Biomedical subjects

P J Bugelski

Publications and source records attributed to P J Bugelski.

At least 19 recordsLinked to original sources

PubMed2001

Modification of the Fc region of a primatized IgG antibody to human CD4 retains its ability to modulate CD4 receptors but does not deplete CD4(+) T cells in chimpanzees.

Keliximab, a Primatized IgG1 CD4 mAb, was reconfigured to an IgG4 antibody. The gamma4 constant region was further modified by substituting glutamic acid for serine at position 235 in the CH2 domain (IgG4-E), to remove residual binding to Fcgamma receptors, and substitution of serine with proline at position 228 in the hinge region (IgG4-PE) for greater stability. Pharmacokinetic analysis in rats gave a t(1/2) of approximately 4 days for IgG4-E and 9 days for IgG4-PE, consistent with a greater stability of the IgG4-PE molecule. The effects on T cell subsets were assessed in chimpanzees given escalating doses of IgG4-PE: 0.05 mg/kg on Day 16, 1.5 mg/kg dose on Day 43, and 15 mg/kg on Day 85. Receptor modulation was observed at the two highest doses, but no depletion of T cells at any dose. The in vitro and in vivo results demonstrate the potential of this IgG4-PE mAb for use in human trials.

Amino Acid Substitution↗

PubMed2001

Comparison of adipose tissue changes following administration of rosiglitazone in the dog and rat.

Rosiglitazone (BRL-49653-C), a thiazolidinedione, is a potent agonist for the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Thiazolidinediones have been reported to induce adipocyte differentiation in vitro and there is limited data on their effects in vivo. The objective of this study was to compare the effects of rosiglitazone on adipocyte differentiation between dogs and rats. Morphological (light and ultrastructural) and morphometric evaluations were conducted on perirenal adipose tissue from dogs that have been treated for 1 month with 0.4, 5, 60 mg/kg/day and rats treated for the same period with 80 mg/kg/day. There was a dose-related change in the phenotype of white adipose tissue in dogs, reflected by an increase in nuclear numerical density (up to threefold) and cytoplasmic area fraction (up to 2.1-fold). In addition, there was an enlargement of the nuclei and a reduction in the size of the white adipocyte lipid vacuoles. Ultrastructural changes included an increase in the number of mitochondria per adipocyte. In the rat, similar changes were seen in nuclear numerical density (1.5-fold increase) and cytoplasmic area fraction (2.2-fold increase). There were also increased numbers of mitochondria per cell in white adipocytes giving them similar numbers of mitochondria to brown adipocytes. In the brown adipocytes, there was a reduction in cytoplasmic area fraction with a corresponding increase in the size of the lipid filled vacuoles in other words there was a converging of the phenotypes of the white and brown adipose tissues.

Adipose Tissue↗

PubMed2001

Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys.

BACKGROUND: Allergic respiratory diseases are characterized by large numbers of eosinophils and their reactive products in airways and blood; these are believed to be involved in progressive airway damage and remodeling. IL-5 is the principal cytokine for eosinophil maturation, differentiation, and survival. Mepolizumab (SB-240563), a humanized monoclonal antibody (mAb) specific for human IL-5, is currently in clinical trials for treatment of asthma. OBJECTIVE: The purpose of this study was to characterize the pharmacologic activity and long-term safety profile of an anti--human IL-5 mAb to support clinical trials in asthmatic patients. METHODS: Naive and Ascaris suum -sensitive cynomolgus monkeys received various dose levels of mepolizumab and were monitored for acute and chronic pharmacologic and toxic responses. RESULTS: To support preclinical safety assessment, cynomolgus monkey IL-5 was cloned, expressed, and characterized. Although monkey IL-5 differs from human IL-5 by 2 amino acids (Ala27Gly and Asn40His), mepolizumab has comparable inhibitory activity against both monkey IL-5 and human IL-5. In A suum--sensitive monkeys, single doses of mepolizumab significantly reduced blood eosinophilia, eosinophil migration into lung airways, and levels of RANTES and IL-6 in lungs for 6 weeks. However, mepolizumab did not affect acute bronchoconstrictive responses to inhaled A suum. In an IL-2--induced eosinophilia model (up to 50% blood eosinophilia), 0.5 mg/kg mepolizumab blocked eosinophilia by >80%. Single-dose and chronic (6 monthly doses) intravenous and subcutaneous toxicity studies in naive monkeys found no target organ toxicity or immunotoxicity up to 300 mg/kg. Monkeys did not generate anti-human IgG antibodies. Monthly mepolizumab doses greater than 5 mg/kg caused an 80% to 100% decrease in blood and bronchoalveolar lavage eosinophils lasting 2 months after dosing, and there was no effect on eosinophil precursors in bone marrow after 6 months of treatment. Eosinophil decreases correlated with mepolizumab plasma concentrations (half-life = 13 days). CONCLUSION: These studies demonstrate that chronic antagonism of IL-5 by mepolizumab in monkeys is safe and has the potential, through long-term reductions in circulating and tissue-resident eosinophils, to be beneficial therapy for chronic inflammatory respiratory diseases.

Animals↗

PubMed2001

Immunomodulatory effects of anti-CD4 antibody in host resistance against infections and tumors in human CD4 transgenic mice.

Anti-CD4 antibodies, which cause CD4(+) T-cell depletion, have been shown to increase susceptibility to infections in mice. Thus, development of anti-CD4 antibodies for clinical use raises potential concerns about suppression of host defense mechanisms against pathogens and tumors. The anti-human CD4 antibody keliximab, which binds only human and chimpanzee CD4, has been evaluated in host defense models using murine CD4 knockout-human CD4 transgenic (HuCD4/Tg) mice. In these mice, depletion of CD4(+) T cells by keliximab was associated with inhibition of anti-Pneumocystis carinii and anti-Candida albicans antibody responses and rendered HuCD4/Tg mice susceptible to P. carinii, a CD4-dependent pathogen, but did not compromise host defense against C. albicans infection. Treatment of HuCD4/Tg mice with corticosteroids impaired host immune responses and decreased survival for both infections. Resistance to experimental B16 melanoma metastases was not affected by treatment with keliximab, in contrast to an increase in tumor colonization caused by anti-T cell Thy1.2 and anti-asialo GM-1 antibodies. These data suggest an immunomodulatory rather than an overt immunosuppressive activity of keliximab. This was further demonstrated by the differential effect of keliximab on type 1 and type 2 cytokine expression in splenocytes stimulated ex vivo. Keliximab caused an initial up-regulation of interleukin-2 (IL-2) and gamma interferon, followed by transient down-regulation of IL-4 and IL-10. Taken together, the effects of keliximab in HuCD4/Tg mice suggest that in addition to depleting circulating CD4(+) T lymphocytes, keliximab has the capability of modulating the function of the remaining cells without causing general immunosuppression. Therefore, keliximab therapy may be beneficial in controlling certain autoimmune diseases.

Animals↗

PubMed2001

Maximization of signal derived from cDNA microarrays.

Microarray technology is a powerful tool for generating expression data on a large number of genes simultaneously. However, as for any assay, it must be reproducible to give confidence in the results. Using a classical statistical method--the factorial design of experiments--we have assessed the effects of different experimental factors in our system. Significant effects on signal were seen when the standard components were substituted with a different enzyme, fluorescent label, or RNA purification method. This has led to the implementation of an improved procedure that maximizes signal without affecting the variability of the system, thus increasing the signal-to-noise ratio. In addition, we were able to quantify the variability between microarrays and replicates within microarrays.

Analysis of Variance↗

PubMed2000

Nonlinear Dynamics in the Progression of Atherosclerotic Fatty Streaks: Morphometric Analysis.

Atherosclerotic lesions are heterogeneous in terms of their cellular and lipid composition. While heterogeneity can be the result of stochastic noise, an alternate hypothesis is that the differences observed among individual lesions arise from deterministic chaos. Five New Zealand white rabbits were fed a diet containing 0.15% cholesterol for 6 months. Segments of the aorta were fixed in formalin, stained en bloc with Nile red (NR) and filipin (F), and en face fluorescence microscopy was used to map the distribution of lipids in fatty streaks (FS). The smallest lesions detected stained only with filipin. Larger lesions stained with both Nile red and filipin and two distinct regions of Nile red staining, NR-orange (rich in polar lipids) and NR-yellow (rich in neutral lipids) were observed. Digital overlays revealed a "nested" arrangement of F, NR-orange, and NR-yellow. The lesions also showed marked heterogeneity in their lipid composition. Thus, although initially similar, as FS increased in size, their composition became divergent, suggesting that the ultimate composition of a FS was highly sensitive to its initial composition. Sensitivity to initial condition is one of the hallmarks of deterministic systems. To determine if FS were self-similar, another hallmark of deterministic chaos, the borders of the different regions defined by NR and F staining were subjected to fractal analysis. For each lesion, the borders of the F, NR-orange, and NR-yellow regions were found to be fractal. Return maps were constructed for the differently stained regions. Analysis of the entire 104-lesion data set showed that although the data could be described by a four-parameter logistic model, the population was not chaotic. However, return maps drawn for the maxima of the NR-orange stained regions demonstrated chaos. Taken together, the data suggest that deterministic chaos plays a role in the evolution of atheromatous disease but, in common with most biologic systems, as the lesions progress, chaotic behavior is dampened.

Journal Article↗

PubMed2000

A strategy for primary high throughput cytotoxicity screening in pharmaceutical toxicology.

PURPOSE: Recent advances in combinatorial chemistry and high throughput screens for pharmacologic activity have created an increasing demand for in vitro high throughput screens for toxicological evaluation in the early phases of drug discovery. METHODS: To develop a strategy for such a screen, we have conducted a data mining study of the National Cancer Institute's Developmental Therapeutics Program (DTP) cytotoxicity database. RESULTS: Using hierarchical cluster analysis, we confirmed that the different tissues of origin and individual cell lines showed differential sensitivity to compounds in the DTP Standard Agents database. Surprisingly, however, approaching the data globally, linear regression analysis showed that the differences were relatively minor. Comparison with the literature on acute toxicity in mice showed that the predictive power of growth inhibition was marginally superior to that of cell death. CONCLUSIONS: This datamining study suggests that in designing a strategy for high throughput cytotoxicity screening: a single cell line, the choice of which may not be critical, can be used as a primary screen; a single end point may be an adequate measure and a cut off value for 50% growth inhibition between 10(-6) and 10(-8) M may be a reasonable starting point for accepting a cytotoxic compound for scale up and further study.

Animals↗

PubMed2000

Preclinical development of keliximab, a Primatized anti-CD4 monoclonal antibody, in human CD4 transgenic mice: characterization of the model and safety studies.

The preclinical safety assessment of biopharmaceuticals necessitates that studies be conducted in species in which the products are pharmacologically active. Monoclonal antibodies are a promising class of biopharmaceuticals for many disease indications; however, by design, these agents tend to have limited species cross-reactivity and tend to only be active in primates. Keliximab is a human-cynomolgus monkey chimeric (Primatized) monoclonal antibody with specificity for human and chimpanzee CD4. In order to conduct a comprehensive preclinical safety assessment of this antibody to support chronic treatment of rheumatoid arthritis in patients, a human CD4 transgenic mouse was used for chronic and reproductive toxicity studies and for genotoxic studies. In addition, immunotoxicity studies were conducted in these mice with Candida albicans, Pneumocystis carinii and B16 melanoma cells to assess the effects of keliximab on host resistance to infection and immunosurveillance to neoplasia. The results of these studies found keliximab to be well tolerated with the only effects observed being related to its pharmacologic activity on CD4+ T lymphocytes. The use of transgenic mice expressing human proteins provides a useful alternative to studies in chimpanzees with biopharmaceutical agents having limited species cross-reactivity.

Animals↗

PubMed2000

Repeated acetaminophen dosing in rats: adaptation of hepatic antioxidant system.

Repeated dosing of acetaminophen (paracetamol) to rats is reported to decrease their sensitivity to its hepatotoxic effects, which are associated with oxidative stress and glutathione depletion. We determined if repeated acetaminophen dosing produced adaptive response of key antioxidant system enzymes. Male rats (Sprague-Dawley, 10 weeks) were given 800, 1200, or 1600 mg/kg/day acetaminophen by oral gavage for 4 days. Liver was assayed for oxidative stress and antioxidant markers: malondialdehyde (MDA), thiobarbituric acid reactive substance (TBARS), total antioxidant status (TAS), glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PD), catalase (CAT), and superoxide dismutase (SOD), and alanine transaminase (ALT) as a marker of hepatocellular injury. Acetaminophen at 1200/1600 mg/kg decreased GSH 26/47%, GPx 21/26%, CAT 35/28%, SOD 21/12%; and TAS 28/18% (correlated with CAT, r=0.91; SOD, r=0.66; GPx, r=0.45). Despite antioxidant deficiencies, and no TBARS change, MDA decreased 26%/33%/37% at 800/1200/1600 mg/kg, which correlated with increased GR (61%/62%/76%, r=0.77) and G6PD (130%/110%/190%, r=0.78). Both MDA (r=0.68) and G6PD (r=0.71) correlated with hepatic ALT, which decreased 27%/43%/48%, respectively. Resistance to acetaminophen hepatotoxicity produced by repeated exposure is partially attributable to upregulation of hepatic G6PD and GR activity as an adaptive and protective response to oxidative stress and glutathione depletion.

Acetaminophen↗

PubMed1999

Selective inhibition of phospholipases by atiprimod, a macrophage targeting antiarthritic compound.

Azaspiranes are cationic amphiphilic compounds that are active in a number of models of autoimmune disease and transplantation. Repeated administration of cationic amphiphiles induces phospholipid accumulation in a variety of species. The present study was conducted to explore the mechanism of phospholipid accumulation in rats caused by treatment with the novel azaspirane, SK&F 106615 (atiprimod). Atiprimod inhibited the activities of partially purified phospholipases A(2) and C, but not D, in a noncompetitive manner in vitro. Treatment of rats for 28 days with 10 mg/kg/day of atiprimod increased the contents of arachidonate-containing molecular species within plasmalogen subclasses of hepatic phosphatidylcholine and phosphatidylethanolamine. In contrast, diacyl-linked species were not affected, indicating a selective effect upon an hepatic plasmalogen-selective phospholipase A(2). Taken together, the data suggest that the beneficial effects of atiprimod in autoimmune diseases may involve inhibition of phospholipase A(2) and C activities. Further, the data suggest that atiprimod is a selective inhibitor of plasmalogen-selective phospholipase A(2) in vivo.

Animals↗

PubMed1999

Atiprimod (SK&F 106615), a novel macrophage targeting agent, enhances alveolar macrophage candidacidal activity and is not immunosuppressive in Candida-infected mice.

Azaspiranes are novel macrophage-targeting agents with activity in preclinical animal models of autoimmune disease and transplantation. The purpose of this work was to determine the effects of atiprimod (SK&F 106615), an azaspirane being developed for the treatment of rheumatoid arthritis, on rat pulmonary alveolar macrophage (AM) function and immunocompetance in Candida-infected mice. AM from rats treated with 20 mg/kg/day of atiprimod for 15 days demonstrated enhanced killing of Candida albicans ex vivo. Concentration-dependent increases in candidacidal activity were also observed as early as one hour after exposure in vitro in AM from untreated normal rats. Treatment of AM with atiprimod in vitro did not increase particulate-stimulated superoxide production or phagocytosis of Candida but decreased their ability to concentrate acridine orange, indicating an increase in lysosomal pH. Increased candidacidal activity was inhibited by superoxide dismutase and catalase, suggesting a role for reactive oxygen intermediates (ROI). Atiprimod also increased free radical-mediated killing of Candida in the presence of H2O2, iron and iodide in a cell-free system. These findings indicated that treatment with atiprimod increased the candidacidal activity of rat AM in a free radical-dependent manner. The data also suggested that atiprimod did not increase ROI production by AM, but rather increased the efficiency of radical-mediated killing. This increase may be caused by cyclization of atiprimod, facilitating electron transfer and peroxidation of lipid membranes. In vivo studies in Candida-infected CBA mice showed that atiprimod (10 mg/kg/day), did not compromise immune function in the infected mice and could be differentiated from prototypical immunosuppressive compounds used for treatment of autoimmune diseases.

Acridine Orange↗

PubMed1997

A novel technique for mapping the lipid composition of atherosclerotic fatty streaks by en face fluorescence microscopy.

We introduce here a new fluorescence microscopy technique for en face analysis of the atherosclerotic fatty streaks (FS). This technique is semiquantitative and has the sensitivity and resolution to map lipids to individual cells in FS less than 100 microns in diameter. New Zealand White rabbits were fed an atherogenic diet for up to 26 weeks. Aortas were fixed in formalin and stained en bloc with the fluorescent dyes Nile red and filipin. Fluorescent staining was validated by correlating microfluorimetric and biochemical measurements of the lipid content in FS. To determine the cell types associated with the different staining patterns, FS were also evaluated by transmission electron microscopy (TEM) and immunohistochemistry (IH). Correlation of microfluorimetry, TEM, IH, and biochemical data indicated that regions rich in non-esterified cholesterol stained with filipin and fluoresced blue owing to accumulations of lipid vesicles and/or cholesterol crystals. Regions rich in neutral and polar lipids stained with Nile red and fluoresced yellow or orange, respectively, owing to accumulations of lipids in both macrophages and smooth muscle cells (SMC). Digital overlays of the filipin and Nile red images revealed that larger lesions (> 0.5 mm diameter) had a "nested" distribution of lipids, with a blue (filipin) fringe surrounding an orange (Nile red) fringe surrounding a yellow (Nile red) center.

Animals↗

PubMed1996

Characterization of CD4-gp120 activation intermediates during human immunodeficiency virus type 1 syncytium formation.

The mechanism by which cells expressing HIV envelope glycoproteins progress from binding CD4+ cells to syncytia formation is not entirely understood. The purpose of these investigations was to use physical and biochemical tools (temperature shifts, soluble CD4, protease inhibitors, and a battery of anti-CD4 monoclonal antibodies) to isolate discrete steps during syncytia formation. Previously (Fu et al., J Virol 1993;67:3818), we found that preincubation of cells stably expressing HIV-1 gp 160 (TF228.1.16) with CD4+ SupT1 cells at 16 degrees C, a temperature that is nonpermissive for syncytia formation, resulted in an increased rate of syncytia formation when the cocultures were shifted to the syncytia-permissive temperature of 37 degrees C. We have since found that syncytia formation is further enhanced by shifting the cocultures from 16 to 4 degrees C prior to incubation at 37 degrees C. Together, these data suggest that two discrete states, which we term the first and second activation intermediates (FAI and SAI), are involved in syncytia formation. We have found that acquisition of the FAI (by preincubation at 16 degree C) is sensitive to some serine protease inhibitors (PI), soluble CD4 (sCD4), shedding of gp120, and anti-CD4 monoclonal antibodies (MAb) directed toward the CDR-1/2 and CDR-3 regions of domain 1 on CD4. Expression of the FAI (formation of syncytia by shifting from 16 to 37 degrees C) remains sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2 but is less sensitive to MAb that bind CDR-3 and is insensitive to PI. Similarly, acquisition of the SAI (shifting cocultures from 16 to 4 degrees C), is sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2. In contrast, expression of the SAI (shifting cocultures from 16 to 4 to 37 degrees C) is sensitive only to MAb directed toward CDR-1/2 and cannot be blocked by sCD4, shedding of gp120, or PI. These data allow us to propose that syncytia formation, mediated by HIV-1 envelope glycoproteins, proceeds by a multistep cascade.

Antibodies, Monoclonal↗

PubMed1996

Relationships between antibodies against human soluble complement receptor 1 (hsCR1) from various species.

The relationships between antibodies against human soluble complement receptor 1 (hsCR1) were studied in rodents, dogs, nonhuman primates, and humans. An antibody response occurred in all species except humans. The anti-hsCR1 antibodies from the various species were characterized to determine if they recognize similar epitopes on the hsCR1 molecule. Dog and monkey sera, positve for hsCR1 binding, were used as blocking antibodies against mouse anti-hsCR1 monoclonal antibodies as well as mouse and rat anti-hsCR1-positive sera. Human sera (blood group antisera: anti-Knops, anti-McCoy, anti-Knops/McCoy, anti-Swain-Langley) and serum from one burn patient (who became seropositive despite ever receiving treatment with hsCR1) were also used to test blocking of mouse, rat, dog, and monkey anti-hsCR1. Characterization of anti-hsCR1 antibodies from different species demonstrated that hsCR1 causes divergent antibody responses among animals. While mouse, rat, and dog antibodies cross inhibit binding by approximately 50%, monkey antibodies recognize primarily different epitopes of the hsCR1 molecule. Moreover, human antibodies binding hsCR1 are completely different from the animal antibodies, including monkey. This study indicates that although hsCR1 is immunogenic in animals, there is a difference in response between species, particularly between nonprimates and primates, and finally, that this antibody response is not predictive for humans.

Animals↗

PubMed1995

Effect of TNF alpha production inhibitors BRL 61063 and pentoxifylline on the response of rats to poly I:C.

BRL 61063 is a novel xanthine phosphodiesterase (PDE) type IV inhibitor with selective inhibitory activity for tumor necrosis factor (TNF) alpha production. This compound inhibits TNF alpha production by activated human blood monocytes in vitro and in animal models of endotoxemia and influenza infection. Inhibition of TNF alpha may be beneficial in many diseases; however, little is known about potential adverse effects of such inhibition on host defense. In an ex vivo study, we examined the effect of BRL 61,063 on the microbicidal and tumoricidal activity of pulmonary lavage cells during a local inflammatory response in rats challenged with Poly I:C. Pentoxifylline, a PDE inhibitor which also blocks TNF alpha production, was used for comparison. Treatment with BRL 61063 or pentoxifylline did not block the inflammatory response to Poly I:C or the activation of bronchoalveolar lavage (BAL) cells but reduced the level of tumoricidal activity attained. At the dosages used, pentoxifylline was more inhibitory than BRL 61063. Drug treatment did not prevent further stimulation of tumoricidal activity by LPS in vitro. LPS-stimulated cells from BRL 61063-treated rats reached a level of activation similar to the control group while the LPS-stimulated activity of BAL cells from pentoxifylline treated rats remained lower than control. Although pentoxifylline was more inhibitory for tumoricidal activity than BRL 61063, the latter was a more potent inhibitor of TNF alpha release as measured in vivo in LPS-challenged rats. This finding indicates that TNF alpha is not the main mediator involved in the activation of pulmonary macrophage tumoricidal function. Treatment with either BRL 61063 or pentoxifylline had little or no effect on the Poly I:C-induced candidacidal activity of BAL cells indicating that these compounds are unlikely to compromise non-specific host defense against infection.

Animals↗

PubMed1995

Biochemical and functional analysis of rat bronchoalveolar macrophages containing chemically induced phospholipid inclusions.

Cationic amphiphilic drugs (CADs) are structurally characterized by hydrophobic ring structures and hydrophilic side chains. Studies have demonstrated that repeated administration of CADs to experimental animals and humans may induce phospholipid (PL) accumulation within the cells of various tissues. The immunomodulatory azaspiranes are novel CADs with beneficial effects in a number of animal models of autoimmune disease and transplantation. Although the mechanism of action of these compounds is unclear, efficacy in all of the disease models is accompanied by the generation of suppressor cell (SC) activity in various lymphoid organs. SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4,5]decane-2-propanamine+ ++ hydrochloride) and two analogs, SK&F 106615 and SK&F 103811, were compared with chlorphentermine and chloroquine for their ability to induce PL accumulation and SC activity. Oral administration of SK&F 105685 and SK&F 106615 caused PL accumulation in bronchoalveolar lavage macrophages (AM) but to a far lesser extent (three- to fivefold) than chlorphentermine. Neither the immunologically unreactive azaspirane SK&F 103811 nor chloroquine affected PL levels. AM from rats treated with SK&F 105685 or SK&F 106615 expressed more potent SC activity than chlorphentermine. Thus, SC activity did not correlate with the extent of PL accumulation. Neither SK&F 103811 nor chloroquine induced SC activity. AM from SK&F 105685-treated rats had an enhanced ability to kill the opportunistic pathogen Candida albicans in vitro indicating that there was no impairment of macrophage-dependent host defense mechanisms.

Animals↗

PubMed1995

Ultrastructural evidence of an interaction between Env and Gag proteins during assembly of HIV type 1.

Assembly and budding of retroviruses is believed to involve a complex interaction of envelope and capsid proteins at the host cell membrane. The nature of these interactions is, however, incompletely understood. Studies of the topography of the surface of HIV-1 have shown that the envelope glycoprotein projections (knobs) are arranged in a T = 7 levo rotational symmetry. Similarly, an icosahedral structure has been suggested for the p17 matrix of HIV-1. In an effort to investigate whether there is a structural interaction between these molecules, virions whose maturation was blocked by an inhibitor of HIV protease were studied using cytochemistry, morphometry, and 2D fast Fourier transform image enhancement. Analysis of the relationship between core morphology and the topographic distribution of envelope glycoprotein projections on HIV-1 provided structural evidence of an interaction between Env and Gag proteins. Furthermore, image enhancement revealed a periodic substructure in the Pr55gag plaque. Taken together, the data suggest an interaction between Pr55gag and the gp120-gp41 complex during assembly and budding of HIV-1. This interaction may, in part, contribute to determining the amount of Env glycoprotein that will be incorporated into a virion, and therefore play a role in the biology of HIV-1.

HIV Core Protein p24↗

PubMed1995

A single amino acid change in the cytoplasmic domain of the simian immunodeficiency virus transmembrane molecule increases envelope glycoprotein expression on infected cells.

We have described a virus termed CP-MAC, derived from the BK28 molecular clone of simian immunodeficiency virus, that was remarkable for its ability to infect Sup-T1 cells with rapid kinetics, cell fusion, and CD4 down-modulation (C. C. LaBranche, M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, and J. A. Hoxie, J. Virol. 68:5509-5522, 1994 [Erratum 68:7665-7667]). Compared with BK28, CP-MAC exhibited a number of changes in its envelope glycoproteins, including a highly stable association between the external (SU) and transmembrane (TM) molecules, a more rapid electrophoretic mobility of TM, and, of particular interest, a marked increase in the level of envelope protein expression on the surface of infected cells. These changes were shown to be associated with 11 coding mutations in the env gene (5 in SU and 6 in TM). In this report, we demonstrate that a single amino acid mutation of a Tyr to a Cys at position 723 (Y723C) in the TM cytoplasmic domain of CP-MAC is the principal determinant for the increased expression of envelope glycoproteins on the cell surface. When introduced into the env gene of BK28, the Y723C mutation produced up to a 25-fold increase in the levels of SU and TM on chronically infected cells, as determined by fluorescence-activated cell sorter analysis with monoclonal and polyclonal antibodies. A similar effect was observed when a Tyr-to-Cys change was introduced at the analogous position (amino acid 721) in the SIVmac239 molecular clone, which, unlike BK28 does not contain a premature stop codon in its TM cytoplasmic tail. Substituting other amino acids, including Ala, Ile, and Ser, at this position produced increases in surface envelope glycoproteins that were similar to that observed for the Cys substitution, while a Tyr-to-Phe mutation produced a smaller increase. These results could not be accounted for by differences in the kinetics or efficiency of envelope glycoprotein processing or by shedding of SU from infected cells. However, immunoelectron microscopy demonstrated that the Y723C mutation in BK28 produced a striking redistribution of cell surface envelope molecules from localized patches to a diffuse pattern that covered the entire plasma membrane. This finding suggests that mutation of a Tyr residue in the simian immunodeficiency virus TM cytoplasmic domain may disrupt a structural element that can modulate envelope glycoprotein expression on the surface of infected cells.

Amino Acid Sequence↗