To demonstrate that CK2β can be activated in A549 cells, we assessed CK2β activation at different time points after stimulation with spermine (a polyamine that regulates CK2 activity through direct interaction with the β subunit of the holoenzyme 40) or with the broad kinase activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In spermine-stimulated cells, CK2β activation was increased at 2 h and 4 h but had returned to the basal level at 6 h and 8 h (Figure 1a, left). TPA treatment caused stronger kinase activation than did spermine, as compared to the respective controls. TPA-induced CK2β activation reached its highest level at 4 h and had progressively decreased at 6 h and 8 h (Figure 1a, right). These results show that CK2β can indeed be activated in the cell line used for this study.

To assess whether CK2β is activated by influenza virus, we incubated A549 cells with medium containing the human A/Puerto Rico/8/34 influenza virus (multiplicity of infection [MOI] = 5) for 1 h at 37°C and then analyzed CK2β activation at different time points by using an antibody that detects serine phosphorylation at position 209 of CK2β. CK2β activation in uninoculated cells was assigned a value of 100% (baseline control). CK2β activation did not differ between inoculated and control cells at any point during hours 2 to 20 after inoculation (Figure 1b), suggesting that CK2β is not activated during influenza virus replication.

We next assessed CK2β activation during the 1-h inoculation period, when virus binds to cells and is internalized. Cells were incubated with virus-containing medium (MOI = 5) at 4°C for 20 min. The medium was then replaced with pre-warmed medium and cells were incubated at 37°C. CK2β activation was analyzed 0, 30, 45, and 60 min after start of inoculation period. At 45 min and 60 min after infection, CK2β activation was approximately 65% and 80%, respectively, greater than that in control cells (Figure 1c). Therefore, CK2β activation is upregulated very early in the viral life cycle and is inhibited when virus replication starts.

To evaluate the effect of CK2β on IVA replication, we silenced CK2β expression in A549 cells by using a gene-specific siRNA. To ensure that the amount of siRNA duplex used did not negatively influence cell viability, we first performed a cell proliferation assay 24 h, 48 h, and 72 h post-transfection (p.t.). CK2β siRNA did not reduce the number of viable cells or affect cell morphology, as compared to those of control cells, at any time point (data not shown), suggesting that the amount of siRNA used is not toxic to the cells.

Total amount of the CK2β protein was assayed 24 h, 48 h, and 72 h p.t. by Western blot analysis. In cells transfected with CK2β siRNA, CK2β expression was substantially less than that in control-transfected cells at 24 h p.t. and was almost completely absent at 48 h p.t. (Figure 2a), demonstrating the efficiency of this gene-specific siRNA. The amount of CK2β protein started to increase again at 72 h p.t. (Figure 2a), indicating that siRNA-mediated inhibition of CK2β gene expression is transient. Additionally, to determine whether CK2β gene silencing negatively affects expression of the α-subunit of CK2, we assayed CK2α protein levels at the same 3 time points. CK2α expression was not reduced in CK2β siRNA-transfected cells at 24 h, 48 h, or 72 h p.t. (Figure 2b). Because CK2β expression was most significantly reduced 48 h p.t., cells were inoculated with virus 48 h p.t. and further incubated for 24 h. At first, we used a relatively high MOI of 5, as A549 cells are less susceptible to IVA infection than commonly used cell lines such as MDCK cells. It is noteworthy that most of the cells remained viable after a 24-h inoculation at this MOI. Virus titers in the cell supernatants differed substantially 24 h post-inoculation (p.i.). The mean virus titer in CK2β siRNA-transfected cells was 3 times that in control siRNA-transfected cells (Figure 3a). The difference in virus titers was more pronounced when cells were inoculated with a very low virus dose (MOI = 0.1 and 0.001). At MOI = 0.1, the mean virus titer was approximately 5 times as high in cells transfected with CK2β siRNA as in control siRNA-transfected cells (Figure 3a). At MOI = 0.001, the difference exceeded 1 log10 (Figure 3a).

We next confirmed that CK2β activity influences the outcome of viral infection. To assess the effect of CK2 inhibition, we incubated cells with 5 μM 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT, a potent and specific CK2 inhibitor 41) or with solvent (DMSO) before and during virus inoculation (MOI = 5). To determine the effect of CK2β activation, we treated cells with 300 μM spermine or with H₂O before and during virus inoculation. At 24 h p.i., the mean virus titer in DMAT-treated cells increased as compared to controls (Figure 3b). Conversely, cells treated with the CK2β activator spermine had a mean virus titer approximately half that of the control titer at 24 h p.i. (Figure 3b). These results again suggest that activation of CK2β is inconsistent with efficient virus replication.

Having shown that 1) CK2β activation is inhibited during IVA replication and 2) CK2β silencing results in increased virus titers, we next studied the effect of CK2β gene silencing on the initial step of the viral replication cycle: virus entry. We transfected cells with either control or CK2β siRNA for 48 h. Cells were then inoculated with a high dose of virus (MOI = 50), incubated at 4°C for 30 min to block virus entry while binding to the cell surface, and incubated at 37°C for 30 min to allow virus entry. The cell membranes were then stained with an antibody specific for epidermal growth factor receptor (EGFR) and examined by confocal microscopy. Virus entry was slowed in control siRNA-transfected cells but not in CK2β siRNA-transfected cells, where they had accumulated at the nuclear membrane (predominantly) and in the nucleus 30 min after temperature shift (Figure 4). Although most viruses localized at the nuclear membrane of control siRNA-transfected cells, significantly less viral antigen was detected within the nucleus, and a large proportion of the virus population still remained in the cytoplasm and at the cell membrane (small fraction) (Figure 4). Therefore, internalization and nuclear import of virus are substantially greater when CK2β protein is absent or reduced.

We next examined whether CK2β gene silencing affects viral protein synthesis. Cells were transfected with either control or CK2β siRNA for 48 h, inoculated (MOI = 1) for 9 h or 24 h, and lysed for Western blot analysis. Total amount of viral protein was detected by using an antibody specific for IVA of the H1N1 subtype. As NP was the most heavily expressed of the viral proteins, we quantified the NP bands. The value obtained at 9 h p.i from cells transfected with control siRNA was assigned a value of 100%. NP production increased in both control and CK2β siRNA-transfected cells between 9 h and 24 h p.i. (Figure 5a).

However, a substantially larger amount of viral NP protein was synthesized in CK2β siRNA-transfected cells than in control-transfected cells at both time points (Figure 5a). While the expression of a late viral protein (matrix protein, M1) differed only slightly at 9 h p.i. between cells transfected with CK2β and control siRNA, M1 expression was higher in CK2β siRNA-transfected cells than in control-transfected cells at 24 h p.i. (Figure 5a).

To confirm that cells transfected with control siRNA and with CK2β siRNA differed in their viral protein content, we measured expression of NP protein in transfected cells at 24 h p.i. (MOI = 1) by FACS. Markedly more NP was produced in cells transfected with CK2β siRNA than in control siRNA-transfected cells (Figure 5b). 64% of the cell population transfected with CK2β siRNA expressed NP, whereas only 38% of the control siRNA-transfected cells were NP⁺ (Figure 5b). This finding supports the results of our Western blot analysis and shows higher production of viral NP protein in CK2β siRNA-transfected cells.

Next, we investigated the contribution of CK2β to the intracellular localization of the viral RNP complexes. Cells were transfected with either control or CK2β siRNA for 48 h and then inoculated (MOI = 1) for 6 h and 8 h, the time points at which optimal nuclear export of RNP usually occurs 293242. Viral RNP complexes were stained with an anti-NP antibody, and CK2β protein was stained with anti-CK2β antibody. Confocal microscopy demonstrated a reduced amount of CK2β protein at 6 h and 8 h p.i in cells transfected with CK2β siRNA. In cells transfected with control siRNA, the majority of CK2β was found in the cytoplasm (Figure 6). At 6 h p.i. in cells transfected with CK2β siRNA, the viral RNP complexes were localized predominantly in the nucleus, with a small fraction in the cytoplasm. However, there was no substantial difference in nuclear RNP export in cells transfected with CK2β and control siRNA. Further, at 8 h p.i. viral RNP complexes were distributed in both the cytoplasm and the nuclei of control and CK2β siRNA-transfected cells (Figure 6), showing that the nuclear-cytoplasmic transport of viral RNP complexes is not affected by CK2β or by its absence.

We used a minigenome (RNP-like) system to evaluate whether CK2-mediated phosphorylation of PA and NP influences viral polymerase activity. Cells were transfected with either control or CK2β siRNA for 48 h and then co-transfected with the pol I-driven plasmid encoding the luciferase gene and pol I/pol II-responsive plasmids that express the viral PB2, PB1, PA, and NP proteins. After incubation for 24 h, luciferase activity (indicative of viral polymerase activity) was assayed in cell extracts. Transfection without the PB1 plasmid revealed negligible background luciferase expression. Inhibition of CK2β expression did not substantially influence viral polymerase activity, which was only slightly greater after CK2β siRNA treatment than after control siRNA treatment (Figure 7). The results indicate that the CK2β-mediated phosphorylation of PA and NP is not required for regulation of viral polymerase activity.

The human epithelial lung cell line A549 was purchased from the American Type Culture Collection (Rockville, MD) and was cultured in FK12 medium containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotics (penicillin/streptomycin). Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS) and 1% (v/v) antibiotics (penicillin/streptomycin). All cells were cultivated at 37°C in a humidified atmosphere containing 5% CO₂. The H1N1-subtype human influenza virus A/Puerto Rico/8/34 (PR/8) was generated by 8-plasmid reverse genetics 59 and used at the indicated multiplicity of infection (MOI; determined in MDCK cells) in the presence of TPCK trypsin (1:6000).

Confluent monolayers of MDCK cells in 35-mm dishes were inoculated with 10-fold dilutions of PR/8 influenza virus in DMEM with 3% bovine serum albumin (BSA) and antibiotics and incubated at 37°C for 1 h. The inoculum was removed, and cells were washed with PBS and overlaid with 2-fold DMEM containing 1.8% bacto-agar, 0.2% BSA, and 2 μg/ml TPCK trypsin. After 3 days of incubation at 37°C, cells were stained with 0.1% crystal violet in 10% formaldehyde solution and the plaques were counted.

To determine the effect of CK2 inhibition, confluent monolayers of A549 cells in 35-mm dishes were treated with 5 μM 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT; Calbiochem, San Diego, CA) or with solvent (DMSO) (Sigma-Aldrich, St. Louis, MO) 1 h prior to infection. To determine the effect of CK2β activation, cells were treated with 300 μM spermine (Sigma-Aldrich) or with H₂O 1 h prior to infection. Cells were then inoculated at MOI = 5 in the presence of DMAT or DMSO. Virus-containing supernatants were harvested 24 h post-infection (p.i.) and virus titers were determined by plaque assay.

Human CK2β siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) is a pool of 3 target-specific 20- to 25-nt siRNAs designed to silence CK2β gene expression in human cells. A549 cells (grown in 35-mm dishes) were transfected with siRNA duplex (1 μg) by using Lipofectamine 2000 (Invitrogen, New Brunswick, NJ). The control siRNA (Santa Cruz Biotechnology) is a nontargeting 20- to 25-nt siRNA that does not cause specific degradation of any known cellular mRNA. Cells were transfected for 48 h, washed with FK12 medium without supplements, then inoculate at the indicated MOIs and incubated for different time periods, depending on the purpose of the experiment. Transfection efficiency monitored by fluorescein-conjugated control siRNA (Santa Cruz Biotechnology) varied up to 85% (flow cytometry analysis) according to the maximum protein level reduction.

A549 cells were grown in 96-well dishes and transfected with 1 μg of CK2β siRNA or control siRNA for 24 h, 48 h, or 72 h at 37°C, 5% CO₂. The cell medium was then replaced with fresh medium. After further incubation for 1 h, the medium was replaced with 200 μl of FK12 medium containing 10% (v/v) FBS and tetrazolium bromide (MTT; 175 μg mL^-1, Sigma-Aldrich). After 90 min, the incubation medium was replaced with PBS/4% paraformaldehyde (PFA), and cells were fixed for 30 min at room temperature (RT). Cells were air dried and, after addition of isopropanol (150 μL/well), plates were vigorously agitated for 10 min. The plates were photometrically analyzed at 550 nm in an enzyme-linked immunosorbent assay reader (Bio-Rad Microplate Reader, Model 680). Sixteen samples from each group were measured, and the mean and standard error was calculated. Cell morphology was analyzed by confocal laser scanning microscopy after transfection with either siRNA.

Cell lysate was cleared by centrifugation, and total protein concentration was determined by the Bradford assay before separation by SDS-PAGE. A rabbit polyclonal antibody that detects phosphorylated CK2β (P-S209) (1:200; abcam, Cambridge, MA) was used to determine CK2β activity. Expression of CK2α and CK2β were detected with anti-CK2α (1:200; Calbiochem) and anti-CK2β (1:500; Calbiochem) mouse monoclonal antibody, respectively. Anti-β-actin mouse monoclonal antibody (1:500; Santa Cruz Biotechnology) was used to detect β-actin expression as a protein-loading control. Proteins recognized by mAbs were further analyzed with horseradish peroxidase-conjugated, species-specific secondary antibodies (1:2000; Jackson ImmunoResearch, West Grove, PA) and a standard enhanced chemiluminescence reaction (Amersham Biosciences, Piscataway, NJ). Specific bands were quantified by using the PC-BAS software package (Fuji, Kanagawa, Japan). To detect synthesis of viral proteins, cells were incubated with a goat anti-IVA (H1N1) polyclonal antibody (1:500; Biodesign International, Saco, ME).

To detect ERK activation, cells were transfected with either control or CK2β siRNA for 48 h and were then infected at an MOI of 5 and further incubated for 4, 6, or 8 h. After Western blot analysis, ERK activation was measured with a mAb specific for phosphorylated ERK (1:500; Santa Cruz Biotechnology). ERK2 (1:500; Santa Cruz Biotechnology) served as a protein loading control.

After 48 h of transfection with either siRNA, cells were infected at MOI = 1 and incubated for 24 h. Cells were detached with trypsin, fixed in PBS/4% PFA, permeabilized with 1% Triton X-100, and incubated with FITC-conjugated mouse anti-NP mAb (clone IA52, 1:500; Argene Inc) in PBS/3% BSA for 30 min on ice. The percentage of NP-expressing cells was determined by FACS analysis using (FACSCalibur, BD Biosciences, Rockville, MD).

A549 cells grown on collagen-coated glass coverslips (BD, Franklin Lakes, NJ) were transfected with either control or CK2β siRNA for 48 h, then infected at MOI = 1 and incubated further for 6 h and 8 h. Cells were washed with PBS at the indicated time points p.i. and fixed with 4% PFA in PBS for 30 min at room temperature or overnight at 4°C. Cells were permeabilized with 1% Triton X-100 in PBS for 10 min and incubated with a goat anti-NP antibody, clone G150 (1:100; St Jude Children's Research Hospital) and anti-CK2β mouse monoclonal antibody (1:100; Calbiochem) in PBS/3% BSA for 1 h for analysis of the nuclear export of RNP complexes and the amount of expressed CK2β protein. The AlexaFluor488-conjugated goat anti-mouse antibody and AlexaFluor594-conjugated donkey anti-goat antibody (1:200; Molecular Probes, Invitrogen, Carlsbad, CA) were used as secondary antibodies. Cells were washed with PBS and then with double-distilled water and mounted with para-phenylene diamine (Sigma-Aldrich) containing 500 nM TO-PRO-3 (Molecular Probes, Invitrogen) for nuclear staining. Fluorescence was visualized with a confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany).

To study virus entry, cells were infected at a MOI = 50, chilled at 4°C for 30 min, and incubated at 37°C for 30 min. Cells were permeabilized, fixed, and incubated with a combination of goat anti-IVA (H1N1) polyclonal antibody (1:100) (Biodesign International) and mouse anti-epidermal growth factor receptor (EGFR) mAb (1:100; Santa Cruz Biotechnology). Alexa594-conjugated donkey anti-goat and AlexaFluor488-conjugatedgoat anti-mouse antibodies (Molecular Probes) were used as secondary antibodies.

Subconfluent monolayers of A549 cells (7.5 × 10⁵ cells in 35-mm dishes) were transfected with CK2β siRNA or control siRNA for 48 h. Cells were then transfected with 2 μg of luciferase reporter plasmid (enhanced green fluorescent protein (EGFP) open reading frame in pHW72-EGFP substituted with the luciferase gene60) and a mixture of the PB2 (1 μg), PB1 (1 μg), PA (1 μg), and NP (2 μg) plasmids of A/Puerto-Rico/8/34 (H1N1) virus. After 24 h, cell extracts were prepared in 500 μL of lysis buffer, and luciferase levels were assayed with a luciferase assay reagent kit (Promega, Madison, WI) and BD Monolight 3010 luminometer (BD Biosciences).