title: Mutational analyses of the influenza A virus polymerase subunit PA reveal distinct functions related and unrelated to RNA polymerase activity.
abstract: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia, United States of America.
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Liang
authors.forename: Shamika
authors.initials: S
authors.lastname: Danzy
authors.forename: Luan Danh
authors.initials: LD
authors.lastname: Dao
authors.forename: Tristram G
authors.initials: TG
authors.lastname: Parslow
authors.forename: Yuying
authors.initials: Y
authors.lastname: Liang
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004285:Dogs; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity / Q000502:physiology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D030781:Organisms, Genetically Modified; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D014774:Virulence / Q000235:genetics
chemical_list: C061290:PA protein, influenza viruses; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases