The Experts below are selected from a list of 312 Experts worldwide ranked by ideXlab platform
Bryan T. Eaton - One of the best experts on this subject based on the ideXlab platform.
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Fine mapping of a surface-accessible, immunodominant site on the bluetongue virus major core Protein VP7.
Virology, 1994Co-Authors: Lin-fa Wang, Denis B. Scanlon, Jacki A. Kattenbelt, James O. Mecham, Bryan T. EatonAbstract:Abstract The 349-amino-acid major core Protein VP7 of bluetongue virus (BTV) is both the most abundant viral structural Protein and the major immunogenic serogroup-reactive viral antigen. Previous studies indicated that a conformation-dependant antigenic site, defined by the VP7-specific monoclonal antibody 20E9/B7/G2(20E9), was accessible from the virus surface and that the binding of the monoclonal antibody to this epitope could be blocked specifically by antisera raised against different serotypes of bluetongue virus, suggesting it is a serogroup-specific immunodominant epitope. Using a combination of three different mapping strategies, we have located the 20E9 binding site at the N-terminus of the molecule, between amino acids 30 and 48. The fine mapping of the 20E9 immunodominant epitope will facilitate structure-function analyses of the major core Protein and provide new opportunities to improve existing BTV serodiagnosis methods based on this immunogenic site.
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Major core Protein VP7 of Australian bluetongue virus serotype 15: sequence and antigenicity divergence from other BTV serotypes
Journal of General Virology, 1994Co-Authors: Lin-fa Wang, Jacki A. Kattenbelt, Allan R. Gould, L.i. Pritchard, Gary Crameri, Bryan T. EatonAbstract:Full-length cDNA of the RNA genome segment coding for the major core Protein VP7 of Australian bluetongue virus serotype 15 (BTV-15) has been isolated by reverse transcription-PCR cloning. Comparative analysis indicated that the BTV-15 VP7 sequence had diverged significantly from that of other members of the BTV serogroup. At the amino acid level, BTV-15 VP7 exhibited sequence identities of 80 to 84% with VP7 molecules of other serotypes, significantly lower than the sequence identities of between 93 and 100% observed among other serotypes characterized to date. This was consistent with previous observations that there were significant immunological differences between BTV-15 and other BTV serotypes and that monoclonal antibodies raised against BTV-1 VP7 failed to react with BTV-15 VP7. Recombinant BTV-15 VP7 Protein produced from Escherichia coli was largely insoluble, but maintained its immunogenicity. Polyclonal mouse sera raised against the recombinant VP7 Protein reacted strongly with VP7 of BTV-15, but weakly with that of BTV-1.
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High level expression of the major core Protein VP7 and the non-structural Protein NS3 of bluetongue virus in yeast: use of expressed VP7 as a diagnostic, group-reactive antigen in a blocking ELISA
Virus research, 1991Co-Authors: John C. Martyn, Allan R. Gould, Bryan T. EatonAbstract:The major core Protein VP7 and a non-structural Protein NS3 of bluetongue virus serotype 1 have been synthesized from recombinant plasmids using both an in vitro transcription/translation system and a yeast expression system. Bluetongue virus genes were transcribed under the control of the bacteriophage SP6 promoter and the regulatable yeast metallothionein promoter. An indirect ELISA showed that expression of NS3 in yeast was inducible with 1 mM CuSO4 and VP7 synthesis was constitutive but could be further induced. The preferred procedure for antigen extraction from yeast was sonication for VP7 and SDS/NaOH treatment for NS3. Yeast-expressed VP7 antigen and a monoclonal antibody were used in a blocking ELISA to distinguish sera raised against bluetongue virus serotypes from those generated to viruses of the epizootic haemorrhagic disease serogroup.
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A bluetongue serogroup-reactive epitope in the amino terminal half of the major core Protein VP7 is accessible on the surface of bluetongue virus particles.
Virology, 1991Co-Authors: Bryan T. Eaton, Allan R. Gould, John C. Martyn, Alex D. Hyatt, Barbara E.h. Coupar, John R. WhiteAbstract:Abstract Immunoelectron microscopy has been used to confirm that the core Protein VP7 is accessible on the surface of bluetongue virus (BTV) particles. Monospecific antibodies generated to vaccinia virus-expressed VP7 and an anti-VP7 monoclonal antibody (MAb 20E9) bound to native virus particles and were localized by Protein A-gold. In contrast, MAb 20E9 labeled directly with gold failed to gain access and bind, suggesting that VP7 is neither adventitiously adsorbed to the virion surface nor exposed in a manner such as protrusion through the outer capsid. Thus the surface layer of BTV may be considered as a net which only partially obscures the underlying core particle. Sequencing of VP7 revealed it to be an extremely hydrophobic Protein, 350 amino acids in length with cysteine residues at positions 15, 65, and 154. Examination of VP7 in the cytosol of cells infected with either BTV or a vaccinia virus recombinant expressing VP7 indicated that the Protein may exist as an oligomer, whose constituent monomers are not linked by intermolecular disulfide bonds. The cysteine residues in sodium dodecyl sulfate (SDS)-denatured, dithiothreitol (DTT)-treated VP7 were labeled with the fluorescent iodoacetamide AEDANS and the Protein was cleaved by V8 protease. The size of the labeled peptides and knowledge of the location of potential V8 cleavage sites suggested that the enzyme cleaved VP7 at three locations (glutamic acid residues at positions 61, 104 (or 108), and 132 (or 134 or 135). Analysis of the fluorescent peptides generated by V8 protease cleavage of VP7 labeled with AEDANS in the absence of DTT (i.e., with any putative intramolecular disulfide bonds intact) suggested that the cysteine at position 154 was the only one accessible to AEDANS. The cysteines at positions 15 and 65 may therefore be linked via a disulfide bond. Denaturation of VP7 with SIDS did not eliminate the capacity of the Protein to bind MAb 20E9. However, the sensitivity of the epitope to reduction and acetylation and its resistance to either of these processes alone suggest that it may be located near a disulfide bond linking cysteines at positions 15 and 65. Confirmation that the epitope lay in the amino-terminal half of the VP7 came from immunoelectron microscopy experiments in which thin sections of bacteria expressing the complete VP7 and the amino-terminal half were probed with MAb 20E9 and Protein A-gold.
Polly Roy - One of the best experts on this subject based on the ideXlab platform.
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Assembly and Intracellular Localization of the Bluetongue Virus Core Protein VP3
Journal of virology, 2005Co-Authors: Alak Kanti Kar, Nao Iwatani, Polly RoyAbstract:The bluetongue virus (BTV) core Protein VP3 plays a crucial role in the virion assembly and replication process. Although the structure of the Protein is well characterized, much less is known about the intracellular processing and localization of the Protein in the infected host cell. In BTV-infected cells, newly synthesized viral core particles accumulate in specific locations within the host cell in structures known as virus inclusion bodies (VIBs), which are composed predominantly of the nonstructural Protein NS2. However, core Protein location in the absence of VIBs remains unclear. In this study, we examined VP3 location and degradation both in the absence of any other viral Protein and in the presence of NS2 or the VP3 natural associate Protein, VP7. To enable real-time tracking and processing of VP3 within the host cell, a fully functional enhanced green fluorescent Protein (EGFP)-VP3 chimera was synthesized, and distribution of the fusion Protein was monitored in different cell types using specific markers and inhibitors. In the absence of other BTV Proteins, EGFP-VP3 exhibited distinct cytoplasmic focus formation. Further evidence suggested that EGFP-VP3 was targeted to the proteasome of the host cells but was dispersed throughout the cytoplasm when MG132, a specific proteasome inhibitor, was added. However, the distribution of the chimeric EGFP-VP3 Protein was altered dramatically when the Protein was expressed in the presence of the BTV core Protein VP7, a normal partner of VP3 during BTV assembly. Interaction of EGFP-VP3 and VP7 and subsequent assembly of core-like particles was further examined by visualizing fluorescent particles and was confirmed by biochemical analysis and by electron microscopy. These data indicated the correct assembly of EGFP-VP3 subcores, suggesting that core formation could be monitored in real time. When EGFP-VP3 was expressed in BTV-infected BSR cells, the Protein was not associated with proteasomes but instead was distributed within the BTV inclusion bodies, where it colocalized with NS2. These findings expand our knowledge about VP3 localization and its fate within the host cell and illustrate the assembly capability of a VP3 molecule with a large amino-terminal extension. This also opens up the possibility of application as a delivery system.
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Preliminary crystallographic study of bluetongue virus capsid Protein, VP7.
Journal of molecular biology, 1992Co-Authors: A.k. Basak, David I. Stuart, Polly RoyAbstract:Bluetongue virus serotype 10 (BTV-10) VP7, expressed by insect cells infected with the recombinant baculovirus, has been purified and crystallized. Two crystal forms suitable for X-ray analysis have been obtained. Type I crystals belong to space group P6(3)22 with a = b = 95.2 A, c = 181.0 A, alpha = beta = 90 degrees gamma = 120.0 degrees, and contain a single subunit in the crystallographic asymmetric unit. They diffract to dmin = 3.0 A. Type II crystals belong to space group P2(1) with a = 69.4 A, b = 97.1 A, c = 71.4 A, beta = 109.0 degrees, and contain a trimer in the crystallographic asymmetric unit. They diffract to dmin = 2.1 A. These results, together with solution studies, show that the molecule is a trimer.
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Expression of the major core antigen VP7 of African horsesickness virus by a recombinant baculovirus and its use as a group-specific diagnostic reagent.
Journal of General Virology, 1992Co-Authors: T. Chuma, José Manuel Sánchez-vizcaíno, H. Le Blois, M. Diaz-laviada, Polly RoyAbstract:The major core Protein, VP7, of African horsesickness virus serotype 4 (AHSV-4), the aetiological agent of a recent outbreak of the disease in southern Europe, was expressed in insect cells infected with a recombinant baculovirus containing a cloned copy of the relevant AHSV gene (S7). Analyses of its biochemical and antigenic properties confirmed the authenticity of the Protein expressed. The high-level expression of VP7 under the control of the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus induced disc-shaped crystals in infected insect cells. This enabled us to purify the Protein by a one-step ultracentrifugation procedure and to utilize it for the detection of antibodies raised in horses to various serotypes of AHSV. A serological relationship between AHSV and two other orbiviruses, bluetongue virus and epizootic haemorrhagic disease virus, was also demonstrated.
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The complete sequence of the group-specific antigen, VP7, of African horsesickness disease virus serotype 4 reveals a close relationship to bluetongue virus
Journal of General Virology, 1991Co-Authors: Polly Roy, T. Hirasawa, M. Fernandez, V. M. Blinov, J. M. Sanchez-vixcain RodriqueAbstract:The complete sequence of the S7 RNA that codes for the major group-specific coat Protein, VP7, of African horsesickness virus serotype 4 (AHSV-4) was determined from cDNA analyses and found to be 1179 nucleotides in length. One single open reading frame of 353 codons was observed defining a Protein of M r 38107 with a net charge of -1·5 at neutral pH. Comparison of the AHSV-4 VP7 sequence with that of bluetongue virus serotype 10 revealed an overall similarity of 44%, with the amino- and carboxy-terminal regions exhibiting the greatest levels of homology. In addition, potential secondary structures of the terminal sequences of the S7 RNA segments of AHSV-4 and BTV serotypes 10, 13 and 17 are presented.
Timothy F. Kowalik - One of the best experts on this subject based on the ideXlab platform.
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bluetongue virus evolution sequence analyses of the genomic s1 segments and major core Protein VP7
Virology, 1991Co-Authors: Timothy F. KowalikAbstract:The S1 segments, encoding the group-specific antigen, VP7, from the five United States prototype BTV serotypes were cloned as full-length entities. The nucleotide and deduced amino acid sequences of segment S1 of BTV-2 were determined and compared with BTV-10, -11, -13, and -17, completing the sequencing of this cognate gene segment from all five US BTV serotypes. Each segment is 1156 by long and contains an open reading frame encoding the 349-amino acid VP7 Protein. Most (>94%) of the amino acids of VP7 among the serotypes are conserved, including the location (position 255) of a single lysine residue. Secondary structure analyses of VP7 predict a putative eight-stranded β-barrel between amino acid positions 150 and 250, a structure similar to that observed in ssRNA viruses. The S1 genes are flanked by conserved 5' and 3' noncoding regions. Stem-loop structures are predicted at the 3' end of each gene (nucleotide positions 1058–1097). The S1 segments of BTV-2, -10, -11, and -17 have >93% of the nucleotides conserved, while <80% of their bases are identical with BTV-13. Analyses of nucleotide mismatches in each codon position of the VP7 open reading frame, transition frequencies, and evolutionary distances show that of the five, BTV-13 is the most distantly related and that BTV-10 and -17 are the most closely related serotypes. Evolutionary distance calculations of segment L2 from BTV-10, -11, and -17 concur with these observations. Comparison of this relationship with hybridization data of segment M3, which codes for VP5, suggests that BTV-17 has evolved by a combination of genetic drift and genomic reassortment. The data also indicate that the five US BTV serotypes are derived from two distinct gene pools. Evolution distances were used to estimate an evolution rate of 2.2 × 10−3 nucleotide substitution/site/year for BTV segment S1. This rate is similar to the genes of retroviruses and implies an absence of RNA polymerase proofreading activity for dsRNA viruses.
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Bluetongue virus evolution: sequence analyses of the genomic S1 segments and major core Protein VP7.
Virology, 1991Co-Authors: Timothy F. KowalikAbstract:The S1 segments, encoding the group-specific antigen, VP7, from the five United States prototype BTV serotypes were cloned as full-length entities. The nucleotide and deduced amino acid sequences of segment S1 of BTV-2 were determined and compared with BTV-10, -11, -13, and -17, completing the sequencing of this cognate gene segment from all five US BTV serotypes. Each segment is 1156 by long and contains an open reading frame encoding the 349-amino acid VP7 Protein. Most (>94%) of the amino acids of VP7 among the serotypes are conserved, including the location (position 255) of a single lysine residue. Secondary structure analyses of VP7 predict a putative eight-stranded β-barrel between amino acid positions 150 and 250, a structure similar to that observed in ssRNA viruses. The S1 genes are flanked by conserved 5' and 3' noncoding regions. Stem-loop structures are predicted at the 3' end of each gene (nucleotide positions 1058–1097). The S1 segments of BTV-2, -10, -11, and -17 have >93% of the nucleotides conserved, while
Allan R. Gould - One of the best experts on this subject based on the ideXlab platform.
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Expression of the major inner capsid Protein of the epizootic haemorrhagic disease virus in baculovirus and potential diagnostic use
Virus research, 1996Co-Authors: Hadya S. Nagesha, Allan R. Gould, John R. White, Ross A. Lunt, Christine J. DuchAbstract:The RNA 7 encoding the major capsid Protein (VP7) of epizootic haemorrhagic disease virus (EHDV), Australian serotype 2 (strain CS439), was cloned and the complete nucleotide sequence was determined. The coding region contained 1047 nucleotides (nt) capable of encoding a predicted 349 amino acid (aa) polypeptide with a calculated molecular size of 38.087 kDa. When the VP7 gene was expressed in bacterial or yeast expression systems, the expression product showed weak or no reactivity with polyclonal antibodies to EHDV. Therefore, the expression of the VP7 gene in baculovirus was pursued. The expressed EHDV VP7 was similar in antigenicity to that of the native virus as revealed by its reactivity in ELISA with monoclonal antibody (MAb) specific to EHDV. Preliminary ELISA results indicated that the recombinant Protein binds to EHDV antibodies in serum and that these antibodies block the binding of EHDV-specific MAb. The availability of a reliable EHDV recombinant VP7 could enhance our existing assay for detection of EHDV-specific antibodies.
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Major core Protein VP7 of Australian bluetongue virus serotype 15: sequence and antigenicity divergence from other BTV serotypes
Journal of General Virology, 1994Co-Authors: Lin-fa Wang, Jacki A. Kattenbelt, Allan R. Gould, L.i. Pritchard, Gary Crameri, Bryan T. EatonAbstract:Full-length cDNA of the RNA genome segment coding for the major core Protein VP7 of Australian bluetongue virus serotype 15 (BTV-15) has been isolated by reverse transcription-PCR cloning. Comparative analysis indicated that the BTV-15 VP7 sequence had diverged significantly from that of other members of the BTV serogroup. At the amino acid level, BTV-15 VP7 exhibited sequence identities of 80 to 84% with VP7 molecules of other serotypes, significantly lower than the sequence identities of between 93 and 100% observed among other serotypes characterized to date. This was consistent with previous observations that there were significant immunological differences between BTV-15 and other BTV serotypes and that monoclonal antibodies raised against BTV-1 VP7 failed to react with BTV-15 VP7. Recombinant BTV-15 VP7 Protein produced from Escherichia coli was largely insoluble, but maintained its immunogenicity. Polyclonal mouse sera raised against the recombinant VP7 Protein reacted strongly with VP7 of BTV-15, but weakly with that of BTV-1.
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High level expression of the major core Protein VP7 and the non-structural Protein NS3 of bluetongue virus in yeast: use of expressed VP7 as a diagnostic, group-reactive antigen in a blocking ELISA
Virus research, 1991Co-Authors: John C. Martyn, Allan R. Gould, Bryan T. EatonAbstract:The major core Protein VP7 and a non-structural Protein NS3 of bluetongue virus serotype 1 have been synthesized from recombinant plasmids using both an in vitro transcription/translation system and a yeast expression system. Bluetongue virus genes were transcribed under the control of the bacteriophage SP6 promoter and the regulatable yeast metallothionein promoter. An indirect ELISA showed that expression of NS3 in yeast was inducible with 1 mM CuSO4 and VP7 synthesis was constitutive but could be further induced. The preferred procedure for antigen extraction from yeast was sonication for VP7 and SDS/NaOH treatment for NS3. Yeast-expressed VP7 antigen and a monoclonal antibody were used in a blocking ELISA to distinguish sera raised against bluetongue virus serotypes from those generated to viruses of the epizootic haemorrhagic disease serogroup.
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A bluetongue serogroup-reactive epitope in the amino terminal half of the major core Protein VP7 is accessible on the surface of bluetongue virus particles.
Virology, 1991Co-Authors: Bryan T. Eaton, Allan R. Gould, John C. Martyn, Alex D. Hyatt, Barbara E.h. Coupar, John R. WhiteAbstract:Abstract Immunoelectron microscopy has been used to confirm that the core Protein VP7 is accessible on the surface of bluetongue virus (BTV) particles. Monospecific antibodies generated to vaccinia virus-expressed VP7 and an anti-VP7 monoclonal antibody (MAb 20E9) bound to native virus particles and were localized by Protein A-gold. In contrast, MAb 20E9 labeled directly with gold failed to gain access and bind, suggesting that VP7 is neither adventitiously adsorbed to the virion surface nor exposed in a manner such as protrusion through the outer capsid. Thus the surface layer of BTV may be considered as a net which only partially obscures the underlying core particle. Sequencing of VP7 revealed it to be an extremely hydrophobic Protein, 350 amino acids in length with cysteine residues at positions 15, 65, and 154. Examination of VP7 in the cytosol of cells infected with either BTV or a vaccinia virus recombinant expressing VP7 indicated that the Protein may exist as an oligomer, whose constituent monomers are not linked by intermolecular disulfide bonds. The cysteine residues in sodium dodecyl sulfate (SDS)-denatured, dithiothreitol (DTT)-treated VP7 were labeled with the fluorescent iodoacetamide AEDANS and the Protein was cleaved by V8 protease. The size of the labeled peptides and knowledge of the location of potential V8 cleavage sites suggested that the enzyme cleaved VP7 at three locations (glutamic acid residues at positions 61, 104 (or 108), and 132 (or 134 or 135). Analysis of the fluorescent peptides generated by V8 protease cleavage of VP7 labeled with AEDANS in the absence of DTT (i.e., with any putative intramolecular disulfide bonds intact) suggested that the cysteine at position 154 was the only one accessible to AEDANS. The cysteines at positions 15 and 65 may therefore be linked via a disulfide bond. Denaturation of VP7 with SIDS did not eliminate the capacity of the Protein to bind MAb 20E9. However, the sensitivity of the epitope to reduction and acetylation and its resistance to either of these processes alone suggest that it may be located near a disulfide bond linking cysteines at positions 15 and 65. Confirmation that the epitope lay in the amino-terminal half of the VP7 came from immunoelectron microscopy experiments in which thin sections of bacteria expressing the complete VP7 and the amino-terminal half were probed with MAb 20E9 and Protein A-gold.
Harry B. Greenberg - One of the best experts on this subject based on the ideXlab platform.
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Cross-Linking of Rotavirus Outer Capsid Protein VP7 by Antibodies or Disulfides Inhibits Viral Entry
Journal of virology, 2011Co-Authors: Scott T. Aoki, Philip R Dormitzer, Shane D. Trask, Harry B. Greenberg, Barbara S Coulson, Stephen C HarrisonAbstract:Antibodies that neutralize rotavirus infection target outer coat Proteins VP4 and VP7 and inhibit viral entry. The structure of a VP7-Fab complex (S. T. Aoki, et al., Science 324:1444-1447, 2009) led us to reclassify epitopes into two binding regions at inter- and intrasubunit boundaries of the calcium-dependent trimer. It further led us to show that antibodies binding at the intersubunit boundary inhibit uncoating of the virion outer layer. We have now tested representative antibodies for each of the defined structural epitope regions and find that antibodies recognizing epitopes in either binding region neutralize by cross-linking VP7 trimers. Antibodies that bind at the intersubunit junction neutralize as monovalent Fabs, while those that bind at the intrasubunit region require divalency. The VP7 structure has also allowed us to design a disulfide cross-linked VP7 mutant which recoats double-layered particles (DLPs) as efficiently as does wild-type VP7 but which yields particles defective in cell entry as determined both by lack of infectivity and by loss of α-sarcin toxicity in the presence of recoated particles. We conclude that dissociation of the VP7 trimer is an essential step in viral penetration into cells.
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Structure of rotavirus outer-layer Protein VP7 bound with a neutralizing Fab
Science, 2009Co-Authors: Scott T. Aoki, Shane D. Trask, E.c. Settembre, Harry B. Greenberg, Stephen C Harrison, Philip R DormitzerAbstract:Rotavirus outer-layer Protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca2+) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer Protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca2+ sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.
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Rotavirus assembly - interaction of surface Protein VP7 with middle layer Protein VP6.
Archives of virology, 2001Co-Authors: Joanna M. Gilbert, Ningguo Feng, John T. Patton, Harry B. GreenbergAbstract:The interaction between the rotavirus Proteins viral Protein 6 (VP6) and VP7 was examined in several exogenous Protein expression systems. These Proteins associated in the absence of other rotaviral Proteins as demonstrated by a coimmunoprecipitation assay. Deletion analysis of VP7 indicated that truncations of either the mature amino or carboxyl terminus disrupted the proper folding of the Protein and were not able to coimmunoprecipitate VP6. Truncation analysis of VP6 indicated that trimerization of VP6 was necessary, but not sufficient, for VP7 binding. MAb mapping and coimmunoprecipitation interference assays indicate that the VP6 amino acid residues between 271 and 342 are required for VP7 interaction. The interaction of VP6 and VP7 was also examined by the assembly of soluble VP7 onto baculovirus-expressed virus-like particles containing VP2 and VP6. Abrogation of this binding by preincubation of the particles with VP6 MAbs mapped to this same domain of VP6, validated our coimmunoprecipitation results. VP6 IgA MAbs that have been shown to be protective in vivo, but not a nonprotective IgA MAb, can interfere with VP7 binding to VP6. This suggests that these IgA MAbs may protect against rotavirus infection by blocking rotavirus assembly.
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Identification of mutations in the rotavirus Protein VP4 that alter sialic-acid-dependent infection.
Journal of General Virology, 1998Co-Authors: Juan E. Ludert, Y Hoshino, Ningguo Feng, Bruce B. Mason, Juana Angel, Baozhang Tang, Erick M. Mackow, Franco Maria Ruggeri, Harry B. GreenbergAbstract:To explore further the role of VP4 as the rotavirus cell attachment Protein, VP7 monoreassortants derived from the sialic-acid-dependent simian strain RRV and from the sialic-acid-independent human strains D, DS-1 and ST-3 were tested for susceptibility of infectivity of neuraminidase-treated MA-104 cells. Infectivity of RRV x D VP7 and RRV x ST-3 VP7 monoreassortants decreased when sialic acid was removed from the cell surface. However, of three separate RRV x DS-1 VP7 monoreassortants tested, only one was sialic-acid-dependent. Sequence analysis showed that both sialic-acid-independent strains contained a single amino acid change, Lys to Arg, at position 187. In addition, sialic-acid-independent infectivity was seen in one of 14 RRV VP4 neutralization escape mutants tested, and this strain was found to have a Gly to Glu change at amino acid position 150. These results indicate that positions 150 and 187 of VP4 play an important role in early rotavirus-cell interactions.
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Expression of the rotavirus SA11 Protein VP7 in the simple eukaryote Dictyostelium discoideum.
Journal of virology, 1995Co-Authors: Kerry R. Emslie, Philip R Dormitzer, Harry B. Greenberg, J M Miller, M B Slade, K L WilliamsAbstract:The outer capsid Protein of rotavirus, VP7, is a major neutralization antigen and is considered a necessary component of any subunit vaccine developed against rotavirus infection. For this reason, significant effort has been directed towards producing recombinant VP7 that maintains the antigenic characteristics of the native molecule. Using a relatively new expression system, the simple eukaryote Dictyostelium discoideum, we have cloned the portion of simian rotavirus SA11 genome segment 9, encoding the mature VP7 Protein, downstream of a native D. discoideum secretion signal sequence in a high-copy-number extrachromosomal vector. The majority of the recombinant VP7 expressed by transformants was intracellular and was detected by Western immunoblot following gel electrophoresis as two or three bands with an apparent molecular mass of 35.5 to 37.5 kDa. A small amount of VP7 having an apparent molecular mass of 37.5 kDa was secreted. Both the intracellular VP7 and the secreted VP7 were N glycosylated and sensitive to endoglycosidase H digestion. Under nonreducing electrophoresis conditions, over half the intracellular VP7 migrated as a monomer while the remainder migrated with an apparent molecular mass approximately twice that of the monomeric form. In an enzyme-linked immunosorbent assay, intracellular VP7 reacted with both nonneutralizing and neutralizing antibodies. The monoclonal antibody recognition pattern paralleled that found with VP7 expressed in either vaccinia virus or herpes simplex virus type 1 and confirms that D. discoideum-expressed VP7 is able to form the major neutralization domains present on viral VP7. Because D. discoideum cells are easy and cheap to grow, this expression system provides a valuable alternative for the large-scale production of recombinant VP7 Protein.
