Membrane-perturbing domains of HIV type 1 glycoprotein 41.

Structural and functional studies were performed to assess the membrane actions of peptides based on HIV-1 glycoprotein 41,000 (gp41). Previous site-directed mutagenesis of gp41 has shown that amino acid changes in either the N-terminal fusion or N-leucine zipper region depressed viral infection and syncytium formation, while modifications in the C-leucine zipper domain both increased and decreased HIV fusion. Here, synthetic peptides were prepared corresponding to the N-terminal fusion region (FP-I; gp41 residues 519-541), the nearby N-leucine zipper domain (DP-107; gp41 residues 560-597), and the C-leucine zipper domain (DP-178; gp41 residues 645-680). With erythrocytes, FP-I or DP-107 induced dose-dependent hemolysis and promoted cell aggregation; FP-I was more hemolytic than DP-107, but each was equally effective in aggregating cells. DP-178 produced neither hemolysis nor aggregation, but blocked either FP-I- or DP-107-induced hemolysis and aggregation. Combined with previous nuclear magnetic resonance and Fourier transform infrared spectroscopic results, circular dichroism (CD) spectroscopy showed that the alpha-helicity for these peptides in solution decreased in the order: DP-107 >> DP-178 > FP-I. CD analysis also indicated binding of DP-178 to either DP-107 or FP-I. Consequently, DP-178 may inhibit the membrane actions mediated by either FP-I or DP-107 through direct peptide interactions in solution. These peptide results suggest that the corresponding N-terminal fusion and N-leucine zipper regions participate in HIV infection, by promoting membrane perturbations underlying the merging of the viral envelope with the cell surface. Further, the C-leucine zipper domain in "prefusion" HIV may inhibit these membrane activities by interacting with the N-terminal fusion and N-leucine zipper domains in unactivated gp41. Last, exogenous DP-178 may bind to the N-terminal and N-leucine zipper domains of gp41 that become exposed on HIV stimulation, thereby preventing the fusogenic actions of these gp41 regions leading to infection.

Membrane interactions of the synthetic N-terminal peptide of HIV-1 gp41 and its structural analogs.

Structural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519-541), and also FP analogs (FP520V/E with Val-->Glu at residue 520; FP527L/R with Leu-->Arg at 527; FP529F/Y with Phe-->Tyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection.

Conformational mapping of a viral fusion peptide in structure-promoting solvents using circular dichroism and electrospray mass spectrometry.

The N-terminal domain of human immunodeficiency virus (HIV)-1 glycoprotein 41,000 (FP; residues 1-23; NH2-AVGIGALFLGFLGAAGSTMGARS-CONH2) is involved in the fusion and cytolytic processes underlying viral-cell infection. Here, we use circular dichroism (CD) spectroscopy, along with electrospray ionization (ESI) mass spectrometry and tandem (MS/MS) mass spectrometry during the course of hydrogen/deuterium exchange, to probe the local conformations of this synthetic peptide in two membrane mimics. Since amino acids that participate in defined secondary structure (i.e., alpha-helix or beta-sheet) exchange amido hydrogens more slowly than residues in random structures, deuterium exchange was combined with CD spectroscopy to map conformations to specific residues. For FP suspended in the highly structure-promoting solvent hexafluoroisopropanol (HFIP), CD spectra indicated high alpha-helix and disordered structures, whereas ESI and MS/MS mass spectrometry indicated that residues 5-15 were alpha-helical and 16-23 were disordered. For FP suspended in the less structure-promoting solvent trifluoroethanol (TFE), CD spectra showed lower alpha-helix, with ESI and MS/MS mass spectrometry indicating that only residues 9-15 participated in the alpha-helix. These results compare favorably with previous two-dimensional nuclear magnetic resonance studies on the same peptide.

Cytotoxic activity of the amino-terminal region of HIV type 1 Nef protein.

Myristoylated 21- and 25-residue N-terminal peptides of the Nef protein of HIV-1 lysed human erythrocytes and were cytotoxic toward a human CD4+ T cell line, CEM, and primary human peripheral blood mononuclear cells (PBMCs). The corresponding nonmyristoylated N-terminal peptides were only very weakly hemolytic and cytotoxic. A myristoylated peptide consisting of residues 31-50 of Nef was neither hemolytic nor cytotoxic. Alteration of the tryptophan residue at position 13 to a serine did not change the hemolytic and cytotoxic activity. Studies of the ultraviolet fluorescence of the tryptophan at position 5 in the peptide, using an artificial membrane system and fluorescence-quenching agents that inserted into the bilayer at different levels, suggested that myristoylation results in this residue being brought into contact with the upper hydrocarbon region of the lipid bilayer of the cell membrane. This tryptophan is flanked by a number of polar residues that would maintain it in this position, resulting in a considerable increase in disorder in the upper regions of the lipid bilayer, leading to its destabilization and to lysis. The cytotoxic activity of the myristoylated Nef fragments may, in part, explain the killing and deletion of cells, especially in lymphoid tissues, during HIV infection.

The amino-terminal peptide of HIV-1 glycoprotein 41 fuses human erythrocytes.

The ability of synthetic peptides based on the amino-terminus of HIV-1 glycoprotein 41,000 (gp41) to fuse human erythrocytes was investigated. Previous site-directed mutagenesis studies have shown an important role for the N-terminal gp41 domain in HIV-fusion, in which replacement of hydrophobic amino acids with polar residues inhibits viral infection and syncytia formation. Here, a synthetic peptide (FP; 23 amino acid residues 519-541) corresponding to the N-terminus of HIV-1 gp41, and also a FP analog (FP526L/R) with Arg replacing Leu-526, were prepared with solid phase techniques. The lipid mixing and leakage of resealed ghosts triggered by these peptides were examined with fluorescence quenching techniques. Peptide-induced aggregation of human erythrocytes was studied using Coulter counter sizing and scanning electron microscopy (SEM). Using resealed erythrocyte ghosts at physiologic pH, FP induces rapid lipid mixing between red cell membranes at doses previously shown to hemolyze intact cells. FP also causes leakage from resealed ghosts, and promotes the formation of multicelled aggregates with whole erythrocytes. Contrarily, similar FP526L/R concentrations did not induce red cell lysis, lipid mixing, leakage or aggregation. Since the fusogenic potency of FP and FP526L/R parallels earlier gp41 mutagenesis studies showing that substitution of Arg for Leu-526 blocks fusion activity, these data suggest that the N-terminal gp41 domain in intact HIV participates in fusion.

Antivirals that target the amino-terminal domain of HIV type 1 glycoprotein 41.

Functional and structural studies were made to assess whether a class of antiviral agents targets the N-terminal domain of the glycoprotein 41,000 (gp41) of human immunodeficiency virus type 1 (HIV-1). Previous experiments have shown that the amino-terminal peptide (FP-I; 23 amino acids, residues 519-541) of HIV-1 gp41 is cytolytic to both human erythrocytes (non-CD4+ cells) and Hut-78 cells (CD4+ lymphocytes). Accordingly, FP-I-induced hemolysis may be used as a surrogate assay for evaluating the role of the N-terminal gp41 domain in HIV-cell interactions. Here, we studied the blocking of FP-I-induced lysis of erythrocytes by the following anti-HIV agents: (1) IgG [i.e., anti-(518-541) IgG] raised to an immunoconjugate of Arg-FP-I, (2) apolipoprotein A-1 (apo A-1) and a peptide based on apo A-1, (3) dextran sulfate, (4) gp41 peptide (residues 637-666), and (5) anionic human serum albumins. Dose-response curves indicated that their relative potency in inhibiting FP-I-induced hemolysis was approximately correlated with their previously reported anti-HIV activity. Electron spin resonance (ESR) studies showed that FP-I spin labeled at the N-terminal alanine binds to anti-(518-541) IgG, dextran sulfate, and anionic albumins. The high in vitro antiviral activity and low cytotoxicity of these agents suggest that blocking membrane-FP-I interactions offers a novel approach for AIDS therapy or prophylaxis.

The amino-terminal peptide of HIV-1 gp41 interacts with human serum albumin.

Structural and functional studies were made to assess interactions between human serum albumin (HSA) and the amino-terminal peptide (FP-I; 23-residue peptide 519-541) of glycoprotein 41,000 (gp41) of human immunodeficiency virus type-1 (HIV-1). Circular dichroism (CD) spectroscopy indicated that the peptide binds to albumin with dominant alpha-helical character. Peptide binding to albumin was also examined using FP-I spin labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). Electron spin resonance (ESR) spectra of FP-II bound to HSA at 38 degrees C indicated that the spin label at the amino-terminal residue (Ala-519) was motionally restricted. The ESR spectrum of 12-nitroxide stearate (12-NS)-labeled HSA was identical to that obtained with FP-II, indicating that the reporter groups for the 12-NS and FP-II probes are similarly bound to albumin. Contrarily, ESR spectra of HSA labeled with FP-III indicated high mobility for the reporter group (Met-537) at the aqueous-protein interface. This suggests that the N-terminal gp41 peptide binds as an alpha helix (residues 519-536) to fatty acid sites on HSA, such that Ala-519 of the peptide residues in the interior of the protein while Met-537 lies outside the protein in aqueous solution. It is also of interest that addition of HSA to human red blood cells dramatically reduced the ability of FP-I to induce hemolysis, presumably through peptide-albumin binding that inhibited FP-I interactions with red cell membranes. The significance of these results focuses on the following three points. The first is that high serum levels of albumin may limit the efficacy of anti-HIV therapies using peptides based on the N-terminal gp41 domain. The second is that the elucidation of FP-I and HSA interactions with physical techniques may provide clues on the molecular features underlying viral FP-I combination with receptors on the target cell surface. Last, the affinity of albumin for the N-terminal gp41 peptide may play a subordinate role in the blocking of HIV infectivity in vitro that has been reported for chemically modified albumins.

The amino-terminal peptide of HIV-1 glycoprotein 41 lyses human erythrocytes and CD4+ lymphocytes.

Functional studies assessed the cytolytic activity of the amino terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of the Human Immunodeficiency Virus Type-1 (HIV-1). Synthetically prepared FP-I efficiently hemolyzed human red blood cells at 37 degrees C, with 40% lysis at 32 microM. Kinetic studies indicated that FP-I induced maximal hemolysis in 30 min, probably through tight binding of the peptide with the red cell membrane. The Phe-Leu-Gly-Phe-Leu-Gly (residues 526-531) motif in FP-I apparently plays a critical role in lysis of red cells, since no hemolytic activity was observed for an amino-acid-substituted FP-I in which the unique Phe-Leu-Gly-Phe-Leu-Gly was converted to Ala-Leu-Gly-Ala-Leu-Gly. As neither smaller constituent peptides (e.g., residues 519-524 and residues 526-536) nor a N-terminal flanking peptide (e.g., residues 512-523) induced red cell hemolysis, the entire 23-residue (519-541) sequence of FP-I may be required for hemolytic activity. FP-I was also cytolytic with CD4(+)-bearing Hut-78 cells, with 40% lysis at approx. 150 microM. These results are consistent with an earlier hypothesis that the N-terminal peptide of gp41 may partially contribute to the in vivo cytopathic actions of HIV-1 infection (Gallaher, W.R. (1987) Cell 50, 327-328).

The amino-terminal peptide of HIV-1 glycoprotein 41 interacts with human erythrocyte membranes: peptide conformation, orientation and aggregation.

Structural studies assessed interactions between the amino-terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of Human Immunodeficiency Virus Type-1 (HIV-1) and human erythrocyte membranes and simulated membrane environments. Peptide binding was examined at sub-hemolytic (approx. less than 5 microM) and hemolytic (greater than or equal to 5 microM) doses (Mobley et al. (1992) Biochem. Biophys. Acta 1139, 251-256), using circular dichroism (CD) and Fourier-transform infrared (FTIR) measurements with FP-I, and electron spin resonance (ESR) studies employing FP-I spin-labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). In the sub-lytic regime, FP-I binds to both erythrocyte lipids and dispersions of SDS with high alpha-helicity. Further, ESR spectra of FP-II labeled erythrocyte ghosts indicated peptide binding to both lipid and protein. In ghost lipids, FP-II was monomeric and exhibited low polarity and rapid, anisotropic motion about its long molecular axis (i.e., alpha-helical axis), with restricted motion away from this axis. The spin-label at the amino-terminal residue (Ala-519) is insensitive to the aqueous broadening agent chromium oxalate and buried within the hydrophobic core of the membrane; the angle that the alpha-helix (residues 519-536) makes to the normal of the bilayer plane is either 0 degree or 40 degrees. Contrarily, ESR spectra of ghost lipids labeled with sub-lytic doses of FP-III indicated high mobility and polarity for the reporter group (Met-537) at the aqueous-membrane interface, as well as extreme sensitivity to chromium oxalate. At lytic FP-I doses, CD and FTIR showed both alpha-helix and beta-structure for peptide in ghost lipids or detergent, while ESR spectra of high-loaded FP-II in ghost membranes indicated peptide aggregates. Membrane aggregates of FP-I may be involved in hemolysis, and models are suggested for N-terminal gp41 peptide participation in HIV-induced fusion and cytolysis.

Thermotropic lipid phase separation in the human immunodeficiency virus.

The presence of thermodependent lipid domains in the envelope of the human immunodeficiency virus (HIV) was studied. HIV was propagated in Hut-78 cells and purified by differential-gradient centrifugation. Since the virus was highly infectious in cell culture and Western blots of detergent-inactivated HIV showed envelope proteins when exposed to sera containing anti-HIV antibodies, this viral preparation was not deficient in 'spike' or 'knob' particles. Electron spin resonance (ESR) studies of intact HIV labeled with 5-nitroxide stearate (5-NS) indicated that a temperature-dependent lipid phase separation occurs with a high onset at approx. 42 degrees C and a low onset at approx. 15 degrees C. Cooling below 42 degrees C induces 5-NS clustering. Similar phase separations with high onsets at approx. 37-38 degrees C were previously identified in 5-NS labeled human erythrocytes (cholesterol/phospholipid (C/P) molar ratio = 0.90) and cholesterol-loaded (C/P = 0.85-0.98) rat liver plasma membranes. These were attributed to a temperature-sensitive redistribution of endogenous lipid components such that 5-NS is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains at low temperatures. Since HIV has a lipid envelope with a similarly high C/P of 0.88 (Aloia et al. (1988) Proc. Natl. Acad. Sci. USA 85, 900-904), cholesterol-rich and cholesterol-poor domains also probably exist in HIV at physiologic temperatures. The reduced stability and infectivity of HIV noted on heating above 42 degrees C may be due, in part, to the abolition of these thermodependent domains.

Lipid composition and fluidity of the human immunodeficiency virus.

Lipid analyses of the human immunodeficiency virus (HIV) propagated in Hut 78 cells indicated a low total lipid/protein ratio, a high cholesterol/phospholipid molar ratio, and major phospholipids consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylserine; comparable lipid profiles were noted for human erythrocytes and other RNA viruses. Electron spin resonance (ESR) studies of HIV labeled with 5-nitroxide stearate (N-oxy-4',4'-dimethyloxazolidine derivative of ketostearate) showed a low "fluidity" at 37 degrees C, similar to other enveloped RNA viruses and erythrocytes and probably due to the high cholesterol/phospholipid ratio. Ethanol (50%) completely disrupts the envelope, contributing to the rapid inactivation of HIV by ethanol. Contrarily, heating to 57 degrees C causes much less fluidization, and this heating may play a role in the slower viral inactivation at high temperatures. Should a critical minimum ordering in the HIV envelope be necessary for viral stability and infectivity, manipulating the lipid composition or fluidizing the HIV membrane, or both, may provide an untried therapeutic approach.

Thermotropic lipid phase separations in human erythrocyte ghosts and cholesterol-enriched rat liver plasma membranes.

Electron spin resonance (ESR) studies of human erythrocyte ghosts labeled with 5-nitroxide stearate, I(12,3), indicate that a temperature-dependent lipid phase separation occurs with a high onset at 38 degrees C. Cooling below 38 degrees C induces I(12,3) clustering. Similar phase separations were previously identified in human platelet and cholesterol-loaded [cholesterol/phospholipid molar ratio (C/P) = 0.85] rat liver plasma membranes [L.M. Gordon et al., 1983; J. Membrane Biol. 76; 139-149]; these were attributed to redistribution of endogenous lipid components such that I(12,3) is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains. Further enrichment of rat liver plasma membranes to C/P ratios of 0.94-0.98 creates an "artificial" system equivalent to human erythrocyte ghosts (C/P = 0.90), using such criteria as probe flexibility, temperature dependent I(12,3) clustering; and polarity of the probe environment. Consequently, cholesterol-rich and -poor domains probably exist in both erythrocyte ghosts and high cholesterol liver membranes at physiologic temperatures. The temperature dependence of cold-induced hypertonic lysis of intact human erythrocytes was examined by incubating cells in 0.9 M sucrose for 10 min at 1 degree C intervals between 9 and 46 degrees C (Stage 1), and then subjecting them to 0 degrees C for 10 min (Stage 2). Plots of released hemoglobin are approx. sigmoidal, with no lysis below 18 degrees C and maximal lysis above 40 degrees C. The protective effect of low temperatures during Stage 1 may be due to the formation of cholesterol-rich domains that alter the bilayer distribution and/or conformation of critical membrane-associated proteins.

Thermotropic lipid phase separations in human platelet and rat liver plasma membranes.

Electron spin resonance (ESR) studies were conducted on human platelet plasma membranes using 5-nitroxide stearate, I(12,3). The polarity-corrected order parameter S and polarity-uncorrected order parameters S(T parallel) and S(T perpendicular) were independent of probe concentration at low I(12.3)/membrane protein ratios. At higher ratios, S and S(T perpendicular) decreased with increasing probe concentration while S(T parallel) remained unchanged. This is the result of enhanced radical interactions due to probe clustering. A lipid phase separation occurs in platelet membranes that segregates I(12,3) for temperatures less than 37 degrees C. As Arrhenius plots of platelet acid phosphatase activity exhibit a break at 35 to 36 degrees C, this enzyme activity may be influenced by the above phase separation. Similar experiments were performed on native [cholesterol/phospholipid ratio (C/P) = 0.71] and cholesterol-enriched [C/P = 0.85] rat liver plasma membranes. At 36 degrees C, cholesterol loading reduces I(12,3) flexibility and decreases the probe ratio at which radical interactions are apparent. The latter effects are attributed to the formation of cholesterol-rich lipid domains, and to the inability of I(12,3) to partition into these domains because of steric hinderance. Cholesterol enrichment increases both the high temperature onset of the phase separation occurring in liver membranes from 28 degrees to 37 degrees C and the percentage of probe-excluding, cholesterol-rich lipid domains at elevated temperatures. A model is discussed attributing the lipid phase separation in native liver plasma membranes to cholesterol-rich and -poor domains. As I(12,3) behaves similarly in cholesterol-enriched liver and human platelet plasma membranes, cholesterol-rich and -poor domains probably exist in both systems at physiologic temperatures.

Respiration of 14CO2 by intact animals of various species given L-[1-14C]fucose or D-[1-14C]arabinose.

The production of 14CO2 from L-[1-14C]fucose and D-[1-14C]arabinose been studied in five mammalian species. Cats, guinea pigs, mice, and rabbits respired about 22% of the label of L-[1-14C]fucose or of D-[1-14C]arabinose within 6 h after intraperitoneal injection of the sugar. Rats respired only 1.5% of the L-fucose label and 5% of the D-arabinose label in the same time period. Liver homogenates from cat, guinea pig, and rabbit produced significantly more 14CO2 from L-[1-14C]fucose or D-[1-14C]arabinose than mouse or liver homogenates. Unlike those of the other species, guinea pig liver homogenates had very low L-fucose dehydrogenase activity. The results suggest that substantial catabolism of L-fucose and D-arabinose occurs in the tissues of some animal species. Investigators wishing to employ L-fucose as a tracer of glycoprotein metabolism must, therefore, ensure that the species that they employ does not metabolize L-fucose to products interfering with their studies.

Thyroid hormone levels in the developing obese-hyperglycemic syndrome.

Examination of the thyroid hormone levels of C57BL/6J ob/ob mice and their lean littermates between the ages of 6 days and 22 weeks revealed that obese mice have significantly reduced hormone concentrations between 10 and 21 days of age. Thereafter, the values remained equal to, or above those or their lean littermates. The present data indicate that the hypometabolism and abnormal thermal regulation of weanling mice could be a result of diminished thyroid hormone levels, but that other mechanisms must be responsible for the persistence of these abnormalities in adult ob/ob mice.

Carcass and liver composition following acute oxytetracycline treatment of ob/ob mice.

Fully mature (24-week old) C57BL/6J ob/ob mice and their lean littermates received daily oxytetracycline injections (50 or 100 mg/kg) during a 10 day period. The effects of the drug on the glucose, IRI, corticosterone levels, and on hepatic and body composition of ad libitum fed obese mice were compared with those of food-restricted and ad libitum fed lean and obese control animals. When compared with food-restricted obese mice, drug treatment led to substantial reductions of serum glucose, serum IRI, carcass fat, and hepatic lipid content, while it increased lean body mass and liver glycogen concentration. Similarly, oxytetracycline decreased body weight, and serum glucose in lean mice, but the drug had no substantial effect on circulating IRI levels or on the lipid content of carcass. A significant increase in hepatic lipid was observed in drug-treated lean mice. No effects of the drug on basal corticosterone levels were noted in either phenotype. These data support previous findings showing the effectiveness of oxytetracycline to reverse many of the metabolic abnormalities of ob/ob mice. In addition, the present results suggest that the drug acts by independently altering abnormal metabolism in many target organs, including pancreas, adipose tissue, liver, and muscle, rather than by merely reducing circulating insulin levels or by generally increasing insulin sensitivity.