Roniviridae

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Peter J. Walker - One of the best experts on this subject based on the ideXlab platform.

  • RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
    2016
    Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. Walker
    Abstract:

    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

  • RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
    PloS one, 2011
    Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. Walker
    Abstract:

    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.

  • Glycosylation of gp116 and gp64 envelope proteins of yellow head virus of Penaeus monodon shrimp
    Journal of General Virology, 2010
    Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Roger D. Pearson, Tristan P. Wallis, Jeffrey J. Gorman, Wojtek P. Michalski, Peter J. Walker
    Abstract:

    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.

Chumporn Soowannayan - One of the best experts on this subject based on the ideXlab platform.

  • RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
    2016
    Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. Walker
    Abstract:

    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

  • RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
    PloS one, 2011
    Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. Walker
    Abstract:

    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.

  • Glycosylation of gp116 and gp64 envelope proteins of yellow head virus of Penaeus monodon shrimp
    Journal of General Virology, 2010
    Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Roger D. Pearson, Tristan P. Wallis, Jeffrey J. Gorman, Wojtek P. Michalski, Peter J. Walker
    Abstract:

    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.

  • Structural and functional characterization of yellow head virus proteins
    2009
    Co-Authors: Chumporn Soowannayan
    Abstract:

    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.

John Ziebuhr - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of White Bream Virus Reveals a Novel Genetic Cluster of Nidoviruses
    Journal of virology, 2006
    Co-Authors: Heike Schütze, Sonja Bayer, Rachel Ulferts, Barbara Schelle, Harald Granzow, Bernd Hoffmann, Thomas C. Mettenleiter, John Ziebuhr
    Abstract:

    The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

  • major genetic marker of nidoviruses encodes a replicative endoribonuclease
    Proceedings of the National Academy of Sciences of the United States of America, 2004
    Co-Authors: Konstantin A Ivanov, Alexander E Gorbalenya, Tobias Hertzig, Mikhail Rozanov, Sonja Bayer, Volker Thiel, John Ziebuhr
    Abstract:

    Coronaviruses are important pathogens that cause acute respiratory diseases in humans. Replication of the ≈30-kb positive-strand RNA genome of coronaviruses and discontinuous synthesis of an extensive set of subgenome-length RNAs (transcription) are mediated by the replicase-transcriptase, a barely characterized protein complex that comprises several cellular proteins and up to 16 viral subunits. The coronavirus replicase-transcriptase was recently predicted to contain RNA-processing enzymes that are extremely rare or absent in other RNA viruses. Here, we established and characterized the activity of one of these enzymes, replicative nidoviral uridylate-specific endoribonuclease (NendoU). It is considered a major genetic marker that discriminates nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) from all other RNA virus families. Bacterially expressed forms of NendoU of severe acute respiratory syndrome coronavirus and human coronavirus 229E were revealed to cleave single-stranded and double-stranded RNA in a Mn2+-dependent manner. Single-stranded RNA was cleaved less specifically and effectively, suggesting that double-stranded RNA is the biologically relevant NendoU substrate. Double-stranded RNA substrates were cleaved upstream and downstream of uridylates at GUU or GU sequences to produce molecules with 2′-3′ cyclic phosphate ends. 2′-O-ribose-methylated RNA substrates proved to be resistant to cleavage by NendoU, indicating a functional link with the 2′-O-ribose methyltransferase located adjacent to NendoU in the coronavirus replicative polyprotein. A mutagenesis study verified potential active-site residues and allowed us to inactivate NendoU in the full-length human coronavirus 229E clone. Substitution of D6408 by Ala was shown to abolish viral RNA synthesis, demonstrating that NendoU has critical functions in viral replication and transcription.

  • The 3C-Like Proteinase of an Invertebrate Nidovirus Links Coronavirus and Potyvirus Homologs
    Journal of virology, 2003
    Co-Authors: John Ziebuhr, Sonja Bayer, Jeff A. Cowley, Alexander E Gorbalenya
    Abstract:

    Gill-associated virus (GAV), a positive-stranded RNA virus of prawns, is the prototype of newly recognized taxa (genus Okavirus, family Roniviridae) within the order Nidovirales. In this study, a putative GAV cysteine proteinase (3C-like proteinase [3CLpro]), which is predicted to be the key enzyme involved in processing of the GAV replicase polyprotein precursors, pp1a and pp1ab, was characterized. Comparative sequence analysis indicated that, like its coronavirus homologs, 3CLpro has a three-domain organization and is flanked by hydrophobic domains. The putative 3CLpro domain including flanking regions (pp1a residues 2793 to 3143) was fused to the Escherichia coli maltose-binding protein (MBP) and, when expressed in E. coli, was found to possess N-terminal autoprocessing activity that was not dependent on the presence of the 3CLpro C-terminal domain. N-terminal sequence analysis of the processed protein revealed that cleavage occurred at the location 2827LVTHE↓VRTGN2836. The trans-processing activity of the purified recombinant 3CLpro (pp1a residues 2832 to 3126) was used to identify another cleavage site, 6441KVNHE↓LYHVA6450, in the C-terminal pp1ab region. Taken together, the data tentatively identify VxHE↓(L,V) as the substrate consensus sequence for the GAV 3CLpro. The study revealed that the GAV and potyvirus 3CLpros possess similar substrate specificities which correlate with structural similarities in their respective substrate-binding sites, identified in sequence comparisons. Analysis of the proteolytic activities of MBP-3CLpro fusion proteins carrying replacements of putative active-site residues provided evidence that, in contrast to most other 3C/3CLpros but in common with coronavirus 3CLpros, the GAV 3CLpro employs a Cys2968-His2879 catalytic dyad. The properties of the GAV 3CLpro define a novel RNA virus proteinase variant that bridges the gap between the distantly related chymotrypsin-like cysteine proteinases of coronaviruses and potyviruses.

Benjamin W. Neuman - One of the best experts on this subject based on the ideXlab platform.

  • identification and characterization of a ribose 2 o methyltransferase encoded by the ronivirus branch of nidovirales
    Journal of Virology, 2016
    Co-Authors: Cong Zeng, Yi Wang, Yingke Tang, Xu Jin, Shilei Wang, Chengpeng Fan, Ying Sun, Lei Qin, Benjamin W. Neuman
    Abstract:

    UNLABELLED The order Nidovirales currently comprises four virus families: Arteriviridae, Coronaviridae (divided into the subfamilies Coronavirinae and Torovirinae), Roniviridae, and the recently recognized Mesoniviridae RNA cap formation and methylation have been best studied for coronaviruses, with emphasis on the identification and characterization of two virus-encoded methyltransferases (MTases) involved in RNA capping, a guanine-N7-MTase and a ribose-2'-O-MTase. Although bioinformatics analyses suggest that these MTases may also be encoded by other nidoviruses with large genomes, such as toroviruses and roniviruses, no experimental evidence has been reported thus far. In this study, we show that a ronivirus, gill-associated virus (GAV), encodes the 2'-O-MTase activity, although we could not detect 2'-O-MTase activity for the homologous protein of a torovirus, equine torovirus, which is more closely related to coronaviruses. Like the coronavirus 2'-O-MTase, the roniviral 2'-O-MTase harbors a catalytic K-D-K-E tetrad that is conserved among 2'-O-MTases and can target only the N7-methylated cap structure of adenylate-primed RNA substrates. However, in contrast with the coronavirus protein, roniviral 2'-O-MTase does not require a protein cofactor for stimulation of its activity and differs in its preference for several biochemical parameters, such as reaction temperature and pH. Furthermore, the ronivirus 2'-O-MTase can be targeted by MTase inhibitors. These results extend our current understanding of nidovirus RNA cap formation and methylation beyond the coronavirus family. IMPORTANCE Methylation of the 5'-cap structure of viral RNAs plays important roles in genome replication and evasion of innate recognition of viral RNAs by cellular sensors. It is known that coronavirus nsp14 acts as an N7-(guanine)-methyltransferase (MTase) and nsp16 as a 2'-O-MTase, which are involved in the modification of RNA cap structure. However, these enzymatic activities have not been shown for any other nidoviruses beyond coronaviruses in the order Nidovirales In this study, we identified a 2'-O-methyltransferase encoded by ronivirus that shows common and unique features in comparison with that of coronaviruses. Ronivirus 2'-O-MTase does not need a protein cofactor for MTase activity, whereas coronavirus nsp16 needs the stimulating factor nsp10 for its full activity. The conserved K-D-K-E catalytic tetrad is identified in ronivirus 2'-O-MTase. These results extend our understanding of nidovirus RNA capping and methylation beyond coronaviruses and also strengthen the evolutionary and functional links between roniviruses and coronaviruses.

Jeff A. Cowley - One of the best experts on this subject based on the ideXlab platform.

  • RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
    2016
    Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. Walker
    Abstract:

    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

  • RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
    PloS one, 2011
    Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Wojtek P. Michalski, Peter J. Walker
    Abstract:

    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.

  • Glycosylation of gp116 and gp64 envelope proteins of yellow head virus of Penaeus monodon shrimp
    Journal of General Virology, 2010
    Co-Authors: Chumporn Soowannayan, Jeff A. Cowley, Roger D. Pearson, Tristan P. Wallis, Jeffrey J. Gorman, Wojtek P. Michalski, Peter J. Walker
    Abstract:

    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.

  • The 3C-Like Proteinase of an Invertebrate Nidovirus Links Coronavirus and Potyvirus Homologs
    Journal of virology, 2003
    Co-Authors: John Ziebuhr, Sonja Bayer, Jeff A. Cowley, Alexander E Gorbalenya
    Abstract:

    Gill-associated virus (GAV), a positive-stranded RNA virus of prawns, is the prototype of newly recognized taxa (genus Okavirus, family Roniviridae) within the order Nidovirales. In this study, a putative GAV cysteine proteinase (3C-like proteinase [3CLpro]), which is predicted to be the key enzyme involved in processing of the GAV replicase polyprotein precursors, pp1a and pp1ab, was characterized. Comparative sequence analysis indicated that, like its coronavirus homologs, 3CLpro has a three-domain organization and is flanked by hydrophobic domains. The putative 3CLpro domain including flanking regions (pp1a residues 2793 to 3143) was fused to the Escherichia coli maltose-binding protein (MBP) and, when expressed in E. coli, was found to possess N-terminal autoprocessing activity that was not dependent on the presence of the 3CLpro C-terminal domain. N-terminal sequence analysis of the processed protein revealed that cleavage occurred at the location 2827LVTHE↓VRTGN2836. The trans-processing activity of the purified recombinant 3CLpro (pp1a residues 2832 to 3126) was used to identify another cleavage site, 6441KVNHE↓LYHVA6450, in the C-terminal pp1ab region. Taken together, the data tentatively identify VxHE↓(L,V) as the substrate consensus sequence for the GAV 3CLpro. The study revealed that the GAV and potyvirus 3CLpros possess similar substrate specificities which correlate with structural similarities in their respective substrate-binding sites, identified in sequence comparisons. Analysis of the proteolytic activities of MBP-3CLpro fusion proteins carrying replacements of putative active-site residues provided evidence that, in contrast to most other 3C/3CLpros but in common with coronavirus 3CLpros, the GAV 3CLpro employs a Cys2968-His2879 catalytic dyad. The properties of the GAV 3CLpro define a novel RNA virus proteinase variant that bridges the gap between the distantly related chymotrypsin-like cysteine proteinases of coronaviruses and potyviruses.