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K Wiegers

Publications and source records attributed to K Wiegers.

13 recordsLinked to original sources
PubMed2005

High mutation rate in dopa-responsive dystonia: detection with comprehensive GCHI screening.

Mutations in GTP cyclohydrolase I (GCHI) are found in 50 to 60% of cases with dopa-responsive dystonia (DRD). Heterozygous GCHI exon deletions, undetectable by sequencing, have recently been described in three DRD families. We tested 23 individuals with DRD for the different mutation types by conventional and quantitative PCR analyses and found mutations, including two large exon deletions, in 87%. The authors attribute this high mutation rate to rigorous inclusion criteria and comprehensive mutational analysis.

Adolescent↗
PubMed2002

Exon deletions in the GCHI gene in two of four Turkish families with dopa-responsive dystonia.

Most cases of dopa-responsive dystonia (DRD) are thought to be caused by mutations in the GCHI gene; however, by sequencing, mutations are found in only 40% to 60%. Recently, a single report identified, via Southern blot analysis, a large genomic GCHI deletion in a "mutation-negative" case. This report describes four families with DRD, two of which carry large deletions, thus confirming that deletions are an important subtype of GCHI mutations. These deletions were detected by quantitative duplex PCR that is amenable to DNA diagnostics.

Adult↗
PubMed2001

Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly.

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

3T3 Cells↗
PubMed2000

A conformational switch controlling HIV-1 morphogenesis.

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) proceeds in two steps. Initially, an immature virus with a spherical capsid shell consisting of uncleaved Gag polyproteins is formed. Extracellular proteolytic maturation causes rearrangement of the inner virion structure, leading to the conical capsid of the infectious virus. Using an in vitro assembly system, we show that the same HIV-1 Gag-derived protein can form spherical particles, virtually indistinguishable from immature HIV-1 capsids, as well as tubular or conical particles, resembling the mature core. The assembly phenotype could be correlated with differential binding of the protein to monoclonal antibodies recognizing epitopes in the HIV-1 capsid protein (CA), suggesting distinct conformations of this domain. Only tubular and conical particles were observed when the protein lacked spacer peptide SP1 at the C-terminus of CA, indicating that SP1 may act as a molecular switch, whose presence determines spherical capsid formation, while its cleavage leads to maturation.

Animals↗
PubMed1999

Cyclophilin A incorporation is not required for human immunodeficiency virus type 1 particle maturation and does not destabilize the mature capsid.

The cellular protein cyclophilin A (CypA) is packaged into human immunodeficiency virus type 1 (HIV-1) virions through a specific interaction with the capsid (CA) domain of the Gag polyprotein. CypA is important for infectivity, but its role in viral replication is currently unknown. Previous reports suggested that CypA promotes uncoating or enhances maturation. We analyzed the morphology and capsid stability of HIV-1 variants defective in CypA binding and of virus grown in the presence of cyclosporin. Both cyclosporin treatment and alteration of Gly89 or Pro90 in the CypA-binding site of CA caused a 5- to 20-fold decrease in CypA incorporation. Virus produced from cyclosporin-treated cells and variants G89V and G89A were 10- to 100-fold less infectious but exhibited normal virion morphologies with regular cone-shaped capsids. Irregular capsid morphologies and lower infectivities were observed for some other variants in the CypA-binding region. Decreased CypA incorporation did not reduce the kinetics of intracellular polyprotein processing or of virus release. No increase in immature particles was observed. These results suggest that CypA does not promote virion maturation. Furthermore, detergent stripping of virus particles with various CypA contents revealed no difference in capsid stability. Based on these results and those reported in the accompanying paper, it appears likely that CypA also is not an uncoating factor. Alternative models for CypA function are discussed.

Capsid↗
PubMed1998

Sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual Gag polyprotein cleavage sites.

Retroviruses are produced as immature particles containing structural polyproteins, which are subsequently cleaved by the viral proteinase (PR). Extracellular maturation leads to condensation of the spherical core to a capsid shell formed by the capsid (CA) protein, which encases the genomic RNA complexed with nucleocapsid (NC) proteins. CA and NC are separated by a short spacer peptide (spacer peptide 1 [SP1]) on the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein and released by sequential PR-mediated cleavages. To assess the role of individual cleavages in maturation, we constructed point mutations abolishing cleavage at these sites, either alone or in combination. When all three sites between CA and NC were mutated, immature particles containing stable CA-NC were observed, with no apparent effect on other cleavages. Delayed maturation with irregular morphology of the ribonucleoprotein core was observed when cleavage of SP1 from NC was prevented. Blocking the release of SP1 from CA, on the other hand, yielded normal condensation of the ribonucleoprotein core but prevented capsid condensation. A thin, electron-dense layer near the viral membrane was observed in this case, and mutant capsids were significantly less stable against detergent treatment than wild-type HIV-1. We suggest that HIV maturation is a sequential process controlled by the rate of cleavage at individual sites. Initial rapid cleavage at the C terminus of SP1 releases the RNA-binding NC protein and leads to condensation of the ribonucleoprotein core. Subsequently, CA is separated from the membrane by cleavage between the matrix protein and CA, and late release of SP1 from CA is required for capsid condensation.

Amino Acid Sequence↗
PubMed1994

Mapping of antigenic domains in poliovirus VP1 involved in structural rearrangements during virus morphogenesis and antigenic alterations of the virion.

Monoclonal antibodies (mAbs) directed against linear epitopes of the structural polypeptide VP1 of poliovirus type 1, Mahoney (PV1M), were used as sensitive tools to evaluate the accessibility of certain amino acid residues, both during virus morphogenesis and after conformational transitions of the capsid resulting from heat treatment (H- or 80S particles) and cell-receptor interaction (A- or 135S particles). Antibody binding sites were mapped by immunoblotting of VP1 fragments after procaryotic expression and by introduction of nested sets of deletions into recombinant VP1. The binding sites clustered at the amino- and carboxy-termini of the polypeptide, respectively. In 14S particles the amino-terminal sites were accessible for our mAbs, most likely from the inner surface of the particle. The carboxy-terminal sites became inaccessible during formation of pentamers from protomers. As shown by differential reaction of the mAbs, the amino-terminus of VP1 becomes externalized up to residues 41-55, whereas residues 56-67 remain buried during transition to both 80S and 135S particles. Carboxy-terminal residues 280-286 also become accessible to antibody binding on the surface of the altered particles. Since these residues are part of the canyon cleft of VP1, a structural rearrangement indicated by these mAbs is apparently associated with the loss of binding ability of 135S particles to the cellular receptor, which could explain the loss of infectivity of these particles.

Antibodies, Monoclonal↗
PubMed1993

Revertants of poliovirus escape mutants: new insights into antigenic structures.

Poliovirus variants that escape neutralization by monoclonal antibodies (mAbs) have previously been selected and characterized in order to determine antigenic sites on the surface of the virion. Phenotypic revertants of poliovirus type 1 escape mutants were selected within all three antigenic sites (sites 1, 2, and 3) on the basis of their reactivity with the selecting mAb. The phenotypic and genotypic properties of these revertants were determined by binding and neutralization assays. Sequencing of the viral RNA revealed different types of reversions. Besides reversion to wild-type genotype, we found phenotypic revertants which had amino acid substitutions differing from wild type, thus revealing amino acids that are also tolerated by the antibody. In another type of revertant, alterations in other parts of the epitope were found, providing a refined resolution of a particular antibody recognition site. Most of the revertants regained the property to be neutralized by the mAb. However, in one case they remained resistant to neutralization despite the fact that binding to the selecting antibody was reestablished. These results indicate that virus neutralization might be achieved by different mechanisms depending on the particular mAb.

Amino Acid Sequence↗
PubMed1992

Molecular basis of antigenic structures of poliovirus: implications for their evolution during morphogenesis.

Neutralizing monoclonal antibodies against poliovirus type 1 were obtained after conventional immunization or combined in vivo-in vitro immunization. Antibody binding sites were determined by sequence analysis of neutralization-resistant mutants. Site 3 variants had several amino acid substitutions in previously unidentified positions for neutralization resistance. Evidence for a linkage of subsites 3a and 3b is presented. Some site 3b antibodies as defined previously precipitated 14S subunits, although with reduced titers.

Antibodies, Monoclonal↗
PubMed1990

A new antigenic site of poliovirus recognized by an intertypic cross-neutralizing monoclonal antibody.

A monoclonal antibody (mAb 7J6) neutralizing poliovirus type 2 (PV2) and poliovirus type 1 (PV1) was obtained after immunization of BALB/c mice with infectious PV2, strain MEF-1. Preincubation of mAb 7J6 with PV1 inhibited its binding to PV2 and vice versa. Neutralization-resistant variants of PV2 and PV1 were selected. Nucleotide sequencing of the RNAs of some variants revealed mutations in the loop of amino acid residues 239 to 245 in VP2 and in the loop of amino acid residues 195 to 207 in VP3. This is the first evidence that these two loops contribute to a neutralization antigenic site (N-Ag) for poliovirus. Moreover, this new site on PV2 induced intertypic cross-neutralizing antibodies.

Amino Acid Sequence↗
PubMed1989

N-AgIB of poliovirus type 1: a discontinuous epitope formed by two loops of VP1 comprising residues 96-104 and 141-152.

Analysis of resistant mutants to neutralizing monoclonal antibodies revealed a discontinuous neutralization epitope on VP1 of poliovirus type 1, Mahoney. The epitope has the unique property of being also part of a sequential epitope within neutralization antigenic site I (N-AgI). It is formed by residues in the loop 96-104 connecting the B and C strand and in the loop 141-152 connecting the D and E strand of VP1. Because of strong analogy to neutralization immunogen IB (NImIB) of human rhinovirus 14 (HRV-14) we have called this site N-AgIB of poliovirus type 1.

Amino Acid Sequence↗
PubMed1988

Evidence for a complex structure of neutralization antigenic site I of poliovirus type 1 Mahoney.

We have selected neutralization escape mutants by using a monoclonal antibody (nt-MAb) against a sequential epitope between amino acids 93 through 104 (neutralization antigenic site I) of poliovirus type 1 Mahoney. The majority of mutants were also resistant against five strain-specific nt-MAbs which recognized conformation-dependent epitopes, suggesting that the neutralization antigenic site I must be involved in the formation of such epitopes. An analysis of all mutants by the binding of nt-MAbs and by isoelectric focusing of VP1 allowed discrimination of five classes of mutants. Sequence analysis of mutant RNAs revealed point mutations and deletions in the antibody-binding site.

Antibodies, Monoclonal↗