RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp.
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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2011-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3153931?pdf=render |
| _version_ | 1818525906330714112 |
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| author | Chumporn Soowannayan Jeff A Cowley Wojtek P Michalski Peter J Walker |
| author_facet | Chumporn Soowannayan Jeff A Cowley Wojtek P Michalski Peter J Walker |
| author_sort | Chumporn Soowannayan |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-11T06:15:34Z |
| format | Article |
| id | doaj.art-55f92c51cb374c3e9e6a387adab93529 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-11T06:15:34Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-55f92c51cb374c3e9e6a387adab935292022-12-22T01:18:00ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0168e2215610.1371/journal.pone.0022156RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp.Chumporn SoowannayanJeff A CowleyWojtek P MichalskiPeter J WalkerGill-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.http://europepmc.org/articles/PMC3153931?pdf=render |
| spellingShingle | Chumporn Soowannayan Jeff A Cowley Wojtek P Michalski Peter J Walker RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp. PLoS ONE |
| title | RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp. |
| title_full | RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp. |
| title_fullStr | RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp. |
| title_full_unstemmed | RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp. |
| title_short | RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp. |
| title_sort | rna binding domain in the nucleocapsid protein of gill associated nidovirus of penaeid shrimp |
| url | http://europepmc.org/articles/PMC3153931?pdf=render |
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