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L Pereira

Publications and source records attributed to L Pereira.

At least 73 records · Page 4Linked to original sources
PubMed1996

Role of apical and basolateral membranes in replication of human cytomegalovirus in polarized retinal pigment epithelial cells.

Human retinal pigment epithelial (RPE) cells, which are permissive for human cytomegalovirus (HCMV) replication, were used to evaluate virus infection from apical and basolateral membranes of polarized cells. Tests of HCMV infectivity showed that the apical membrane was 20-30-fold more susceptible to infection than the basolateral membrane; in contrast, both membranes were equally susceptible to infection by herpes simplex virus type 1 (HSV-1). Neutralizing monoclonal antibodies (MAbs) to HCMV glycoprotein B (gB) blocked penetration of virions into polarized RPE cells. This indicated that gB has a function in fusion of the virion envelope with the apical membrane of these cells, as it has with the cell membrane of unpolarized human fibroblasts. In contrast to HSV-1-infected RPE cells, the paracellular permeability of polarized RPE cells changed slowly following infection with HCMV. Confocal microscopy examination of HCMV-infected RPE cells revealed that the pattern of ZO-1 staining was altered at late times. Addition of gB-specific neutralizing MAbs to the apical and basolateral membranes of HCMV-infected RPE cells failed to inhibit plaque development; this indicated that progeny virions infect adjacent cells before disassembly of tight junctions and are sequestered from neutralization during spread across lateral cell membranes. The finding that progeny HCMV virions cross lateral cell membranes, which differ substantially in protein composition from apical membranes, suggests that polarized RPE cells contain multiple receptors for HCMV.

Animals↗
PubMed1996

Mutations in the carboxyl-terminal hydrophobic sequence of human cytomegalovirus glycoprotein B alter transport and protein chaperone binding.

Human cytomegalovirus glycoprotein B (gB) plays a role in the fusion of the virion envelope with the host cell membrane and in syncytium formation in infected cells. Hydrophobic sequences at the carboxyl terminus, amino acids (aa) 714 to 771, anchor gB in the lipid bilayer, but the unusual length of this domain suggests that it may serve another role in gB structure. To explore the function(s) of this region, we deleted aa 717 to 747 (gB deltaI mutation), aa 751 to 771 (gB deltaII mutation), and aa 717 to 772 (gB deltaI-II mutation) and constructed a substitution mutation, Lys-748 to Val (Lys748Val)-Asn749Ala-Pro750Ile (gB KNPm). Mutated forms of gB were expressed in U373 glioblastoma cells and subjected to analysis by flow cytometry, confocal microscopy, and immunoprecipitation. Mutations gB deltaI-II and gB deltaII alone caused secretion of gB into the medium, confirming that aa 751 to 771 function as a membrane anchor. In contrast, mutations gB deltaI and gB KNPm blocked cell surface expression and arrested gB transport in the endoplasmic reticulum (ER). Detailed examination of gB deltaI and gB KNPm with a panel of monoclonal antibodies showed that the mutated forms were indistinguishable from wild-type gB in conformation and formed oligomers; however, they remained sensitive to endoglycosidase H and did not undergo endoproteolytic cleavage. Analysis of protein complexes formed by gB and molecular chaperones in the ER showed that calnexin and calreticulin, lectin-like chaperones, bound equal amounts of uncleaved wild-type gB, gB deltaI, and gB KNPm, but the glucose-regulated proteins 78 (BiP) and 94 formed stable complexes only with the mutated forms, causing their retention in the ER. Our studies show that aa 714 to 750 are key residues in the architecture of gB molecules and that the ER chaperones, which facilitate gB folding and monitor the quality of glycoproteins, detect subtle changes in folding intermediates that are conferred by mutations in this region.

Amino Acid Sequence↗
PubMed1996

Accessory human cytomegalovirus glycoprotein US9 in the unique short component of the viral genome promotes cell-to-cell transmission of virus in polarized epithelial cells.

Human cytomegalovirus (CMV) encodes accessory glycoproteins that are dispensable for virus growth in nonpolarized cells in culture. We report that CMV deletion mutants lacking the gene for accessory glycoprotein US9 in the unique short component of the viral genome are impaired in plaque formation in polarized human retinal pigment epithelial (ARPE-19) cells. Comparison of CMV deletion mutants in US9 with herpes simplex virus type 1 deletion mutants lacking glycoproteins gE and gI showed that both of these mutants are impaired in altering junctional complexes and increasing paracellular permeability in polarized ARPE-19 cells cultured on permeable filter supports. Results of functional studies indicate that CMV US9 and homologs of gE have analogous roles in promoting virus spread across lateral membranes of polarized epithelial cells.

Animals↗
PubMed1995

Mutated forms of human cytomegalovirus glycoprotein B are impaired in inducing syncytium formation.

Human cytomegalovirus (HCMV) glycoprotein B (gB) promotes virion entry into cells by fusing the virion envelope with the cellular membrane. We recently reported that UB cells (U373 glioblastoma cells constructed to produce HCMV gB constitutively) form multinucleate syncytia that are dependent on the density of gB in the plasma membrane. In this report, we describe the properties of a clonal cell line, UB31-B3, that expressed a spontaneously mutated form of gB which lacked the fusion-inducing function of the wild-type molecule, and three UB cell lines that were constructed to investigate the effect of specific mutations in gB on syncytium formation. Flow cytometry analysis with a pool of monoclonal antibodies (mAbs) showed that the UB cells contained a high density of gB, which was associated with the cell surface. Immune precipitation experiments with UB31-B3 cells showed that the mutant gB reacted with all of the mAbs to the ectodomain of gB but with none of those to the cytoplasmic carboxy terminus, and that it was 35 kDa smaller than wild-type gB. Nucleotide sequence analysis showed that a termination codon had been introduced after amino acid lysine at position 669 in the ectodomain of UB31-B3 gB, generating a truncated glycoprotein. UB31-B3 gB was not secreted into the medium and was stably anchored in the plasma membrane, which suggested that a hydrophobic stretch of amino acids from 629 to 652 in the ectodomain may serve as a membrane anchor for this truncated form. Analysis of the UB cell lines expressing deleted forms of gB showed that deletion of all or part of the cytoplasmic and transmembrane domains reduced or abolished syncytium formation. In contrast, deletion of a major neutralizing region in the ectodomain of gB did not alter syncytium formation. Results of these studies indicate that different regions of the gB molecule participate in syncytium formation.

Antibodies, Monoclonal↗
PubMed1995

Thyroid hormones alter Arrhenius kinetics of succinate-2,6-dichloroindophenol reductase, and the lipid composition and membrane fluidity of rat liver mitochondria.

Thyroid-status-dependent changes in lipid/phospholipid profiles in rat liver mitochondria were examined and attempts were made to correlate the observed changes with kinetic parameters of succinate-2,6-dichloroindophenol reductase (SDR) and membrane fluidity. Thyroidectomy caused substantial decrease in the total phospholipid and cholesterol contents; the proportion of all phospholipid components was also decreased significantly (50-100% decrease). The energy of activation, E1, for SDR decreased with a concomitant increase in the membrane fluidity. Treatment with physiologic doses or replacement doses of L-thyroxine resulted in partial restoration of lipid/phospholipid components. However, the SDR activity decreased with a simultaneous elevation in the phase-transition temperature and significant decreases in E1 and E2. Treatment with L-thyroxine restored membrane fluidity to almost euthyroid values. Regression analysis suggested a role for phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol microdomains in modulating the SDR activity. Membrane fluidity was strongly correlated with total phospholipid, cholesterol, diphosphatidylglycerol and phosphatidyl-inositol, and the ratio of cholesterol/total phospholipid (mol/mol).

Animals↗
PubMed1995

Primary structure and developmental expression of Fbn-1, the mouse fibrillin gene.

Previous studies have reported > 10 kilobases of human fibrillin-1 cDNA sequence, but a consensus regarding the 5' end of the transcript remains to be worked out. One approach to developing a clear consensus would be to search for regions of evolutionary conservation in transcripts from a related species such as mouse. As reported here, the mouse fibrillin-1 transcript encodes a highly conserved polypeptide of 2,871 amino acids. The upstream sequence that flanks the ATG is considerably less well conserved, however. Indeed, the ATG codon (which occurs in the context of a Kozak consensus sequence and is located just upstream of a consensus signal peptide) signals the point where human and mouse fibrillin-1 sequences cease to be nearly identical. Together, these results are consistent with previous efforts by Pereira et al. (Pereira, L., D'Alessio, M., Ramirez, F., Lynch, J. R., Sykes, B., Pangilinan, T., and Bonadio, J. (1993) Human Mol. Genet. 2, 961-968) to identify the human fibrillin-1 translational start site. Sequences immediately upstream of the ATG are GC-rich and devoid of TATA and CCAAT boxes, which suggests that the mouse fibrillin-1 gene will be broadly expressed. A survey of expression in mouse embryo tissues is consistent with this hypothesis and suggests two novel functions for fibrillin-associated microfibrils in non-elastic connective tissues.

Amino Acid Sequence↗
PubMed1995

Conformation-defective herpes simplex virus 1 glycoprotein B activates the promoter of the grp94 gene that codes for the 94-kD stress protein in the endoplasmic reticulum.

GRP94 is a major glycoprotein in the endoplasmic reticulum with calcium-binding properties. Recently, GRP94 has been shown to bind to unassembled forms of multimeric proteins and peptides. We report here that GRP94 forms a stable association with the mutated form of the herpes simplex type virus 1 (HSV-1) glycoprotein B, but not with the fully processed viral protein. Both the glycosylated and unglycosylated forms of GRP94 are capable of complexing with the mutated, conformation-defective viral glycoprotein. Cotransfection of expression vectors for gB and grp94 promoter fusion genes revealed that the grp94 promoter is strongly activated by the mutant form of gB. Analysis of the grp94 promoter mutants showed that two regions in the promoter, a highly conserved element referred to as grp core and the CCAAT element most proximal to the TATA element (C1), mediate the induction of grp94 by malfolded protein. We further determined that the grp94 core and C1 element bind to common as well distinct nuclear factors from grp78, a commonly coregulated gene. Through UV cross-linking, site competition, and immunocross-reactivity, we identified that the heteromeric CCAAT-binding protein (CBF) is one component of the grp94 C1 complex.

Animals↗
PubMed1995

A common missense mutation in the adhalin gene in three unrelated Brazilian families with a relatively mild form of autosomal recessive limb-girdle muscular dystrophy.

Autosomal recessive limb-girdle muscular dystrophies (AR LGMD) represent a heterogeneous group of diseases with a wide spectrum of clinical variability, classified phenotypically into two main groups, the most severe forms (Duchenne-like muscular dystrophy, DLMD, or severe childhood autosomal recessive muscular dystrophy, SCARMD) and the milder forms. Four genes causing AR LGMD have been mapped: the 15q (LGMD2a), the 2p (LGMD2b), the 13q locus (LGMD2c) and the adhalin gene on chromosome 17q (LGMD2d). In the present report we have performed linkage analysis with 17q markers in three mild AR LGMD and in four DLMD families with adhalin deficiency and unlinked to 2p, 15q or 13q genes. Linkage was observed only among the mild cases. Patients from these three 17q-linked families showed near or total deficiency of adhalin in muscle biopsies. An identical missense mutation was identified in all three 17q-linked unrelated families. These results indicate that AR LGMD with a mild phenotype is caused by mutations in the adhalin gene. In addition, they demonstrate that there is at least one other locus for DLMD associated with adhalin deficiency.

Base Sequence↗
PubMed1995

Fifteen novel FBN1 mutations causing Marfan syndrome detected by heteroduplex analysis of genomic amplicons.

Mutations in the gene encoding fibrillin-1 (FBN1), a component of the extracellular microfibril, cause the Marfan syndrome (MFS). This statement is supported by the observations that the classic Marfan phenotype cosegregates with intragenic and/or flanking marker alleles in all families tested and that a significant number of FBN1 mutations have been identified in affected individuals. We have now devised a method to screen the entire coding sequence and flanking splice junctions of FBN1. On completion for a panel of nine probands with classic MFS, six new mutations were identified that accounted for disease in seven (78%) of nine patients. Nine additional new mutations have been characterized in the early stages of a larger screening project. These 15 mutations were equally distributed throughout the gene and, with one exception, were specific to single families. One-third of mutations created premature termination codons, and 6 of 15 substituted residues with putative significance for calcium binding to epidermal growth factor (EGF)-like domains. Mutations causing severe and rapidly progressive disease that presents in the neonatal period can occur in a larger region of the gene than previously demonstrated, and the nature of the mutation is as important a determinant as its location, in predisposing to this phenotype.

Base Sequence↗
PubMed1995

Deletion mutants in human cytomegalovirus glycoprotein US9 are impaired in cell-cell transmission and in altering tight junctions of polarized human retinal pigment epithelial cells.

Retinal cytomegalovirus (CMV) disease is one of the major manifestations of viral pathogenesis in immunosuppressed patients with the acquired immunodeficiency syndrome (AIDS). CMV infection of the retina causes directional destruction which begins at the optic nerve head adjacent to the retinal capillaries and progresses, if untreated, to retinal detachment and blindness. Infection does not occur across the basal membrane of the retinal pigment epithelium (RPE), adjacent to the highly vascularized choroid. CMV replicates in polarized RPE cells, and progeny virions cross apical and lateral membranes of RPE cells grown on permeable filter supports, but not basal membranes. Cell-cell junctions of CMV-infected RPE cells are permeabilized, and the tight junction protein zonula occludens (ZO-1) is disassembled; progeny virions then spread to neighboring cells through the lateral cell membranes, which in polarized cells differ significantly in lipid and protein composition from the apical cell membranes. We found that CMV mutants with deletions in US9 and US8/US9 failed to spread from cell to cell, exhibiting a small-plaque phenotype in polarized RPE cells. Immunofluorescence confocal microscopy staining of ZO-1 protein revealed that RPE cells infected with CMV deletion mutants RV35, RV80, and RV61 did not exhibit altered tight junctions, in contrast to RPE cells infected with wild-type strain AD169 virus. Our findings indicate that US9, which is an accessory glycoprotein in infected foreskin fibroblasts, is required for transmission of virus across cell-cell junctions of polarized RPE cells. The relationship between US9 expression and virus transmission across cell-cell boundaries suggests that US9 may directly or indirectly permeabilize tight junction complexes of polarized RPE cells.

Cytomegalovirus↗
PubMed1994

A molecular approach to the stratification of cardiovascular risk in families with Marfan's syndrome.

BACKGROUND: The fibrillin gene encodes a protein in the extracellular matrix, and this protein is widely distributed in elastic tissues. The fibrillin gene is the site of mutations causing Marfan's syndrome. This disorder shows a high degree of clinical variability both between and within families. Each family appears to have a unique mutation in the fibrillin gene, which precludes the routine use of mutation screening for presymptomatic diagnosis of the disorder. The goal of this study was to develop a widely applicable method of molecular diagnosis. METHODS: We used three newly characterized intragenic sites of normal DNA repeat-sequence variation (i.e., polymorphisms) as markers to follow the inheritance pattern of specific copies (alleles) of the fibrillin gene in multiple kindreds with various clinical features of Marfan's syndrome. RESULTS: The polymorphic markers allowed identification of the particular copy of the fibrillin gene that cosegregated with Marfan's syndrome in 13 of the 14 families tested. In 11 families a definite presymptomatic diagnosis of Marfan's syndrome could be made in family members who had only equivocal manifestations of the disorder. In two other families, some family members demonstrated either classic Marfan's syndrome or a milder but closely related phenotype. The copy of the fibrillin gene that cosegregated with classic Marfan's syndrome was not inherited by family members with the latter, atypical, form of the disease. These milder phenotypes, previously diagnosed as Marfan's syndrome, were not associated with aortic involvement. CONCLUSIONS: These results document the usefulness of novel polymorphic DNA repeat sequences in the presymptomatic diagnosis of Marfan's syndrome. Our findings also demonstrate that the various clinical phenotypes seen in selected families may be due not to single fibrillin mutations, but rather to different genetic alterations. These findings underscore the need for a modification of the current diagnostic criteria for Marfan's syndrome in order to achieve accurate risk assessment.

Alleles↗
PubMed1994

Function of human cytomegalovirus glycoprotein B: syncytium formation in cells constitutively expressing gB is blocked by virus-neutralizing antibodies.

We report that U373 glioblastoma cells constitutively producing human cytomegalovirus (HCMV) glycoprotein B (gB), the product of open reading frame UL55 of the HCMV genome, formed syncytia that contained 5 to 25 nuclei. Flow cytometry with a panel of monoclonal antibodies (mAbs) to the extracellular domain of HCMV gB showed that these cells expressed high densities of gB in the plasma membrane. We studied the properties of five clonal UB cell lines and the results are as follows. Reactivity of a panel of mAbs to HCMV gB showed that UB cells forming syncytia expressed gB with all of the conformational and sequential epitopes contained in the viral gB made in HCMV-infected cells. Syncytium formation in UB cells was independent of low pH and proteolytic cleavage of gB and was blocked by drugs that inhibit glycosylation and translocation of gB to the cell surface. Infected UB cells formed more syncytia than infected U373 cells, virions entered UB cells more rapidly, and higher virus titers were produced. Incubation of UB cells with complement-independent neutralizing antibodies to gB, which prevent virion entry into cells, cell-to-cell transmission of infection, and fusion of HCMV-infected glioblastoma cells, significantly reduced syncytium formation in UB cells. These results show that overlapping functional domains on HCMV gB promote fusion of the virion envelope with the cell surface, fusion of infected U373 cells, and syncytium formation in UB cell lines expressing high densities of gB in the plasma membrane. Our findings indicate that the functional regions of gB can be subjected to detailed analysis without constructing viral mutants by expressing mutated forms of gB with site-directed changes that preclude syncytium formation of U373 cells expressing these gene products.

Antibodies, Viral↗
PubMed1994

Function of glycoprotein B homologues of the family herpesviridae.

The herpesviruses of humans and animals are large, enveloped virions with complex genetic organization that cause either lytic or latent infections. Of the many glycoproteins encoded by these viruses and incorporated into the virion envelope, glycoprotein B (gB) is the most highly conserved. Homologues of gB are encoded by the genomes of all the herpesviruses. gB is required for infectivity and functions in penetration of cells by promoting fusion of the virion and plasma membranes. Syncytium formation, a manifestation of fusion activity in virus-infected cells, results from mutations in gB or in certain other genes encoding glycoproteins and integral membrane proteins. The paradox that mutations in gB and other proteins cause the formation of syncytia can be explained by the hypothesis that gB normally participates in a multisubunit protein complex that controls fusion following a cascade of interactions with cell-surface receptors. Syncytium formation by altered forms of the components of this complex would be the result of less well-regulated events. Recent findings on the control of fusion in other virus systems, and on vesicle fusion in the normal cellular secretory pathway, support a model in which gB functions together with other viral proteins to form a hydrophobic fusion pore.

Animals↗
PubMed1993

The fibrillin-Marfan syndrome connection.

A few years ago no one would have suspected that the well-known disorder of connective tissue, Marfan syndrome, could be caused by mutations in a recently discovered extracellular component, fibrillin. Likewise, nobody would have predicted that fibrillin represents a small family of proteins that are associated with several phenotypically overlapping disorders. The fibrillins are integral constituents of the non-collagenous microfibrils, with an average diameter of 10 nm. These aggregates are distributed in the extracellular matrix of virtually every tissue. Microfibrillar bundles provide the external coating to elastin in elastic fibers, and serve an anchoring function in non-elastic tissues. At higher resolution, individual microfibrils have a "beads-on-a-string" appearance resulting from the head-to-tail polymerization of multiple fibrillin aggregates. Structurally, fibrillin contains a series of repeated sequences homologous to the epidermal growth factor calcium-binding motif. Characterization of fibrillin mutations in Marfan syndrome patients, together with the elucidation of the structure of the fibrillin proteins, have provided new insights, and raised new questions, about the function of the 10 nm microfibrils. For example, it is possible that the fibrillins, in addition to serving a structural function, might also be involved in regulating cellular activities and morphogenetic programs. It is fitting that the long search for the Marfan syndrome gene has brought a novel group of proteins to the forefront of extracellular matrix biology.

Connective Tissue↗
PubMed1993

Transport and secretion of truncated derivatives of herpes simplex virus 1 glycoprotein B.

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB) is one of several glycoproteins that compose the virion envelope. In infected cells, gB is a transmembrane glycoprotein that dimerizes and is glycosylated during its transport through the exocytic pathway to the cell surface. To understand the structural requirements for the transport of gB, we analyzed the processing, dimerization, and transport of mutated forms lacking the transmembrane region and the carboxy terminus of the molecule. Our studies showed that conversion of the membrane-anchored form of gB into soluble forms of different lengths resulted in slowed transport rates and incomplete processing. The longer derivatives, gB-(1-690) and gB-(1-690 delta 849), formed dimers but were incompletely transported and were retained within cells. Similar results were obtained with gB-(1-600), which failed to form dimers. In contrast, gB-(1-475), which also failed to form dimers, was transported and secreted from cells. These findings indicate that dimerization is not required for the transport of short, soluble forms of gB through the exocytic pathway provided that conformational regions contained within these shortened molecules are properly assembled.

Animals↗
PubMed1993

Glycoprotein B of human cytomegalovirus promotes virion penetration into cells, transmission of infection from cell to cell, and fusion of infected cells.

Human cytomegalovirus (HCMV) glycoprotein B (gB) is an abundant glycoprotein in the virion envelope that elicits neutralizing antibodies in human infection and in immunized animals. Using a panel of monoclonal antibodies to gB with neutralizing activity, we analyzed in detail the mechanisms by which these antibodies block HCMV infection. Twelve antibodies with complement-independent neutralizing activity were studied for their effect on the attachment of virions to the cell surface, virion penetration into cells, the transmission of infection from cell to cell, and fusion of infected cells. All of the complement-independent antibodies blocked penetration of virions into cells but had no effect on virion attachment to the cell surface. The most potent neutralizing antibodies also limited the spread of infection from cell to cell. Fusion of HCMV-infected U373 glioblastoma cells was blocked by all but two of the neutralizing antibodies to gB, suggesting that fusion of infected cells is similar but not identical to the fusion of the virion envelope with the cell membrane that occurs during virion entry. Our findings provide the first evidence that HCMV gB is a multifunctional glycoprotein that promotes virion penetration into cells, the transmission of infection from cell to cell, and fusion of infected U373 cells.

Antibodies, Monoclonal↗