Interaction of hRBL2 with viral proteins. (A) hRBL2 interacts with viral RNP complexes. The three viral polymerase proteins and c-Myc-tagged NP were expressed in 293T cells in the absence or presence of virus-like RNA (lanes 1 and 2). Nontagged NP was expressed as a negative control (lane 3). One day later, cell lysates were immunoprecipitated with anti-c-Myc beads. Western blot analysis was carried out with anti-hRBL2 and anti-c-Myc antibodies. EGFP expression in transfected cells indicates the functionality of c-Myc-tagged NP protein. (B) hRBL2 interacts with NP, PB2, and PB1. The three polymerase and NP proteins were individually tested for their interaction with hRBL2 as described above. α, anti; IP, immunoprecipitation.

hRBL2 interferes with NP oligomerization. (A) BiFC assay to assess interactions between hRBL2 and vRNP components. The proteins of interest were fused to the N- or C-terminal portions of Kusabira-Green (GN or GC). Interaction between the proteins of interest thus results in fluorescence. hRBL2 interacts with NP but not with the polymerase subunits. (B) hRBL2 inhibits NP oligomerization in a dose-dependent manner. Cells were transfected with plasmids for the expression of PB2, PB1, PA, pPolI-GFP, and GN-NP and GC-NP. NP-NP interaction was detected by microscopy and FACS analysis (middle panel, red curve). This interaction decreased with increasing amounts of hRBL2 (middle panel). Reporter gene expression in transfected cells indicates the functionality of the GN-NP and GC-NP fusion proteins and the biological significance of hRBL2 interference with NP oligomerization.

Identification of cellular proteins that enhance influenza virus replication. Cells were transfected with plasmids encoding the HK483 (H5N1) PB2, PB1, PA, and NP proteins (the PB2 protein possesses glutamic acid at position 627; PB2-627E). Cells were cotransfected with PolI-EGFP for the synthesis of a virus-like RNA and with an avian (quail cell) cDNA expression library. Cells expressing avian proteins that support efficient replication by PB2-627E in mammalian cells at 33°C produce increased amounts of EGFP. GFP-expressing cells were selected by a FACS cell sorter. After three rounds of selection, plasmid DNA was extracted from the cells and sequenced.

Effect of hRBL2 knockdown on influenza virus replication. (A) Knockdown of hRBL2 in 293 cells. 293 cells were transfected with an siRNA specific to hRBL2 (siRBL2) or with a nonspecific control siRNA. Cells were also transfected with a plasmid expressing hRBL2 (pChRBL2) or a control vector. Two days later, hRBL2 expression levels were assessed by Western blot analysis. Beta-actin expression levels served as an internal control. (B) Viral polymerase activity in cells treated with an siRNA to hRBL2. 293 cells were transfected with hRBL2-specific or control siRNAs (si Control) and incubated at 37°C for 48 h. The transfected cells were then infected with strain WSN (MOI of 1). The amounts of vRNA, cRNA, and mRNA were determined at 5 h postinfection as described in the legend of Fig. 2b. (C) Viral M1 protein production in hRBL2 knockdown cells and control cells. Cells were transfected with siRNAs as described above and infected with WSN virus at an MOI of 3. M1 protein levels were assessed as described in the legend of Fig. 2c. (D) Influenza virus titers in hRBL2 knockdown cells and control cells. Cells were treated with siRNAs as described above, infected with WSN virus at an MOI of 0.05, and incubated at 37°C for the indicated time periods. Virus titers were determined in MDCK cells. The error bars represent standard deviations (n = 3). α, anti.

Effect of RBL2 expression on influenza virus replication and growth. (A) ΔN-qRBL2 increases influenza virus replication in a minireplicon assay. 293T cells were transfected with plasmids expressing HK483 PB1, PB2-627K, PA, and NP proteins, pPolI-Luc, and ΔN-qRBL2. Twenty-four hours after incubation at 33°C, luciferase expression was detected. (B) Viral polymerase activity in cells expressing hRBL2 or qRBL2. 293 cells were transfected with plasmids expressing qRBL2 or hRBL2 or a control vector. Twenty-four hours later, cells were infected with strain WSN (MOI of 1). Three hours later, RNA was extracted and quantified by real-time RT-PCR with primer sets specific for NP vRNA, cRNA, or mRNA. These values were normalized to beta-actin. The error bars represent standard deviations (n = 3). (C) Viral M1 protein production in cells overexpressing hRBL2. 293 cells expressing hRBL2 or a control vector were infected with strain WSN (MOI of 3). At the indicated hours postinfection (hpi), cell lysates were subjected to Western blot analysis with antibodies against M1 and beta-actin. The values show the ratio of M1 to actin normalized to control cells 4 h after infection. (D) Influenza virus titers in 293 cells overexpressing hRBL2 or qRBL2. 293 cells overexpressing hRBL2 (square), qRBL2 (triangle), or a control vector (circle) were infected with strain WSN (MOI of 0.05). The cells were incubated at 37°C for the indicated time periods. Virus titers in the supernatant were determined by plaque assays in MDCK cells. The error bars represent standard deviations (n = 3). α, anti.