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.

Figure 1. Mutational analysis of residues 497–518 of PA on viral growth.

(A) Expression level of the PA mutants in the transfected 293T cells was analyzed by western blot analysis using anti-PA polyclonal antibody. (B) Plaque formation on MDCK cells by wild-type (WT), recombinant mutant L1 (K497A, T498A), and L8 (N513A, D514A). (C) Growth curve analysis of WT, L1, and L8 on MDCK cells at moi of 0.001.

Figure 2. Mutational analysis of residues 497–518 of PA on viral RNA synthesis.

(A) The effects of PA mutants on viral RNA synthesis were analyzed in a 5-plasmid system. Fold induction of luciferase activity over the control was shown in log scale. Results shown are the average of at least 3 independent experiments with error bars representing standard deviation. (B) The levels of all three viral RNA species were analyzed by primer extension assay.

Figure 3. Nuclear localization of PA constructs.

COS1 cells were transfected with expression vector of wild-type or mutant PA proteins, with or without (-PB1, -PB2) expression vectors of PB1 and PB2. Immunofluorescence analysis was conducted to detect the PA protein by monoclonal antibody 3G5 (kindly provided by Dr. Palese, Mount Sinai), with nucleus stained with DAPI.

Figure 4. Characterization of additional single alanine substitution at charged or bulky residues in the C-terminal region.

(A) Plaque formation on MDCK cells. Effects of PA mutations on the polymerase activity. (B) Growth curve analysis of recombinant viruses on MDCK cells at moi of 0.001. (C) The effects of PA mutants on viral RNA synthesis were analyzed in a 5-plasmid system. Fold induction of luciferase activity over the control was shown in log scale. Results shown are the average of at least 3 independent experiments with error bars representing standard deviation. The levels of all three viral RNA species were analyzed by primer extension assay.

Figure 5. PA J10 mutant does not affect the formation of VLPs but decreases viral RNA packaging efficiency.

The 293T cells were transfected with the 17-plasmid system to reconstitute the replication-defective VLPs, in which a full-length vRNA construct was replaced with a corresponding vRNA segment that encodes GFP and is packaged efficiently (PA-GFP, PB1-GFP, and PB2-GFP) , with either the WT or J10 mutant PA (for both the RNA and protein-encoding vectors). The cells were metabolically labeled with ³⁵S for 24 h. (A) Cell lysates were immunoprecipitated with anti-H1N1 antiserum and separated by SDS-PAGE. The most abundant viral proteins, M1 and NP, are highlighted by arrows. C, negative control of which the PA plasmids were omitted from the transfection. (B) Supernatants were VLP-enriched by either chicken erythrocytes or anti-H1N1 antiserum and separated by SDS-PAGE. Only the chicken erythrocytes enriched VLPs are showing here. The viral proteins are highlighted by arrows. C, negative control of which the PA plasmids were omitted from the transfection. (C) Comparison of WT and J10 PA proteins in reporter vRNA packaging. The supernatants containing the replication-defective VLPs, prepared in the absence of metabolic labeling, were collected at 48 h post-transfection and used to infect fresh MDCK cells, with the helper virus at moi of 0.1. The infected MDCK cells were analyzed by flow cytometry for GFP expression at 18 hpi. The GFP-transferring unit per ml is shown.

Figure 6. Localization of the mutants on the PA 3-D structure.

The structure of the PA C-terminal region (PDB ID: 2ZNL) is shown in magenta, with the bound PB1 peptide shown in blue. (A) Residues corresponding to J10 and other J10-like mutants (L3, L5, L8, L9, L10, and D478) are highlighted in yellow. K378 is not shown as it is localized to an unresolved region. The position of J10 site is indicated by arrow. (B) Residues corresponding to L1, L2, and L4 mutations, which affect PA nuclear localization, are highlighted in yellow. (C) Residues of L6 mutant, which is localized to the nucleus but defective in all viral RNA synthesis, are highlighted in yellow.