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Robert Blumenthal

Publications and source records attributed to Robert Blumenthal.

36 records · Page 2Linked to original sources
PubMed2004

An inhibitor of glycosphingolipid metabolism blocks HIV-1 infection of primary T-cells.

OBJECTIVE: HIV-1 uses CD4 and chemokine receptors to enter cells. However, other target membrane components may also be involved. This study examines the role of glycosphingolipids (GSL) in HIV-1 entry into primary lymphocytes and its modulation by an inhibitor of GSL biosynthesis. METHODS: CD4 lymphocytes purified from normal or the p-group subtype individuals that were defective in Gb3 synthesis were treated with a GSL biosynthesis inhibitor, 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP). The PPMP-treated cells were tested for HIV-1 replication by measuring p24 antigen production for 7-14 days post-infection and for susceptibility to HIV-1 Env-mediated fusion monitored by a fluorescent dye transfer assay. The effects of PPMP treatment on HIV-1 binding to CD4 lymphocytes were also examined by measuring HIV-1 p24. RESULTS: CD4 lymphocytes from p donors that are devoid of Gb3, but have elevated levels of GM3 were highly susceptible to HIV-1 fusion/entry. Pre-treatment of primary human CD4 lymphocytes from normal or p-sub-group type with PPMP, significantly reduced HIV-1 replication with no change in CD4 and CXCR4 levels. Inhibition of HIV-1 infection was due to the block in HIV-1 Env-mediated plasma membrane fusion. Binding of HIV-1 to CD4 lymphocytes was not affected by PPMP treatment. CONCLUSION: Manipulation of glycosphingolipid metabolic pathways may alter susceptibility of CD4 lymphocytes to HIV-1 entry.

CD4-Positive T-Lymphocytes↗
PubMed2004

The C- and the N-terminal regions of glycoprotein 41 ectodomain fuse membranes enriched and not enriched with cholesterol, respectively.

To infect target cells, HIV-1 employs a virally encoded transmembrane protein (gp41) to fuse its viral envelope with the target cell plasma membrane. We describe the gp41 ectodomain as comprised of N- and C-terminal subdomains, each containing a heptad repeat as well as a fusogenic region, whose organization is mirrored by the intervening loop region. Recent evidence indicates that the gp41 directed fusion reaction proceeds to initial pore formation prior to gp41 folding into its low energy hairpin conformation. This implies that exposed regions of the gp41 ectodomain are responsible for the bulk of the fusion work, probably through direct protein-membrane interactions. Prevalent fusion models contend that the gp41 ectodomain initially interacts with the target cell surface through its highly hydrophobic N terminus, which is believed to insert into the target membrane, thereby linking the virus to the target cell. This arrangement allows the N-terminal subdomain to interact with the target cell surface, whereas the C-terminal subdomain remains proximal to the virion, allowing interaction with the viral envelope. The composition of the viral envelope and the target cell surface differ due to the virus budding from raft microdomains. We show here that constructs corresponding to the C-terminal subdomain specifically destabilize ordered and cholesterol rich membranes (33 molar %), whereas the N-terminal subdomain is more effective in fusing both unordered cholesterol-free membranes and those containing lower amounts of cholesterol (10 molar %). Moreover we show that, in the context of the C-terminal subdomain, the heptad repeat contributes helical structure, which may describe the enhanced inhibitory effect of the C-terminal subdomain relative to the C-terminal heptad repeat (C34) alone. Our results are discussed in light of recent findings that showcase the role of exposed gp41 regions in effecting membrane fusion.

Amino Acid Sequence↗
PubMed2004

Functional expression of CD4, CXCR4, and CCR5 in glycosphingolipid-deficient mouse melanoma GM95 cells and susceptibility to HIV-1 envelope glycoprotein-triggered membrane fusion.

We had previously reported that glycosphingolipids (GSL) support human immunodeficiency virus type 1 (HIV-1) entry. In this study, we further examined this issue by expressing HIV-1 receptors in GSL-deficient GM95 cells. GM95 cells expressing low levels of CD4 and CXCR4 or CCR5 did not support HIV-1 Env-mediated fusion. However, higher expression of these receptors rendered GM95 cells highly susceptible to fusion with cells expressing appropriate HIV-1 envelope glycoproteins (HIV-1 Envs). The GM95 cells exhibited a different fusion phenotype when compared with GSL(+) NIH3T3 cells bearing similar receptor levels. Fusion of GM95 targets expressing higher levels of CD4 and coreceptors occurred at 25 degrees C and was sensitive to cholesterol depletion or disruption of the cytoskeleton. In contrast, the fusion threshold of NIH3T3CD4X4/R5 targets was at >/=28 degrees C as previously reported and was insensitive to cholesterol depletion or cytoskeletal network disruption. On the basis of these observations, we propose that target membrane GSLs support HIV-1 Env-mediated fusion at low density of receptors by stabilizing receptor pools in natural targets.

Animals↗
PubMed2004

The role of glycosphingolipids in HIV signaling, entry and pathogenesis.

Although HIV uses CD4 and coreceptors (CCR5 and CXCR4) for productive infection of T cells, glycosphingolipids (GSL) may play ancillary roles in lymphoid and non-lymphoid cells. Interactions of the HIV Envelope Glycoprotein (Env) with GSL may help HIV in various steps of its pathogenesis. Physical-chemical aspects of the interactions between HIV Env and GSL leading to CD4-dependent entry into lymphocytes, the role of GSL in HIV transcytosis, and CD4-independent entry into non-lymphoid cells are reviewed. An overview of signaling properties of HIV receptors is provided with some speculation on how GSL may play a role in these events by virtue of being in membrane rafts. Finally, we summarize how interactions between HIV and coreceptors leading to signaling and/or fusion can be analyzed by the use of various tyrosine kinase and cytoskeletal inhibitors.

CD4 Antigens↗
PubMed2004

Elevated expression of GM3 in receptor-bearing targets confers resistance to human immunodeficiency virus type 1 fusion.

GM3, a major ganglioside of T lymphocytes, promotes human immunodeficiency virus type 1 (HIV-1) entry via interactions with HIV-1 receptors and the viral envelope glycoprotein (Env). Increased GM3 levels in T lymphocytes and the appearance of anti-GM3 antibodies in AIDS patients have been reported earlier. In this study, we investigated the effect of GM3 regulation on HIV-1 entry by utilizing a mouse cell line (B16F10), which expresses exceptionally high levels of GM3. Strikingly, B16 cells bearing CD4, CXCR4, and/or CCR5 were highly resistant to CD4-dependent HIV-1 Env-mediated membrane fusion. In contrast, these targets supported membrane fusion mediated by CD4-requiring HIV-2, SIV, and CD4-independent HIV-1 Envs. Coreceptor function was not impaired by GM3 overexpression as indicated by Ca(2+) fluxes mediated by the CXCR4 ligand SDF-1alpha and the CCR5 ligand MIP-1beta. Reduction in GM3 levels of B16 target cells resulted in a significant recovery of CD4-dependent HIV-1 Env-mediated fusion. We propose that GM3 in the plasma membrane blocks HIV-1 Env-mediated fusion by interfering with the lateral association of HIV-1 receptors. Our findings offer a novel mechanism of interplay between membrane lipids and receptors by which host cells may escape viral infections.

Animals↗
PubMed2003

Role of the fusion peptide and membrane-proximal domain in HIV-1 envelope glycoprotein-mediated membrane fusion.

The N-terminal fusion peptide and the interfacial sequence preceding the transmembrane anchor of HIV-1 gp41 are required for viral fusion. Studies with synthetic peptides indicated that these regions function by destabilizing membranes, which is regarded as a crucial step in the membrane fusion reaction. However, it is not clear whether membrane destabilization is induced by these sequences in the intact gp41. We address this question by examining fusion and destabilization of membranes expressing HIV-1(IIIB) wild-type Env and two mutant Envs. (1) A Glu residue at position 2 of the gp41 fusion peptide is substituted for Val (V2E) to produce one mutant. (2) Residues 665-682 in the membrane-proximal domain are deleted to form the other. The process of membrane destabilization was monitored by the influx of Sytox, an impermeant fluorescent dye, into the Env-expressing cells following the interaction with CD4-CXCR4 complexes, and fusion was monitored by observing dye transfer between Env-expressing cells and appropriate target cells. We also monitored the conformational changes in the Envs following their interactions with CD4 and CXCR4 by immunofluorescence using an anti-gp41 mAb that reacts with the six-helix bundle. In contrast to the wild type, both Env mutants did not mediate cell fusion. The V2E Env did not mediate membrane destabilization. However, the Env with an unmodified fusion peptide but with a deletion of residues 665-682 in the membrane-proximal domain did mediate membrane destabilization. The wild type and both mutant Envs undergo conformational changes detected by the anti-gp41 six-helix bundle mAbs. Our results suggest that in intact HIV-1 Env the membrane-proximal domain is not required for membrane perturbations, but rather enables the bending of gp41 that is required for viral and target membranes to come together. Moreover, the observation that the Delta665-683 Env self-inserts its fusion peptide but does not cause fusion suggests that self-insertion of the fusion peptide is not sufficient for HIV-1 Env-mediated fusion.

Amino Acid Sequence↗
PubMed2003

The HIV Env-mediated fusion reaction.

The current general model of HIV viral entry involves the binding of the trimeric viral envelope glycoprotein gp120/gp41 to cell surface receptor CD4 and chemokine co-receptor CXCR4 or CCR5, which triggers conformational changes in the envelope proteins. Gp120 then dissociates from gp41, allowing for the fusion peptide to be inserted into the target membrane and the pre-hairpin configuration of the ectodomain to form. The C-terminal heptad repeat region and the leucine/isoleucine zipper region then form the thermostable six-helix coiled-coil, which drives the membrane merger and eventual fusion. This model needs updating, as there has been a wealth of data produced in the last few years concerning HIV entry, including target cell dependencies, fusion kinetic data, and conformational intermediates. A more complete model must include the involvement of membrane microdomains, actin polymerization, glycosphingolipids, and possibly CD4 and chemokine signaling in entry. In addition, kinetic experiments involving the addition of fusion inhibitors have revealed some of the rate-limiting steps in this process, adding a temporal component to the model. A review of these data that may require an updated version of the original model is presented here.

HIV Envelope Protein gp120↗
PubMed2003

C-terminal octylation rescues an inactive T20 mutant: implications for the mechanism of HIV/SIMIAN immunodeficiency virus-induced membrane fusion.

T20, a synthetic peptide corresponding to a C-terminal segment of the envelope glycoprotein (gp41) of human and simian immunodeficiency viruses, is a potent inhibitor of viral infection. We report here that C-terminal octylation of simian immunodeficiency virus gp41-derived T20 induces a significant increase in its inhibitory potency. Furthermore, when C-terminally octylated, an otherwise inactive mutant in which the C-terminal residues GNWF were replaced by ANAA has potency similar to that of the wild type T20. This effect cannot be explained by a trivial inhibitory effect of the octyl group added to the peptides, because the N-terminally octylated peptides have the same activity as the non-octylated parent peptides. The effects caused by octylation on the oligomerization, secondary structure, and membrane-interaction properties of the peptides were investigated. Our results shed light on the mechanism of inhibition by T20 and provide experimental support for the existence of a pre-hairpin intermediate.

Amino Acid Sequence↗
PubMed2003

Membrane fusion.

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Animals↗
PubMed2003

Progressive truncations C terminal to the membrane-spanning domain of simian immunodeficiency virus Env reduce fusogenicity and increase concentration dependence of Env for fusion.

The simian immunodeficiency virus (SIV) transmembrane (TM) protein, gp41, has multiple functions, which include anchoring the glycoprotein complex in the lipid envelope of the virus and mediating fusion of the virus and host cell membranes. Recently, a series of mutants of the SIVmac239 TM protein that have truncations at the carboxyl terminus of the membrane-spanning domain (MSD) have been characterized (J. T. West, P. Johnston, S. R. Dubay, and E. Hunter, J. Virol. 75:9601-9612, 2001). These mutants retained membrane anchorage but demonstrated reduced fusogenicity and infectivity as the MSD length was shortened. We have established a novel three-color fluorescence assay, which allows qualitative confocal and quantitative flow cytometric analyses, to further characterize the nature of the fusion defect in five of the MSD mutants: TM185, TM186, TM187, TM188, and TM189. Our analysis showed that each mutant could mediate complete lipid and aqueous dye transfer at early time points after effector and target cell mixing. No hemifusion with only lipid dye flux was detected. However, another intermediate fusion stage, which appears to involve small-fusion-pore formation that allowed small aqueous dye transfer but prevented the exchange of large cytoplasmic components, was identified infrequently in mutant-Env-expressing cell and target cell mixtures. Quantitative flow cytometric analysis of these mutants demonstrated that the TM187, TM188, and TM189 mutants were significantly more fusogenic than TM185 and TM186 but remained significantly impaired compared to the wild type. Moreover, fusion efficiency showed an increased dependence on the expression level of glycoproteins, suggesting that, for these mutants, formation of an active fusion complex was an increasingly stochastic event.

Animals↗
PubMed2002

Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics.

HIV entry inhibitors include coreceptor antagonists and the fusion inhibitor T-20. T-20 binds the first helical region (HR1) in the gp41 subunit of the viral envelope (Env) protein and prevents conformational changes required for membrane fusion. HR1 appears to become accessible to T-20 after Env binds CD4, whereas coreceptor binding is thought to induce the final conformational changes that lead to membrane fusion. Thus, T-20 binds to a structural intermediate of the fusion process. Primary viruses exhibit considerable variability in T-20 sensitivity, and determinants outside of HR1 can affect sensitivity by unknown mechanisms. We studied chimeric Env proteins containing different V3 loop sequences and found that gp120coreceptor affinity correlated with T-20 and coreceptor antagonist sensitivity, with greater affinity resulting in increased resistance to both classes of entry inhibitors. Enhanced affinity resulted in more rapid fusion kinetics, reducing the time during which Env is sensitive to T-20. Reduced coreceptor expression levels also delayed fusion kinetics and enhanced virus sensitivity to T-20, whereas increased coreceptor levels had the opposite effect. A single amino acid change (K421D) in the bridging sheet region of the primary virus strain YU2 reduced affinity for CCR5 and increased T-20 sensitivity by about 30-fold. Thus, mutations in Env that affect receptor engagement and membrane fusion rates can alter entry inhibitor sensitivity. Because coreceptor expression levels are typically limiting in vivo, individuals who express lower coreceptor levels may respond more favorably to entry inhibitors such as T-20, whose effectiveness we show depends in part on fusion kinetics.

3T3 Cells↗
PubMed2002

Quantitative measurement of fusion of HIV-1 and SIV with cultured cells using photosensitized labeling.

The fusion of HIV and SIV with biological membranes was studied by photosensitized activation of a hydrophobic probe, [(125)I]iodonaphthylazide ([(125)I]INA), by a fluorescent lipid which is situated in the target membrane. Photosensitized labeling of viral envelope-resident proteins occurs only upon their insertion into target membranes. Photosensitized labeling as a result of HIV-1 Env-mediated cell fusion showed the same kinetics as aqueous dye transfer. We have for the first time measured kinetics of HIV and SIV virus-cell fusion. HIV-1(MN) virions were about 10x less fusion active than SIVmne virions. SIV inactivated by aldrithiol-2 retained fusion activity similar to that seen with untreated virus. The relatively slow time course of SIV-cell fusion (t(1/2) = 19 min) indicates that the fusion events are stochastic. This feature provides a basis for understanding the mode of action of HIV/SIV entry inhibitors that target transition states.

2,2'-Dipyridyl↗
PubMed2002

Acid-induced changes in thermal stability and fusion activity of influenza hemagglutinin.

The conformational and thermal stability of full-length hemagglutinin (HA) of influenza virus (strain X31) has been investigated using a combination of differential scanning calorimetry (DSC), analytical ultracentrifugation, fluorescence, and circular dichroism (CD) spectroscopy as a function of pH. HA sediments as a rosette comprised of 5-6 trimers (31-35 S) over the pH range of 7.4-5.4. The DSC profile of HA in the native state at pH 7.4 is characterized by a single cooperative endotherm with a transition temperature (Tm) of 66 degrees C and unfolding enthalpy (DeltaH(cal)) of 800 kcal x (mol of trimer)(-1). Upon acidification to pH 5.4, there is a significant decrease in the transition temperature (from 66 to 45 degrees C), unfolding enthalpy [from 800 to 260 kcal x (mol of trimer)(-1)], and DeltaH(cal)/DeltaH(vH) ratio (from 3.0 to approximately 1.3). Whereas the far- and near-UV ellipticities are maintained over this pH range, there is an acid-induced increase in surface hydrophobicity and decrease in intrinsic tryptophanyl fluorescence. The major contribution to the DSC endotherm arises from unfolding HA1 domains. The relationship between acid-induced changes in thermal stability and the fusion activity of HA has been examined by evaluating the kinetics and extent of fusion of influenza virus with erythrocytes over the temperature and pH range of the DSC measurements. Surprisingly, X31 influenza virus retains its fusion activity at acidic pH and temperatures significantly below the unfolding transition of HA. This finding is consistent with the notion that the fusion activity of influenza virus may involve structural changes of only a small fraction of HA molecules.

Acids↗
PubMed2002

Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: application to the purification of the fusion protein of the human immunodeficiency virus type 1.

We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.

Chromatography, Affinity↗
PubMed2002

Fluorescent lipid probes in the study of viral membrane fusion.

Fluorescent lipid probes are widely used in the observation of viral membrane fusion, providing a sensitive method to study fusion mechanism(s). Due to the wealth of data concerning liposome fusion, a variety of fusion assays has been designed including fluorescent probe redistribution, fluorescence dequenching, fluorescence resonance energy transfer and photosensitized labeling. These methods can be tailored for different virus fusion assays. For instance, virions can be loaded with membrane dye which dequenches at the moment of membrane merger. This allows for continuous observation of fusion and therefore kinetic information can be acquired. In the case of cells expressing viral envelope proteins, dye redistribution studies of lipidic and water-soluble fluorophores yield information about fusion intermediates. Lipid probes can be metabolically incorporated into cell membranes, allowing observation of membrane fusion in vitro with minimal chance of flip flop, non-specific transfer and formation of microcrystals. Fluorescent lipid probes have been incorporated into liposomes and/or reconstituted viral envelopes, which provide a well-defined membrane environment for fusion to occur. Interactions of the viral fusion machinery with the membrane can be observed through the photosensitized labeling of the interacting segments of envelope proteins with a hydrophobic probe. Thus, fluorescent lipid probes provide a broad repertoire of fusion assays and powerful tools to produce precise, quantitative data in real time required for the elucidation of the complex process of viral fusion.

Animals↗
PubMed2002

Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells.

In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-beta-cyclodextrin (MbetaCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1alpha- and macrophage inflammatory protein 1beta-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MbetaCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.

CD4-Positive T-Lymphocytes↗
PubMedDate not supplied

Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review).

Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.

Animals↗