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Polly Roy - One of the best experts on this subject based on the ideXlab platform.
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structure based modification of bluetongue virus helicase Protein VP6 to produce a viable VP6 truncated btv
Biochemical and Biophysical Research Communications, 2014Co-Authors: Eiko Matsuo, Esther Leon, Steve Matthews, Polly RoyAbstract:Bluetongue virus core Protein VP6 is an ATP hydrolysis dependent RNA helicase. However, despite much study, the precise role of VP6 within the viral capsid and its structure remain unclear. To investigate the requirement of VP6 in BTV replication, we initiated a structural and biological study. Multinuclear nuclear magnetic resonance spectra were assigned on his-tagged full-length VP6 (329 amino acid residues) as well as several truncated VP6 variants. The analysis revealed a large structured domain with two large loop regions that exhibit significant conformational exchange. One of the loops (amino acid position 34–130) could be removed without affecting the overall fold of the Protein. Moreover, using a BTV reverse genetics system, it was possible to demonstrate that the VP6-truncated BTV was viable in BHK cells in the absence of any helper VP6 Protein, suggesting that a large portion of this loop region is not absolutely required for BTV replication.
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defining the structure function relationships of bluetongue virus helicase Protein VP6
Journal of Virology, 2003Co-Authors: Alak Kanti Kar, Polly RoyAbstract:The VP6 Protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the Protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the Protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant Proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK(110)V is directly involved in ATP binding, whereas mutant R(205)Q in the arginine-rich motif ER(205)XGRXXR bound ATP at a level comparable to that of the wild-type Protein. The RNA binding activity was drastically altered in the R(205)Q mutant and was also affected in the K(110)N mutant. Helicase activity was altered in both mutants. The mutation E(157)N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified Protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6.
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sequences within the VP6 molecule of bluetongue virus that determine cytoplasmic and nuclear targeting of the Protein
Journal of Virology, 1996Co-Authors: O B Bansal, M L Hong, S Chatterjee, Polly RoyAbstract:Genome segment 9 of bluetongue virus serotype 10 encodes the minor Protein VP6. The Protein is abundant with basic residues particularly in two regions of the carboxy half of the molecule. A series of amino- and carboxy-terminal deletion mutants was expressed in mammalian cells by using a vaccinia virus T7 polymerase-driven transient expression system, and the intracellular fate of the products was monitored by both immunofluorescence staining and cell fractionation techniques. Data obtained indicated clearly that VP6 has nuclear transportation signals which may be correlated with positively charged domains of the molecule. In the intact molecule, though, these signals are masked and the Protein is retained in the cytoplasm. The biochemical and immunofluorescence data obtained indicate that sequences in the region of residues 33 to 80 of the 328-amino acid Protein are required for the retention of VP6 within the cell cytoplasm while amino acids 303 to 308 in the carboxy-terminal half of the molecule appear to possess nuclear localization capabilities.
H Huismans - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the nucleic acid binding activity of inner core Protein VP6 of African horse sickness virus
Archives of Virology, 2005Co-Authors: P. J. De Waal, H HuismansAbstract:Minor structural Protein VP6 is the putative helicase of African horse sickness virus (AHSV), of the genus Orbivirus in the Reoviridae family. We investigated how the Protein interacts with double-stranded (ds) RNA and other nucleic acids. Binding was assayed using an electrophoretic migration retardation assay and a nucleic acid overlay Protein blot assay. VP6 bound double and single stranded RNA and DNA in a NaCl concentration sensitive reaction. Of six truncated VP6 peptides investigated, two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. The distinction between the peptides appeared to be the pI which ranged from more than 8.0 to just above 6.0. Changing the pH of the binding buffer modified the binding activity. Regardless of assay conditions, only peptides with a specific region of amino acids in common, showed evidence of binding activity. No sequence homology was identified with other binding domains, however, the presence of charged amino acids are assumed to be important for binding activity. The results suggested dsRNA binding in the blot assay was strongly affected by the net charge on the peptide.
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characterization of the gene encoding core Protein VP6 of two african horsesickness virus serotypes
Journal of General Virology, 1996Co-Authors: Pamela J Turnbull, Sharon B Cormack, H HuismansAbstract:The genes encoding the inner core Protein VP6 of African horsesickness virus (AHSV) serotypes 3 and 6 have been cloned and sequenced. The genes are 1169 nucleotides in length and both encode a largely hydrophilic Protein of 369 amino acids. The VP6 amino acid sequence is highly conserved between the two serotypes with an overall similarity of 95%. Comparison of the AHSV VP6 amino acid sequences with those of bluetongue virus serotype 10 VP6 revealed that it is 41 amino acids longer with an overall amino acid identity of 29%. The similarity is mainly confined to a short but highly conserved 13 amino acid region at the N terminus, a short seven amino acid region at the C terminus and a 22 amino acid region close to the C terminus. Within this last region is a smaller 11 amino acid region from 318 to 328 with a 91% similarity to the Rep helicase of Escherichia coli.
John T. Patton - One of the best experts on this subject based on the ideXlab platform.
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group a human rotavirus genomics evidence that gene constellations are influenced by viral Protein interactions
Journal of Virology, 2008Co-Authors: Erica Heiman, Mario Barro, Zenobia F Taraporewala, Tamara Barmagen, Sarah M Mcdonald, John T. PattonAbstract:Group A human rotaviruses (HRVs) are the major cause of severe viral gastroenteritis in infants and young children. To gain insight into the level of genetic variation among HRVs, we determined the genome sequences for 10 strains belonging to different VP7 serotypes (G types). The HRVs chosen for this study, D, DS-1, P, ST3, IAL28, Se584, 69M, WI61, A64, and L26, were isolated from infected persons and adapted to cell culture to use as serotype references. Our sequencing results revealed that most of the individual Proteins from each HRV belong to one of three genotypes (1, 2, or 3) based on their similarities to Proteins of genogroup strains (Wa, DS-1, or AU-1, respectively). Strains D, P, ST3, IAL28, and WI61 encode genotype 1 (Wa-like) Proteins, whereas strains DS-1 and 69M encode genotype 2 (DS-1-like) Proteins. Of the 10 HRVs sequenced, 3 of them (Se584, A64, and L26) encode Proteins belonging to more than one genotype, indicating that they are intergenogroup reassortants. We used amino acid sequence alignments to identify residues that distinguish Proteins belonging to HRV genotype 1, 2, or 3. These genotype-specific changes cluster in definitive regions within each viral Protein, many of which are sites of known Protein-Protein interactions. For the intermediate viral capsid Protein (VP6), the changes map onto the atomic structure at the VP2-VP6, VP4-VP6, and VP7-VP6 interfaces. The results of this study provide evidence that group A HRV gene constellations exist and may be influenced by interactions among viral Proteins during replication.
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full genome based classification of rotaviruses reveals a common origin between human wa like and porcine rotavirus strains and human ds 1 like and bovine rotavirus strains
Journal of Virology, 2008Co-Authors: Jelle Matthijnssens, Erica Heiman, Sarah M Mcdonald, Max Ciarlet, Ingrid Arijs, Thomas Delbeke, Enzo A Palombo, Miren Iturrizagomara, Piet Maes, John T. PattonAbstract:Group A rotavirus classification is currently based on the molecular properties of the two outer layer Proteins, VP7 and VP4, and the middle layer Protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.
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translation enhancer in the 3 untranslated region of rotavirus gene 6 mrna promotes expression of the major capsid Protein VP6
Archives of Virology, 2004Co-Authors: A D Yang, M Barro, Mario Gorziglia, John T. PattonAbstract:The eleven rotavirus mRNAs contain 5'-cap structures and most end with the 3'-consensus sequence 5'-UGACC-3'. The UGACC functions as a common translation enhancer (3'-TE-con) that upregulates viral Protein expression through a process mediated by the nonstructural Protein NSP3. To address the possibility that gene-specific enhancers are also contained in the untranslated regions (UTRs) of the rotavirus mRNAs, we used rabbit reticulocyte lysates to investigate the translation efficiencies of analog RNAs containing viral-specific 5'-and 3'-UTRs and the open reading frame for chloramphenicol acetyltransferase. These experiments combined with the analysis of full-length viral RNAs and RNAs containing 3'-truncations showed that a highly active enhancer was present near the 5'-end of the 139-nucleotide 3'-UTR of the gene 6 mRNA (3'-TEg6). The 3'-TEg6 represents a functionally independent enhancer, as no other portion of the gene 6 mRNA was required for its activity. The 3'-TEg6 differs significantly from the 3'-TE-con in that the gene 6-specific enhancer does not require viral Protein for activity and is formed by a sequence unique to only one of the eleven viral mRNAs. Together, our findings suggest that the 3'-UTR of the gene 6 mRNA contains two TEs, one is gene-specific (3'-TEg6) and the other is common to nearly all rotavirus genes (3'-TE-con). The activity of the 3'-TEg6 is likely important for directing the efficient translation of the gene 6 mRNA at levels sufficient to provide the 780 copies of VP6 necessary for the assembly of each progeny virion.
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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.
Eiko Matsuo - One of the best experts on this subject based on the ideXlab platform.
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structure based modification of bluetongue virus helicase Protein VP6 to produce a viable VP6 truncated btv
Biochemical and Biophysical Research Communications, 2014Co-Authors: Eiko Matsuo, Esther Leon, Steve Matthews, Polly RoyAbstract:Bluetongue virus core Protein VP6 is an ATP hydrolysis dependent RNA helicase. However, despite much study, the precise role of VP6 within the viral capsid and its structure remain unclear. To investigate the requirement of VP6 in BTV replication, we initiated a structural and biological study. Multinuclear nuclear magnetic resonance spectra were assigned on his-tagged full-length VP6 (329 amino acid residues) as well as several truncated VP6 variants. The analysis revealed a large structured domain with two large loop regions that exhibit significant conformational exchange. One of the loops (amino acid position 34–130) could be removed without affecting the overall fold of the Protein. Moreover, using a BTV reverse genetics system, it was possible to demonstrate that the VP6-truncated BTV was viable in BHK cells in the absence of any helper VP6 Protein, suggesting that a large portion of this loop region is not absolutely required for BTV replication.
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Bluetongue virus VP6 acts early in the replication cycle and can form the basis of chimeric virus formation
2009Co-Authors: Eiko Matsuo, P RoyAbstract:A minor core Protein, VP6, of bluetongue virus (BTV) possesses nucleoside triphosphatase, RNA binding, and helicase activities. Although the enzymatic functions of VP6 have been documented in vitro using purified Protein, its definitive role in BTV replication remains unclear. In this study, using a recently developed T7 transcript-based reverse genetics system for BTV, we examined the importance of VP6 in virus replication. We show that VP6 is active early in replication, consistent with a role as part of the transcriptase or packaging complex, and that its action can be provided in trans by a newly developed complementary cell line. Further-more, the genomic segment encoding VP6 was mutated to reveal the cis-acting sequences required for repli-cation or packaging, which subsequently enabled the construction of a chimeric BTV expressing enhanced green fluorescent Protein. These data confirm that one of the 10 genome segments of BTV can be replaced with a chimeric RNA containing the essential packaging and replication signals of BTV and the coding sequence of a foreign gene. Bluetongue virus (BTV), the etiological agent of bluetongue disease of livestock, is a member of the genus Orbivirus of the family Reoviridae. BTV particles have three consecutive layers of Proteins organized into two capsids, an outer capsid of tw
Ian N. Clarke - One of the best experts on this subject based on the ideXlab platform.
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enzyme linked immunosorbent assay based on recombinant human group c rotavirus inner capsid Protein VP6 to detect human group c rotaviruses in fecal samples
Journal of Clinical Microbiology, 1998Co-Authors: V. L. A. James, Paul R. Lambden, E.o. Caul, Ian N. ClarkeAbstract:A recent study showed that 43% of a population in the United Kingdom were seropositive for group C rotavirus. The higher than expected incidence may be due to limited diagnosis of acute human group C rotavirus infections because no routine test is available. Human group C rotavirus infections are routinely diagnosed by electron microscopy (EM) and a negative group A rotavirus enzyme-linked immunosorbent assay (ELISA) result. An antigen-detection ELISA was developed with hyperimmune antibodies raised to human group C rotavirus recombinant VP6 (Bristol strain) expressed in insect cells. The assay was used to screen fecal samples to determine the prevalence of group C rotavirus infection. Samples positive by ELISA were confirmed by EM, polyacrylamide gel electrophoresis of double-stranded RNA, or detection of the VP6 gene by reverse transcription-PCR. Retrospective analysis indicated a 1 to 2% detection rate of positivity among samples from patients with acute diarrhea.
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Seroepidemiology of human group C rotavirus in the UK.
Journal of medical virology, 1997Co-Authors: V. L. A. James, Paul R. Lambden, E.o. Caul, S.j. Cooke, Ian N. ClarkeAbstract:The gene coding for the major inner capsid Protein VP6 of human group C rotavirus was cloned into baculovirus using the pBlueBac2 vector and expressed in insect cells. When cultured in High Five cells, VP6 was expressed at a high level and exported to the cell culture medium. Purified VP6 was used to immunise rabbits. Hyperimmune rabbit serum, which reacted with native human group C rotavirus in infected cells, was used to develop and optimise an EIA for the detection of antibodies to group C rotavirus using the recombinant VP6 as a source of antigen. In a local epidemiological survey of 1000 sera grouped by age, an average of 43% of samples were found to have antibodies to human group C rotavirus with the highest proportion (66%) in the 71-75 year age group. In comparison, 97% of adults and 85% of children had antibodies to recombinant VP6 from the bovine RF strain of group A rotavirus. These results suggest that infection with human group C rotavirus is a common occurrence despite the apparent rarity of reports of human group C rotavirus in clinical samples from patients with gastroenteritis.
