Photomicrographs of A549 cells infected with A/PR/8/34 at an MOI of 7 PFU/cell (bottom) or mock-infected (top) for the indicated hours postinfection (indicated at the top). Scale bar, 100 μm.

Immunoblot analysis of host and influenza virus proteins in mock-infected (M) and influenza virus strain A/PR/8/34 (H1N1)-infected (I) A549 cells. Cells were harvested and lysed with 0.5% NP-40 detergent, nuclei were removed, and cytosolic fractions were dissolved in SDS electrophoresis sample buffer, resolved in s 5 to 15% minigradient SDS-PAGE, transferred to PVDF, and probed with various antibodies. Bands were visualized and intensities measured with an Alpha Innotech FluorChemQ MultiImage III instrument. Molecular weight standards are indicated at the left and ratios of each protein (infected divided by mock infected) are indicated for each protein at the right, along with SILAC-measured ratios (far right). *, no viral proteins were measured by SILAC because they were not present in mock-infected samples.

Molecular pathways of regulated proteins. Proteins and their levels of regulation were imported into the Ingenuity Pathways Analysis (IPA) tool, and interacting pathways were constructed. (A) Overview of 4 networks identified at 95% confidence, each of which contained 10 or more “focus” molecules (molecules significantly up- or downregulated). Each box contains an arbitrary network number (upper) as well as the number of focus molecules within the network (lower bolded number). Lines connecting networks indicate the number of focus molecules present in each attached network. (B) Merged network, containing all molecules present in each of the four individual networks. (C to F) Individual networks with pathway names indicated. Solid lines, direct known interactions; dashed lines, suspected or indirect interactions; red, significantly upregulated proteins; pink, moderately upregulated proteins; gray, proteins identified but not significantly regulated; light green, moderately downregulated proteins; dark green, significantly downregulated proteins; white, proteins known to be in the network but not identified in our study. Molecular classes are indicated in the legends.

Distributions of proteins identified in various experiments. (A and B) Venn diagrams of the numbers of identified proteins from various analyses. (A) Proteins from A/PR/9/34-infected A549 cells were fractionated into the cytosolic plus crude membrane (Cyto/Gel) and nuclei (Nuclei/Gel) fractions, resolved in SDS-PAGE, and then subjected to tryptic digest before 1-D LC/MS. Alternatively, proteins were harvested from cytosolic and crude membrane fractions and digested with trypsin, and then peptides were resolved by 2-D orthogonal LC/MS (2-D LC/MS). Results were compiled from two replicate experiments. (B) Proteins identified by the three separate 2-D LC/MS analyses. Proteins from the two technical replicate analyses of the third 2-D LC/MS run were merged prior to being combined with other data. (C) Frequency distributions of identified proteins in two influenza virus-infected A549 sample sets, with L/H ratios expressed as log₂ values. Positive values represent upregulated host proteins in virus-infected cells; negative values represent downregulated host proteins. Only the distributions of one SDS-PAGE analysis and one 2-D LC/MS analysis are shown for clarity. Note that distributions are not identical, with different peak breadths, and not perfectly normal, with the 2-D LC/MS sample exhibiting several substantially downregulated proteins at approximately −13log₂. Characteristics of all SDS-PAGE and 2-D LC/MS protein distributions, mean log₂ L/H ratios, and standard deviations of log₂ L/H ratios are shown in Table 1.

Gene ontology analyses of upregulated and downregulated proteins. The proteins identified in Tables 2 and 3, as well as nonkeratin proteins in Table 2, were imported into the DAVID gene ontology suite of programs at the NIAID, gene identifications were converted by that program, and ontological functions were determined by GOTERM. (A) Biological processes; (B) functional annotations; (C), cellular components; and (D) molecular functions. The numbers of identified genes associated with each group, identified at a confidence level of 95% are illustrated. *, processes, functions, and cellular components that are removed when keratins are excluded from the input gene list. Additional lists of functional groups, processes, and components at different confidence limits are indicated in Table ST-2 in the supplemental material.