The Experts below are selected from a list of 54 Experts worldwide ranked by ideXlab platform
Peter J. Walker - One of the best experts on this subject based on the ideXlab platform.
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ICTV Virus Taxonomy Profile: Roniviridae.
The Journal of general virology, 2020Co-Authors: Peter J. Walker, Jeff A. Cowley, X Dong, J Huang, N Moody, John ZiebuhrAbstract:The family Roniviridae includes the genus Okavirus for three species of viruses with enveloped, rod-shaped virions. The monopartite, positive-sense ssRNA genome (26-27 kb) contains five canonical long open reading frames (ORFs). ORF1a encodes polyprotein pp1a containing proteinase domains. ORF1b is expressed as a large polyprotein pp1ab by ribosomal frameshifting from ORF1a and encodes replication enzymes. ORF2 encodes the nucleoprotein. ORF3 encodes two envelope glycoproteins. ORFX encodes a putative double membrane-spanning protein. Roniviruses infect shrimp but only yellow head virus is highly pathogenic. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Roniviridae, which is available at ictv.global/report/roniviridae.
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RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
2016Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. WalkerAbstract:Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species Okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His6-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (2) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met11 to Arg60. Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M11PVRRPLPPQPPRNARLI29) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domai
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Molecular Biology and Pathogenesis of Roniviruses
Nidoviruses, 2014Co-Authors: Jeff A. Cowley, Peter J. WalkerAbstract:Currently all available information on the Roniviridae has come from studies of yellow head virus (YHV), gill-associated virus (GAV), and genotypic variants of these viruses detected in disparate populations of the black tiger shrimp species, Penaeus monodon. These viruses are very closely related and are currently classified as the type species GAV of the genus Okavirus, the only recognized genus in the family. This chapter covers what is known of the biology of Okaviruses as well as molecular characteristics that place these viruses within the order Nidovirales and distinguish them from the coronaviruses, toroviruses, and arteriviruses. It also covers virus-host interactions, virion morphology, assembly, and structure, genome organization, mechanism of subgenomic RNA (sgRNA) synthesis structural proteins, and nidovirus evolutionary considerations learned from Okaviruses.
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Nidoviruses - Molecular biology and pathogenesis of roniviruses.
Nidoviruses, 2014Co-Authors: Jeff A. Cowley, Peter J. WalkerAbstract:Currently all available information on the Roniviridae has come from studies of yellow head virus (YHV), gill-associated virus (GAV), and genotypic variants of these viruses detected in disparate populations of the black tiger shrimp species, Penaeus monodon. These viruses are very closely related and are currently classified as the type species GAV of the genus Okavirus, the only recognized genus in the family. This chapter covers what is known of the biology of Okaviruses as well as molecular characteristics that place these viruses within the order Nidovirales and distinguish them from the coronaviruses, toroviruses, and arteriviruses. It also covers virus-host interactions, virion morphology, assembly, and structure, genome organization, mechanism of subgenomic RNA (sgRNA) synthesis structural proteins, and nidovirus evolutionary considerations learned from Okaviruses.
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RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
PloS one, 2011Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. WalkerAbstract:Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species Okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His(6)-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (-) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met(11) to Arg(60). Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M(11)PVRRPLPPQPPRNARLI(29)) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.
John F Atkins - One of the best experts on this subject based on the ideXlab platform.
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Evidence for a novel coding sequence overlapping the 5'-terminal ~90 codons of the Gill-associated and Yellow head Okavirus envelope glycoprotein gene
Virology Journal, 2009Co-Authors: Andrew E Firth, John F AtkinsAbstract:The genus Okavirus (order Nidovirales ) includes a number of viruses that infect crustaceans, causing major losses in the shrimp industry. These viruses have a linear positive-sense ssRNA genome of ~26-27 kb, encoding a large replicase polyprotein that is expressed from the genomic RNA, and several additional proteins that are expressed from a nested set of 3'-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5' end of the envelope glycoprotein encoding sequence, ORF3, in the +2 reading frame. The new ORF has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of ORF3. We propose that translation of the new ORF initiates at a conserved AUG codon separated by just 2 nt from the ORF3 AUG initiation codon, resulting in a novel 86 amino acid protein.
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evidence for a novel coding sequence overlapping the 5 terminal 90 codons of the gill associated and yellow head Okavirus envelope glycoprotein gene
Virology Journal, 2009Co-Authors: Andrew E Firth, John F AtkinsAbstract:The genus Okavirus (order Nidovirales) includes a number of viruses that infect crustaceans, causing major losses in the shrimp industry. These viruses have a linear positive-sense ssRNA genome of ~26-27 kb, encoding a large replicase polyprotein that is expressed from the genomic RNA, and several additional proteins that are expressed from a nested set of 3'-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5' end of the envelope glycoprotein encoding sequence, ORF3, in the +2 reading frame. The new ORF has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of ORF3. We propose that translation of the new ORF initiates at a conserved AUG codon separated by just 2 nt from the ORF3 AUG initiation codon, resulting in a novel 86 amino acid protein.
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Evidence for a novel coding sequence overlapping the 5'-terminal ~90 codons of the Gill-associated and Yellow head Okavirus envelope glycoprotein gene
Virology journal, 2009Co-Authors: Andrew E Firth, John F AtkinsAbstract:The genus Okavirus (order Nidovirales) includes a number of viruses that infect crustaceans, causing major losses in the shrimp industry. These viruses have a linear positive-sense ssRNA genome of approximately 26-27 kb, encoding a large replicase polyprotein that is expressed from the genomic RNA, and several additional proteins that are expressed from a nested set of 3'-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5' end of the envelope glycoprotein encoding sequence, ORF3, in the +2 reading frame. The new ORF has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of ORF3. We propose that translation of the new ORF initiates at a conserved AUG codon separated by just 2 nt from the ORF3 AUG initiation codon, resulting in a novel 86 amino acid protein.
Jeff A. Cowley - One of the best experts on this subject based on the ideXlab platform.
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ICTV Virus Taxonomy Profile: Roniviridae.
The Journal of general virology, 2020Co-Authors: Peter J. Walker, Jeff A. Cowley, X Dong, J Huang, N Moody, John ZiebuhrAbstract:The family Roniviridae includes the genus Okavirus for three species of viruses with enveloped, rod-shaped virions. The monopartite, positive-sense ssRNA genome (26-27 kb) contains five canonical long open reading frames (ORFs). ORF1a encodes polyprotein pp1a containing proteinase domains. ORF1b is expressed as a large polyprotein pp1ab by ribosomal frameshifting from ORF1a and encodes replication enzymes. ORF2 encodes the nucleoprotein. ORF3 encodes two envelope glycoproteins. ORFX encodes a putative double membrane-spanning protein. Roniviruses infect shrimp but only yellow head virus is highly pathogenic. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Roniviridae, which is available at ictv.global/report/roniviridae.
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RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
2016Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. WalkerAbstract:Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species Okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His6-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (2) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met11 to Arg60. Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M11PVRRPLPPQPPRNARLI29) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domai
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Molecular Biology and Pathogenesis of Roniviruses
Nidoviruses, 2014Co-Authors: Jeff A. Cowley, Peter J. WalkerAbstract:Currently all available information on the Roniviridae has come from studies of yellow head virus (YHV), gill-associated virus (GAV), and genotypic variants of these viruses detected in disparate populations of the black tiger shrimp species, Penaeus monodon. These viruses are very closely related and are currently classified as the type species GAV of the genus Okavirus, the only recognized genus in the family. This chapter covers what is known of the biology of Okaviruses as well as molecular characteristics that place these viruses within the order Nidovirales and distinguish them from the coronaviruses, toroviruses, and arteriviruses. It also covers virus-host interactions, virion morphology, assembly, and structure, genome organization, mechanism of subgenomic RNA (sgRNA) synthesis structural proteins, and nidovirus evolutionary considerations learned from Okaviruses.
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Nidoviruses - Molecular biology and pathogenesis of roniviruses.
Nidoviruses, 2014Co-Authors: Jeff A. Cowley, Peter J. WalkerAbstract:Currently all available information on the Roniviridae has come from studies of yellow head virus (YHV), gill-associated virus (GAV), and genotypic variants of these viruses detected in disparate populations of the black tiger shrimp species, Penaeus monodon. These viruses are very closely related and are currently classified as the type species GAV of the genus Okavirus, the only recognized genus in the family. This chapter covers what is known of the biology of Okaviruses as well as molecular characteristics that place these viruses within the order Nidovirales and distinguish them from the coronaviruses, toroviruses, and arteriviruses. It also covers virus-host interactions, virion morphology, assembly, and structure, genome organization, mechanism of subgenomic RNA (sgRNA) synthesis structural proteins, and nidovirus evolutionary considerations learned from Okaviruses.
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RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
PloS one, 2011Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. WalkerAbstract:Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species Okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His(6)-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (-) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met(11) to Arg(60). Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M(11)PVRRPLPPQPPRNARLI(29)) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.
Andrew E Firth - One of the best experts on this subject based on the ideXlab platform.
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Evidence for a novel coding sequence overlapping the 5'-terminal ~90 codons of the Gill-associated and Yellow head Okavirus envelope glycoprotein gene
Virology Journal, 2009Co-Authors: Andrew E Firth, John F AtkinsAbstract:The genus Okavirus (order Nidovirales ) includes a number of viruses that infect crustaceans, causing major losses in the shrimp industry. These viruses have a linear positive-sense ssRNA genome of ~26-27 kb, encoding a large replicase polyprotein that is expressed from the genomic RNA, and several additional proteins that are expressed from a nested set of 3'-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5' end of the envelope glycoprotein encoding sequence, ORF3, in the +2 reading frame. The new ORF has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of ORF3. We propose that translation of the new ORF initiates at a conserved AUG codon separated by just 2 nt from the ORF3 AUG initiation codon, resulting in a novel 86 amino acid protein.
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evidence for a novel coding sequence overlapping the 5 terminal 90 codons of the gill associated and yellow head Okavirus envelope glycoprotein gene
Virology Journal, 2009Co-Authors: Andrew E Firth, John F AtkinsAbstract:The genus Okavirus (order Nidovirales) includes a number of viruses that infect crustaceans, causing major losses in the shrimp industry. These viruses have a linear positive-sense ssRNA genome of ~26-27 kb, encoding a large replicase polyprotein that is expressed from the genomic RNA, and several additional proteins that are expressed from a nested set of 3'-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5' end of the envelope glycoprotein encoding sequence, ORF3, in the +2 reading frame. The new ORF has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of ORF3. We propose that translation of the new ORF initiates at a conserved AUG codon separated by just 2 nt from the ORF3 AUG initiation codon, resulting in a novel 86 amino acid protein.
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Evidence for a novel coding sequence overlapping the 5'-terminal ~90 codons of the Gill-associated and Yellow head Okavirus envelope glycoprotein gene
Virology journal, 2009Co-Authors: Andrew E Firth, John F AtkinsAbstract:The genus Okavirus (order Nidovirales) includes a number of viruses that infect crustaceans, causing major losses in the shrimp industry. These viruses have a linear positive-sense ssRNA genome of approximately 26-27 kb, encoding a large replicase polyprotein that is expressed from the genomic RNA, and several additional proteins that are expressed from a nested set of 3'-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5' end of the envelope glycoprotein encoding sequence, ORF3, in the +2 reading frame. The new ORF has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of ORF3. We propose that translation of the new ORF initiates at a conserved AUG codon separated by just 2 nt from the ORF3 AUG initiation codon, resulting in a novel 86 amino acid protein.
Chumporn Soowannayan - One of the best experts on this subject based on the ideXlab platform.
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RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
2016Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. WalkerAbstract:Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species Okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His6-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (2) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met11 to Arg60. Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M11PVRRPLPPQPPRNARLI29) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domai
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RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
PloS one, 2011Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. WalkerAbstract:Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species Okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His(6)-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (-) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met(11) to Arg(60). Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M(11)PVRRPLPPQPPRNARLI(29)) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.
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Glycosylation of gp116 and gp64 envelope proteins of yellow head virus of Penaeus monodon shrimp
Journal of General Virology, 2010Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Roger D. Pearson, Tristan P. Wallis, Jeffrey J. Gorman, Wojtek P. Michalski, Peter J. WalkerAbstract:Yellow head virus (YHV) is a highly virulent pathogen of Penaeus monodon shrimp that is classified in the genus Okavirus, family Roniviridae, in the order Nidovirales. Separation of virion proteins treated with peptide-N-glycosidase-F (PNGase-F) in SDS-polyacrylamide gels and the use of glycoprotein-specific staining methods indicated that the gp116 and gp64 envelope glycoproteins possess N-linked rather than O-linked glycans. Competitive binding inhibition of lectins with various oligosaccharide specificities indicated that glycans linked to gp64 are mannose-rich, whilst glycans linked to gp116 possess terminal N-acetylgalactosamine and N-acetylglucosamine in addition to terminal mannose-type sugars. Mass spectrometry analyses of peptides generated from YHV proteins before and after deglycosylation with PNGase-F, using combinations of the endoproteinases trypsin, Asp-N and Lys-C, confirmed occupancy of six of the seven potential N-linked glycosylation sites in gp116 and three of the four potential sites in gp64.
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Structural and functional characterization of yellow head virus proteins
2009Co-Authors: Chumporn SoowannayanAbstract:Abstract Yellow head virus (YHV) has caused mass mortalities in Penaeus monodon shrimp farmed throughout Southeast Asia since it was first discovered in the early 1990’s. YHV possesses a positive-sense, single-stranded RNA genome and a rod-shaped enveloped virion. Together with the closely related gill-associated virus (GAV) identified in P. monodon shrimp in Australia, it is classified in the genus Okavirus, family Roniviridae within the order Nidovirales. YHV particles contain only three structural proteins, a nucleocapsid (N) protein (p20) protein and two envelope glycoproteins gp116 and gp64. In this study, the glycosylation status of gp116 and gp64 extracted from YHV virions was characterized in detail, including the identification of active N-linked glycosylation sites and the nature of the attached carbohydrates. This was achieved by optimizing and applying a combination of methods that included SDS-PAGE followed by carbohydrate-specific staining of gels or probing of membrane-bound proteins using lectins with different carbohydrate specificities, enzymatic removal of N-linked carbohydrates and a variety of mass spectrometry techniques. In these analyses, it was found that N-linked glycans are the major contributor to the higher estimated mass of gp116 and gp64 by SDS-PAGE compared to those estimated from their deduced amino acid sequences. Neither gp116 nor gp64 were found to posses O-linked glycans. Mannose residues were identified to be the major glycan component of carbohydrates linked to gp116 and gp64 and are possibly the sole component of carbohydrate linked to gp64. Unlike gp64, other glycans such as terminal N-acetyl--D-galactosamine and N-acetyl--D-glucosamine were identified to be attached to gp116. Assuming that glycosylation processes in shrimp mimic those of vertebrates that are known in more detail, the nature of the glycans attached to gp116 suggests that they might be added and modified during the transportation of the protein from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN). Mass spectrometry analyses of tryptic peptides derived from the native glycoproteins and following their enzymatic deglycosylation, generated approximately 81% (gp116) and 66% (gp64) coverage of their predicted amino acid sequences. Detailed mass spectrometry analyses of peptides derived from the deglycosylated proteins identified that most of the potential N-linked glycosylated site in the virion envelope glycoproteins, 6 of 7 present in gp116 and 3 of 4 present in gp64 were identified to be modified by glycans. In gp116, one site was not identified and in gp64 one site was not utilized. As phosphorylation has been shown to affect nucleocapsid protein (N) functioning in vertebrate nidoviruses, SDS-PAGE using two phosphoprotein-specific staining methods, as well as mass spectrometry methods, were employed to examine whether the YHV N protein present in virions is phosphorylated. The protein staining methods provided contradicting results and no phosphate-containing peptides were identified by mass spectrometry. The apparent absence of phosphate in the N protein was also supported by its isoelectric point (pI ~10) determined by isoelectric focusing and two-dimensional electrophoresis (2-DE) analysis, which was very similar to that predicted (pI = 9.98) from its deduced amino acid sequence. Taken together, the data suggest that the YHV N protein encapsulated within virions is not phosphorylated. The RNA-binding capability of the GAV N protein was assessed using an electrophoretic mobility shift assay (EMSA) technique. Full-length and variously truncated forms of the GAV N protein expressed in bacteria were assessed in the assays. It was found that the full-length recombinant N protein bound to RNA in a sequence non-specific manner. Analysis of the five truncated N protein constructs localized the RNA-binding domain to a 50 amino acid sequence in the N-terminal region residing between Met11 and Arg60. A motif rich in proline and arginine residues, which are commonly found in other RNA-binding proteins, occurred in first 18 amino acids of this region. Although RNA-binding was not sequence-specific, the data suggest that this region of the GAV N protein is the most likely site at which it interacts with and nucleates viral genomic RNA during nucleocapsid formation. A synthetic peptide spanning the 18 amino acid of the putative RNA-binding domain was shown to possess RNA-binding properties similar to the recombinant protein fragment. These results indicated that the 18 amino acid, proline and arginine rich motif (MPVRRPLPPQPPRNARLI) in the N-terminal region of the GAV N protein confers its RNA-binding function. Using an immuno-co-precipitation assay, a host protein was found to interact abundantly with the GAV N protein in infected lymphoid organ cells. Mass spectrometry analysis identified the protein as -actin. Immuno-histochemistical double-labeling methods in conjunction with observations made using confocal and electron microscopy revealed that actin and the N protein were co-located in cytoplasm of infected cells. Electron microscopy suggested that interaction of the two proteins occurs before nucleocapsid envelopment within virions, suggesting that -actin might be involved in transporting the N protein or the nucleocapsid from their sites of synthesis to the rough endoplasmic reticulum where the virion acquires its envelopes. In summary, the research described in this thesis has advanced understanding of the YHV/GAV proteome through the identification of the glycosylation sites in the envelope glycoproteins gp116 and gp64, and demonstrating that nucleocapsid protein encapsulated within virion is unlikely to be phosphorylated. Functional studies have also shown that the nucleocapsid protein binds RNA non-specifically through an 18 amino acid domain near its N-terminus and that it binds and co-localizes with -actin in infected cells, suggesting that -actin may play role in trafficking N protein in infected cells.
