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Mutational Analyses of the Influenza A Virus Polymerase Subunit PA Reveal Distinct Functions Related and Unrelated to RNA Polymerase Activity
Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus prod...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253111/ https://www.ncbi.nlm.nih.gov/pubmed/22238617 http://dx.doi.org/10.1371/journal.pone.0029485 |
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author | Liang, Yuhong Danzy, Shamika Dao, Luan Danh Parslow, Tristram G. Liang, Yuying |
author_facet | Liang, Yuhong Danzy, Shamika Dao, Luan Danh Parslow, Tristram G. Liang, Yuying |
author_sort | Liang, Yuhong |
collection | PubMed |
description | Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus production, indicating that PA may have a novel role independent of its polymerase activity. To further examine the roles of PA in the viral life cycle, we have now generated and characterized additional mutations in regions flanking the J10 site from residues 497 to 518. All tested di-codon mutations completely abolished or significantly reduced viral infectivity, but they did so through disparate mechanisms. Several showed effects resembling those of J10, in that the mutant polymerase supported normal levels of viral RNA synthesis but nonetheless failed to generate infectious viral particles. Others eliminated polymerase activity, in most cases by perturbing the normal nuclear localization of PA protein in cells. We also engineered single-codon mutations that were predicted to pack near the J10 site in the crystal structure of PA, and found that altering residues K378 or D478 each produced a J10-like phenotype. In further studies of J10 itself, we found that this mutation does not affect the formation and release of virion-like particles per se, but instead impairs the ability of those particles to incorporate each of the eight essential RNA segments (vRNAs) that make up the viral genome. Taken together, our analysis identifies mutations in the C-terminal region of PA that differentially affect at least three distinct activities: protein nuclear localization, viral RNA synthesis, and a trans-acting function that is required for efficient packaging of all eight vRNAs. |
format | Online Article Text |
id | pubmed-3253111 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-32531112012-01-11 Mutational Analyses of the Influenza A Virus Polymerase Subunit PA Reveal Distinct Functions Related and Unrelated to RNA Polymerase Activity Liang, Yuhong Danzy, Shamika Dao, Luan Danh Parslow, Tristram G. Liang, Yuying PLoS One Research Article Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus production, indicating that PA may have a novel role independent of its polymerase activity. To further examine the roles of PA in the viral life cycle, we have now generated and characterized additional mutations in regions flanking the J10 site from residues 497 to 518. All tested di-codon mutations completely abolished or significantly reduced viral infectivity, but they did so through disparate mechanisms. Several showed effects resembling those of J10, in that the mutant polymerase supported normal levels of viral RNA synthesis but nonetheless failed to generate infectious viral particles. Others eliminated polymerase activity, in most cases by perturbing the normal nuclear localization of PA protein in cells. We also engineered single-codon mutations that were predicted to pack near the J10 site in the crystal structure of PA, and found that altering residues K378 or D478 each produced a J10-like phenotype. In further studies of J10 itself, we found that this mutation does not affect the formation and release of virion-like particles per se, but instead impairs the ability of those particles to incorporate each of the eight essential RNA segments (vRNAs) that make up the viral genome. Taken together, our analysis identifies mutations in the C-terminal region of PA that differentially affect at least three distinct activities: protein nuclear localization, viral RNA synthesis, and a trans-acting function that is required for efficient packaging of all eight vRNAs. Public Library of Science 2012-01-06 /pmc/articles/PMC3253111/ /pubmed/22238617 http://dx.doi.org/10.1371/journal.pone.0029485 Text en Liang et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Liang, Yuhong Danzy, Shamika Dao, Luan Danh Parslow, Tristram G. Liang, Yuying Mutational Analyses of the Influenza A Virus Polymerase Subunit PA Reveal Distinct Functions Related and Unrelated to RNA Polymerase Activity |
title | Mutational Analyses of the Influenza A Virus Polymerase Subunit PA Reveal Distinct Functions Related and Unrelated to RNA Polymerase Activity |
title_full | Mutational Analyses of the Influenza A Virus Polymerase Subunit PA Reveal Distinct Functions Related and Unrelated to RNA Polymerase Activity |
title_fullStr | Mutational Analyses of the Influenza A Virus Polymerase Subunit PA Reveal Distinct Functions Related and Unrelated to RNA Polymerase Activity |
title_full_unstemmed | Mutational Analyses of the Influenza A Virus Polymerase Subunit PA Reveal Distinct Functions Related and Unrelated to RNA Polymerase Activity |
title_short | Mutational Analyses of the Influenza A Virus Polymerase Subunit PA Reveal Distinct Functions Related and Unrelated to RNA Polymerase Activity |
title_sort | mutational analyses of the influenza a virus polymerase subunit pa reveal distinct functions related and unrelated to rna polymerase activity |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253111/ https://www.ncbi.nlm.nih.gov/pubmed/22238617 http://dx.doi.org/10.1371/journal.pone.0029485 |
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