Figure 5. Effect of influenza virus replication factor-2 (IREF-2) knockdown in infected cells.

(A) Endogenous IREF-2 protein levels in knockdown (KD) HeLa cells. The whole-cell lysates of control KD cells (lane 3), pp32 KD cells (lane 4), APRIL KD cells (lane 5), and both pp32 and APRIL KD cells (termed double-KD cells; lane 6) were subjected to SDS-PAGE followed by western blot analysis with antibodies, as indicated on the right side of the panel. To measure the KD levels, 10% and 30% of the lysates of the control KD cells were also subjected to SDS-PAGE followed by western blot analysis with antibodies, respectively (lanes 1 and 2). (B) Viral RNA levels in infected KD HeLa cells. Total RNA was extracted from mock-infected or infected KD cells at an multiplicity of infection (MOI) of 1 at 3, 6, and 9 hr post infection. vRNA, complementary RNA(cRNA), and viral mRNA derived from segment 5 were quantitatively determined by RT-mediated qPCR (RT-qPCR), as described in 'Materials and methods'. (C) Primary transcription level in infected KD HeLa cells. Total RNA was extracted from mock-infected or infected KD cells at an MOI of 10 for 3 hr in the presence or absence of cycloheximide (CHX). Viral mRNA derived from segment 5 was quantitatively determined by RT-qPCR. (D) Effect of IREF-2 on progeny virus production. Control KD and IREF-2 double-KD A549 cells were infected with FluV (WSN/33 strain) at an MOI of 0.001. The number of infectious progeny viruses produced from control KD and IREF-2 double-KD A549 cells at each time point were plotted (upper panel), and the ratios of progeny viruses produced from IREF-2 double-KD A549 cells compared with those from control KD cells were also represented as a percentage of the control (lower panel). Each quantitative result is presented as the average with the standard deviation from at least three independent experiments. Significance was determined using Student’s t test. n.s.: not significant.

DOI: http://dx.doi.org/10.7554/eLife.08939.010

Figure 5—figure supplement 1. Effect of influenza virus replication factor-2 (IREF-2) on viral reporter RNA syntheses from a reconstituted model replicon.

HEK293T of control and IREF-2 knockdown (KD) cells (approximately, 3 × 10⁵ cells in a 22-mm-diameter dish) were transfected with expression plasmids pCAGGS-PB1, -PB2, -PA, and -NP (15 ng each). In addition, 1.5 ng of either phPolI-vNS-Luc (for expression of vNS-Luc; left panel) or phPolI-cNS-Luc (for expression of cNS-Luc; right panel) was co-transfected as a viral genomic source. At 24 hr post co-transfection, viral reporter levels in both the control KD and the IREF-2 KD cells were quantitatively determined by RT-qPCR as described previously (Kawaguchi et al., 2011) and in 'Materials and methods'. Quantification and standard deviations of the viral reporter RNA levels from IREF-2 KD cells expressed as a percentage of the value from the control KD cells. Each experiment was repeated three times.

DOI: http://dx.doi.org/10.7554/eLife.08939.011