Figure 4. Interaction of influenza virus replication factor-2 (IREF-2) proteins with free forms of viral polymerase trimeric complexes.

(A) Interaction between IREF-2 and viral proteins in infected cells. HEK293T cells were transfected with 10 μg of plasmids expressing GSTnls (lane 1), GST-pp32 (lane 2), GST-APRIL (lane 3), and GST-RAF2p48 (lane 4). At 48 hr post transfection, influenza virus was infected at an multiplicity of infection (MOI) of 3. At 6 hr post infection, the transfected and infected cells were collected, lysed, and subjected to GST pull-down assays, as described in 'Materials and methods'. The pulled-down materials and 3% equivalent of the input samples were subjected to SDS-PAGE followed by western blot analysis with anti-PB1, -PB2, -PA, -NP, and -GST (only the pull-down sample) antibodies. (B) Interaction between IREF-2 and viral proteins in the transfected cells. HEK293T cells were transfected with 5 μg of plasmids expressing GSTnls (lanes 1 and 5), GST-pp32 (lanes 2 and 6), GST-APRIL (lanes 3 and 7), and GST-RAF2p48 (lanes 4 and 8) and also co-transfected with the plasmids for the viral polymerase subunits (5 μg of pCAGGS-PB1, 12.5 μg of pCAGGS-PB2, and 2.5 μg of pCAGGS-PA [lanes 1–4] or 10 μg of pCAGGS-NP for NP expression [lanes 5–8]). Forty-eight hours post transfection, the cotransfected cells were collected, lysed, and subjected to GST pull-down assays. The input samples (3%) and pulled-down materials were subjected to SDS-PAGE followed by western blot analysis. (C) Interaction between IREF-2 and trimeric or binary complexes of vRdRP. HEK293T cells were cotransfected with 5 μg of plasmids expressing GSTnls (lanes 1, 4, and 7), GST-pp32 (lanes 2, 5, and 6), GST-APRIL (lanes 3, 6, and 9), 5 μg of pCAGGS-PB1cFlag (lanes 1–9), 12.5 μg of pCAGGS-PB2 (lanes 1–6), and 2.5 μg of pCAGGS-PA (lanes 1–3 and 7–9). Forty-eight hours post transfection, the lysates from the cotransfected cells were subjected to GST pull-down assays or immunoprecipitation assays with anti-Flag antibody. The precipitated materials were subjected to SDS-PAGE followed by western blot analysis.

DOI: http://dx.doi.org/10.7554/eLife.08939.007

Figure 4—figure supplement 1. Electrophoresis mobility shift assay for influenza virus replication factor-2 (IREF-2) and viral RNA.

Radioactively labeled 53-nt-long model vRNA and complementary RNA(cRNA) probes (v53 and c53; 246.9 cpm/fmol) were synthesized by T7 RNA polymerase using [α-³²P] GTP and isolated by gel excision. Each 500 pM (final concentration) of the labeled viral RNA probes, v53 (lanes 1–7) and c53 (lanes 8–14) was incubated with 10 nM or 50 nM of recombinant NP prepared using the Escherichia. coli expression system (lanes 2, 3, 9, and 10), recombinant pp32 (lanes 4, 5, 11, and 12), and recombinant APRIL (lanes 6, 7, 13, and 14) in 50 mM HEPES-NaOH (pH 7.9), 50 mM KCl, 0.5 U/μl of RNase inhibitor, and 15% (v/v) glycerol at 30°C for 30 min. After incubation, each binding mixture was loaded onto 0.6% agarose gel (buffered with TBE) and separated by electrophoresis (50 V for 3 hr). The gel was dried and visualized by autoradiography.

DOI: http://dx.doi.org/10.7554/eLife.08939.008

Figure 4—figure supplement 2. Interaction between viral RNA-dependent RNA polymerase (vRdRP) complexes and endogenous influenza virus replication factor-2 (IREF-2).

HEK293T cells were transfected with plasmids expressing PB1 (lane 2) or PB1cFlag (lanes 3–5), PB2 (lanes 2–4), and PA (lanes 2, 3, and 5), as indicated. At 48 hr post transfection, the cells were harvested, lysed, and subjected to immunoprecipitation assays using anti-Flag antibody, as described in 'Materials and methods'. Input and precipitated materials were analyzed by SDS-PAGE, followed by western blot analysis using the antibodies indicated on the right side of each panel.

DOI: http://dx.doi.org/10.7554/eLife.08939.009