as cellular responses. The HIV exterior envelope glycoprotein trimers, gp120, and the transmembrane glycoprotein, gp41, mediate entry and are the sole viral targets for neutralizing antibodies. However, as subunit immunogens the envelope glycoproteins do not efficiently elicit antibodies capable of neutralizing the extremely diverse array of viruses circulating in the human population. The preponderance of data suggest that inefficient generation of broadly neutralizing antibodies is due to naturally evolved mechanisms of immune evasion inherent in the unmodified HIV envelope glycoproteins. Because the established modes of anti-viral vaccine development, live-attenuation and virus inactivation have not yet been successful for HIV, we and others have focused on subunit vaccine design. In this review, we describe current approaches of rational modification of the envelope glycoproteins based upon structure, antigenicity, biochemistry and biophysics to alter the properties of the envelope glycoproteins such that, as subunit immunogens, they now better elicit broadly neutralizing antibodies. The application of structure-assisted, rational subunit vaccine design may be a general paradigm for future efforts to develop vaccines against emerging human pathogens.
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highly stable trimers formed by human immunodeficiency virus type 1 envelope glycoproteins fused with the trimeric motif of t4 bacteriophage fibritin
2002Co-Authors: Xinzhen Yang, Juliette Lee, Erin Mahony, Peter D Kwong, Richard T Wyatt, Joseph SodroskiAbstract:The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane proteins. Soluble gp140 glycoproteins composed of the uncleaved ectodomains of gp120 and gp41 form unstable, heterogeneous oligomers, but soluble gp140 trimers can be stabilized by fusion with a C-terminal, trimeric GCN4 motif (X. Yang et al., J. Virol. 74:5716-5725, 2000). To understand the influence of the C-terminal trimerization domain on the properties of soluble HIV-1 envelope glycoprotein trimers, uncleaved, soluble gp140 glycoproteins were stabilized by fusion with another trimeric motif derived from T4 bacteriophage fibritin. The fibritin construct was more stable to heat and reducing conditions than the GCN4 construct. Both GCN4- and fibritin-stabilized soluble gp140 glycoproteins exhibited patterns of neutralizing and nonneutralizing antibody binding expected for the functional envelope glycoprotein spike. Of note, two potently neutralizing antibodies, immunoglobulin G1b12 and 2G12, exhibited the greatest recognition of the stabilized, soluble trimers, relative to recognition of the gp120 monomer. The observed similarities between the GCN4 and fibritin constructs indicate that the HIV-1 envelope glycoprotein ectodomains dictate many of the antigenic and structural features of these fusion proteins. The melting temperatures and ligand recognition properties of the GCN4- and fibritin-stabilized soluble gp140 glycoproteins suggest that these molecules assume conformations distinct from that of the fusion-active, six-helix bundle.
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modifications that stabilize human immunodeficiency virus envelope glycoprotein trimers in solution
2000Co-Authors: Xinzhen Yang, Joseph Sodroski, Peter D Kwong, Lauren Florin, Michael Farzan, Peter Kolchinsky, Richard T WyattAbstract:The functional unit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins is a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modifications that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: disruption of the proteolytic cleavage site between gp120 and gp41, introduction of cysteines that form intersubunit disulfide bonds, and addition of GCN4 trimeric helices. Characterization of these secreted glycoproteins by immunologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing the trimers, the formation of intersubunit disulfide bonds between appropriately placed cysteines, and the ability of the trimers to interact with a helical, C-terminal gp41 peptide (DP178) support a model in which the N-terminal gp41 coiled coil exists in the envelope glycoprotein precursor and contributes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progress in understanding the structure and function of the virion envelope glycoprotein spikes.
Joseph Mendicino - One of the best experts on this subject based on the ideXlab platform.
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udp glcnac gal beta 3galnac mucin glcnac galnac beta 6 n acetylglucosaminyltransferase and udp glcnac gal beta 3 glcnac beta 6 galnac mucin glcnac gal beta 3 n acetylglucosaminyltransferase from swine trachea epithelium
1991Co-Authors: Sreedhara Sangadala, Subramanian Sivakami, Joseph MendicinoAbstract:Two specific beta-N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a beta 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal beta 3GalNAc-mucin to yield Gal beta 3(GlcNAc beta 6)GalNAc-Mucin and a beta 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal beta 3(GlcNAc beta 6)GalNAc-mucin to yield GlcNAc beta 3Gal beta 3 (GlcNAc beta 6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The beta 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal beta 1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal beta 1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the beta 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal beta 1,3GalNAc chains was 0.53 microM; for UDP-N-acetylglucosamine, 12 microM; and for Gal beta 1,3GalNAc alpha NO2 phi, 4 mM. The activity of the beta 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal beta 3GalNAc chains in Cowper's gland mucin glycoprotein. The best substrate for the partially purified beta 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal beta 1,3(GlcNAc beta 6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal beta 1,3GalNAc side chains. The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the beta 6- and beta 3-glucosaminyltransferases were shown to be Gal beta 3(GlcNAc beta 6) GalNAc and GlcNAc beta 3 Gal beta 3(GlcNAc beta 6)GalNAc respectively. Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a beta 6-glucosaminyltransferase converts Gal beta 3GalNAc chains in mucin glycoproteins to Gal beta 3(GlcNAc beta 6)GalNAc chains.(ABSTRACT TRUNCATED AT 400 WORDS)
Nancy Sullivan - One of the best experts on this subject based on the ideXlab platform.
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replicative function and neutralization sensitivity of envelope glycoproteins from primary and t cell line passaged human immunodeficiency virus type 1 isolates
1995Co-Authors: Nancy Sullivan, J Li, Wolfgang Hofmann, Joseph SodroskiAbstract:The structure, replicative properties, and sensitivity to neutralization by soluble CD4 and monoclonal antibodies were examined for molecularly cloned envelope glycoproteins derived from human immunodeficiency virus type 1 (HIV-1) viruses either isolated directly from patients or passaged in T-cell lines. Complementation of virus entry into peripheral blood mononuclear cell targets by primary patient envelope glycoproteins exhibited efficiencies ranging from that observed for the HXBc2 envelope glycoproteins, which are derived from a T-cell line-passaged virus, to approximately fivefold-lower values. The ability of the envelope glycoproteins to complement virus entry roughly correlated with sensitivity to neutralization by soluble CD4. Laboratory-adapted viruses were sensitive to neutralization by monoclonal antibodies directed against the CD4-binding site and the third variable (V3) loop of the gp120 glycoprotein. By comparison, viruses with envelope glycoproteins from primary patient isolates exhibited decreased sensitivity to neutralization by these monoclonal antibodies; for these viruses, neutralization sensitivity correlated with replicative ability. Subinhibitory concentrations of soluble CD4 and a CD4-binding site-directed antibody significantly enhanced the entry of viruses containing envelope glycoproteins from some primary patient isolates. The sensitivity of viruses containing the different envelope glycoproteins to neutralization by soluble CD4 or monoclonal antibodies could be predicted by assays dependent on the binding of the inhibitory molecule to the oligomeric envelope glycoprotein complex but less well by assays measuring binding to the monomeric gp120 glycoprotein. These results indicate that the intrinsic structure of the oligomeric envelope glycoprotein complex of primary HIV-1 isolates, while often less than optimal with respect to the mediation of early events in virus replication, allows a relative degree of resistance to neutralizing antibodies. The interplay of selective forces for higher virus replication efficiency and resistance to neutralizing antibodies could explain the temporal course described for the in vivo emergence of HIV-1 isolates with differing phenotypes.
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effect of amino acid changes in the v1 v2 region of the human immunodeficiency virus type 1 gp120 glycoprotein on subunit association syncytium formation and recognition by a neutralizing antibody
1993Co-Authors: Nancy Sullivan, Markus Thali, C Furman, David D Ho, Joseph SodroskiAbstract:The contributions of the first and second variable regions of the human immunodeficiency virus type 1 gp120 glycoprotein to envelope glycoprotein structure, function, and recognition by a neutralizing antibody were studied. Several mutants with substitutions in the V2 loop demonstrated complete dissociation of the gp120 and gp41 glycoproteins, suggesting that inappropriate changes in V2 conformation can affect subunit assembly. Some glycoproteins with changes in V1 or V2 were efficiently expressed on the cell surface and were able to bind CD4 but were deficient in syncytium formation and/or virus entry. Recognition of gp120 by the neutralizing monoclonal antibody G3-4 was affected by particular substitutions affecting residues 176 to 184 in the V2 loop. These results suggest that the V1/V2 variable regions of the human immunodeficiency virus type 1 gp120 glycoprotein play a role in postreceptor binding events in the membrane fusion process and can act as a target for neutralizing antibodies. Images
