An overlapping protein-coding region in influenza A virus segment 3 modulates the host response

Abstract

Influenza A virus (IAV) infection leads to variable and imperfectly understood pathogenicity. We report that segment 3 of the virus contains a second open reading frame ("X-ORF"), accessed via ribosomal frameshifting. The frameshift product, termed PA-X, comprises the endonuclease domain of the viral PA protein with a C-terminal domain encoded by the X-ORF and functions to repress cellular gene expression. PA-X also modulates IAV virulence in a mouse infection model, acting to decrease pathogenicity. Loss of PA-X expression leads to changes in the kinetics of the global host response, which notably includes increases in inflammatory, apoptotic, and T lymphocyte-signaling pathways. Thus, we have identified a previously unknown IAV protein that modulates the host response to infection, a finding with important implications for understanding IAV pathogenesis.

Fig. 1. Characterization of a ribosomal frameshift signal in IAV segment 3

(A) PA-frame synonymous site conservation in an alignment of 1278 representative full-length PA coding sequences. Shown is the synonymous substitution rate relative to the PA segment average (observed/expected) and the corresponding statistical significance (p-value). (B) Positions of stop codons (blue triangles) in the 3 reading frames in 71 representative PA coding sequences. (C) PA-X ORF structure, showing full-length PA in frame 0 and the X-ORF in frame 1, with experimental mutations indicated. (D, E) Detection of frameshifting in rabbit reticulocyte lysate by SDS-PAGE and autoradiography of IVT reactions. (D) Dual reporter constructs separated by the putative ribosomal frameshifting signal from PR8 segment 3 along with 100 nt 5′- and 3′-flanking sequence. WT: wild-type sequence; FS mutant: shiftsite UUU CGU mutated to UUC AGA; IFC: in-frame control. The calculated frameshifting (FS) efficiencies (mean±SEM, n = 4) are shown. (E) IVT reactions were programmed with full-length segment 3 constructs from the 1918 virus and analyzed before (Total) or after (α-X) immunoprecipitation with rabbit anti-1918 X serum. Lane 5 of the bottom panel shows a WT IVT immunoprecipitated with a preimmune bleed. Polypeptides of interest are indicated.

Fig. 2. PA-X mediated repression of plasmid-driven gene expression

293T cells were co-transfected with reporter plasmids encoding (A) ß-galactosidase or (B-D) IAV NP as well as the indicated ‘effector’ plasmids (400 ng unless indicated otherwise). (A) ß-galactosidase accumulation was measured by enzymatic assay. Values plotted are the mean±SEM of 3 experiments normalized to the level seen with an empty plasmid vector as effector. (B) NP, PA and tubulin (as a loading control) accumulation was measured by western blot and (C) quantified from replicate experiments using LiCor Odyssey software. Note in (B) that the same membrane was reprobed for tubulin; residual NP staining is apparent as a doublet running just above tubulin. (C, D) NP mRNA and 5S rRNA (as a loading control) were measured by urea-PAGE and autoradiography of radioactive primer extension assays. (C) NP mRNA accumulation was quantified by densitometry. Values plotted in (C) are the mean±range of two independent experiments, normalized to the control where PB2 was co-transfected in place of PA.

Fig. 3. Characterization of PA-X mutant viruses

(A) MDCK cells infected with the indicated viruses were metabolically labeled with ³⁵S-methionine from 3-8 HPI. Cell lysates were immunoprecipitated with anti-X or the corresponding preimmune (PreI) serum and bound fractions analysed by SDS-PAGE and autoradiography. Aliquots of in vitro translated PA-X (IVT) were run in parallel as markers. The migration of molecular mass standards are also indicated. (B) Lysates from cells infected with the indicated viruses were analyzed by western blotting for PA. (C) Virus titers from MDCK cells (infected at a multiplicity of infection of ~ 0.001) or embryonated eggs (inoculated with 1000 PFU) were determined by plaque assay. Data are the means±SEM of 2-6 separate inoculations, except for 1918-PTC, which was grown once. (D, E) Lysates from MDCK cells infected with the indicated 1918 RNP viruses and metabolically labeled for 45 minute periods ending at the times shown were (D) analyzed by SDS-PAGE and autoradiography. (E) Actin synthesis at 8 HPI was quantified by densitometry. Data plotted are means±SEM of 3 independent experiments. Two-tailed p-values are indicated: (***) p < 0.0001; (**) p = 0.003; (*) p = 0.01.

Fig. 4. Mutation of PA-X affects IAV pathogenicity in a murine model

(A) Mean weight loss of mice infected with 100 pfu of 1918-WT, -FS, and –PTC viruses (n=15, 15, and 10 respectively). Mice were humanely euthanized when they lost ≥ 25% of their initial body weight; no mice were found dead. Error bars represent ±SEM. (B) Survival proportions of mice infected with 10 (dotted line), 100 (solid line), or 1000 (dashed line) pfu of 1918-WT, -FS, and -PTC viruses. Data for WT and FS in (A) and (B) are pooled results obtained with two independently rescued sets of viruses, one set of which was subjected to high-throughput, full genome sequencing to confirm that the only changes between WT and FS were those deliberately introduced in PA. (C) Mean infectious titers of 1918 viruses in homogenized mouse lungs. Error bars represent the SEM. (D) Expression profile heatmaps generated from whole-lung RNA isolated from mice euthanized at 3, 5, and 8 DPI (n=4 per virus, per timepoint), displaying sequences that showed a ≥ 2-fold difference from mock-infected mouse lungs in at least one experimental group. Each column represents data from an individual experiment, while rows represent unique sequences; row-gene identities are preserved across timepoints. Genes in red are upregulated, those in blue are downregulated, and those in black do not differ as compared to a pool of RNA isolated from 6 mock-infected control mice. (E, F) Gene ontology analysis of sequences whose expression levels differed significantly (p < 0.01, ≥2-fold expression level difference) between 1918-WT and -FS infected lung tissue at 3 DPI with 100 pfu. (E) Top-scoring functional categories. (F) Top pathways within color-coded functional categories.