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ABSTRACT: The oligomeric state and the hydrodynamic properties of human respiratory syncytial virus (HRSV) phosphoprotein (P), a known cofactor of the viral RNA-dependent RNA polymerase (L), and a trypsin-resistant fragment (X) that includes its oligomerization domain were analyzed by sedimentation equilibrium and velocity using analytical ultracentrifugation. The results obtained demonstrate that both P and fragment X are homotetrameric with elongated shapes, consistent with electron micrographs of the purified P protein in which thin rod-like molecules of approximately 12.5 +/- 1.0 nm in length were observed. A new chymotrypsin resistant fragment (Y*) included in fragment X has been identified and purified by gel filtration chromatography. Fragment Y* may represent a minimal version of the P oligomerization domain. Thermal denaturation curves based on circular dichroism data of P protein showed a complex behavior. In contrast, melting data generated for fragments X and particularly fragment Y* showed more homogeneous transitions indicative of simpler structures. A three-dimensional model of X and Y* fragments was built based on the atomic structure of the P oligomerization domain of the related Sendai virus, which is in good agreement with the experimental data. This model will be an useful tool to make rational mutations and test the role of specific amino acids in the oligomerization and functional properties of the HRSV P protein.
Proteins Structure Function and Bioinformatics 09/2008; 72(3):946-58. · 3.39 Impact Factor
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ABSTRACT: Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now describe a system, based on a Sendai virus minigenome, to produce large amounts of heterologous viral glycoproteins in the allantoic cavity of embryonated eggs.
Soluble forms of human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) fusion (F) proteins, devoid of their transmembrane and cytoplasmic domains, were produced in allantoic fluids using the Sendai minigenome system. The first step was rescuing in cell cultures Sendai virus minigenomes encoding the proteins of interest, with the help of wild type Sendai virus. The second step was propagating such recombinant defective viruses, together with the helper virus, in the allantoic cavity of chicken embryonated eggs, and passage to optimize protein production. When compared with the production of the same proteins in the culture supernatant of cells infected with vaccinia recombinants, the yield in the allantoic fluid was 5-10 fold higher. Mutant forms of these soluble proteins were easily constructed by site-directed mutagenesis and expressed in eggs using the same approach.
The simplicity and economy of the Sendai minigenome system, together with the high yield achieved in the allantoic fluid of eggs, makes it an attractive method to express soluble glycoproteins aimed for structural studies.
BMC Biotechnology 02/2007; 7:17. · 2.35 Impact Factor
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ABSTRACT: We have reported previously the expression and purification of an anchorless form of the human respiratory syncytial virus (HRSV) F protein (F(TM-)) representing the ectodomain of the full-length F. F(TM-) molecules are seen as unaggregated cones by electron microscopy but completion of proteolytic cleavage of the F0 monomers in the F(TM-) trimer leads to a change in shape from cones to lollipops that aggregate into rosettes. This aggregation apparently occurs by interaction of the fusion peptides of F(TM-) molecules that are exposed after cleavage. Since exposure of the fusion peptide is a key event in the process of membrane fusion, changes associated with F(TM-) cleavage may reflect those occurring in full-length F during membrane fusion. Deletions or substitutions that changed either the length, charge or hydrophobicity of the fusion peptide inhibited aggregation of F(TM-), and these mutants remained as unaggregated cones after cleavage. In contrast, more conservative changes did not inhibit the change of shape and aggregation of F(TM-). When the same changes were introduced in the fusion peptide of full-length F, only the mutations that inhibited aggregation of F(TM-) prevented membrane fusion. Thus, the conformational changes that follow completion of cleavage of the F(TM-) protein require a functional fusion peptide. These sequence constraints may restrict accumulation of sequence changes in the fusion peptide of HRSV F when compared with other hydrophobic regions of the molecule.
Journal of General Virology 07/2006; 87(Pt 6):1649-58. · 3.36 Impact Factor
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ABSTRACT: Several functions required for the replication of influenza A viruses have been attributed to the viral matrix protein (M1), and a number of studies have focused on a region of the M1 protein designated "helix six." This region contains an exposed positively charged stretch of amino acids, including the motif 101-RKLKR-105, which has been identified as a nuclear localization signal, but several studies suggest that this domain is also involved in functions such as binding to the ribonucleoprotein genome segments (RNPs), membrane association, interaction with the viral nuclear export protein, and virus assembly. In order to define M1 functions in more detail, a series of mutants containing alanine substitutions in the helix six region were generated in A/WSN/33 virus. These were analyzed for RNP-binding function, their capacity to incorporate into infectious viruses by using reverse genetics, the replication properties of rescued viruses, and the morphological phenotypes of the mutant virus particles. The most notable effect that was identified concerned single amino acid substitution mutants that caused significant alterations to the morphology of budded viruses. Whereas A/WSN/33 virus generally forms particles that are predominantly spherical, observations made by negative stain electron microscopy showed that several of the mutant virions, such as K95A, K98A, R101A, and K102A, display a wide range of shapes and sizes that varied in a temperature-dependent manner. The K102A mutant is particularly interesting in that it can form extended filamentous particles. These results support the proposition that the helix six domain is involved in the process of virus assembly.
Journal of Virology 02/2005; 79(2):1262-70. · 5.40 Impact Factor
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ABSTRACT: Anchorless fusion (F) proteins () of human respiratory syncytial virus (RSV) are seen by electron microscopy as unaggregated cones when the proteolytic cleavage at two furin sites required for membrane-fusion activity is incomplete, but aggregate into rosettes of lollipop-shaped spikes following cleavage. To show that this aggregation occurred by interactions of the fusion peptide, a deletion mutant of lacking the first half of the fusion peptide was generated. This mutant remained unaggregated even after completion of cleavage, supporting the notion that aggregation of involved the fusion peptide. As exposure of the fusion peptide is a key event that occurs after activation of F proteins, the uncleaved and cleaved forms of may represent the pre- and post-active forms of RSV F protein. In an analysis of the structural differences between the two forms, their thermostability before and after proteolytic cleavage was examined. In contrast to other viral proteins involved in membrane fusion (e.g. influenza haemagglutinin), the pre-active (uncleaved) and post-active (cleaved) forms of were equally resistant to heat denaturation, assessed by spectrofluorimetry, circular dichroism or antibody binding. These results are interpreted in terms of the proposed structural changes associated with the process of membrane fusion mediated by RSV F protein.
Journal of General Virology 01/2005; 85(Pt 12):3677-87. · 3.36 Impact Factor
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ABSTRACT: The influenza virus uses hemagglutinin (HA) to fuse the viral and cellular membranes. As part of an effort to study the membrane-interacting elements of HA, the fusion peptide, and the C-terminal transmembrane anchor, we have expressed in Escherichia coli the full-length HA(2) chain with maltose-binding protein fused at its N-terminus. The chimeric protein can be refolded in vitro in the presence of specific detergents to yield stable, homogeneous trimers, as determined by analytical ultracentrifugation. The trimers have the so-called "low pH" conformation-the rearranged HA(2) conformation obtained when intact HA(1)/HA(2) is induced to refold by exposure to low pH-as detected by electron microscopy and monoclonalantibody reactivity. These results provide further evidence for the notion that the neutral-pH structure of intact HA is metastable and that binding of protons lowers the kinetic barriers that prevent rearrangement to the minimum-free-energy conformation. The refolded chimeric protein described here is a suitable species for undertaking studies of how the fusion peptide inserts into membranes and assessing the nature of possible intermediates in the fusion process.
Biochemistry 06/2004; 43(19):5902-11. · 3.42 Impact Factor
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ABSTRACT: The Rna14-Rna15 complex is a core component of the cleavage factor IA RNA-processing complex from Saccharomyces cerevisiae. To understand the assembly and RNA-binding properties, we have isolated and characterized the Rna14-Rna15 complex using a combination of biochemical and biophysical methods. Analysis of the purified complex, using transmission electron microscopy, reveals that the two proteins assemble into a kinked rod-shaped structure and that these rods are able to further self-associate. Analytical ultracentrifugation reveals that Rna14 mediates this association and facilitates assembly of an A2B2 tetramer (M(r) 230 000), where relatively compact Rna14-Rna15 heterodimers are in rapid equilibrium with tetramers that have a more extended shape. The Rna14-Rna15 complex, unlike the individual components, binds to an RNA oligonucleotide derived from the 3'-untranslated region of the S.cerevisiae GAL7 gene. Based on these structural and thermodynamic data, we propose that CFIA assembly regulates RNA-binding activity.
Nucleic Acids Research 02/2004; 32(11):3364-75. · 8.03 Impact Factor
